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Background: Vancomycin-resistant Enterococcus faecium (VREfm) has emerged as a nosocomial pathogen worldwide. The dissemination of VREfm is due to both clonal spread and spread of mobile genetic elements (MGEs) such as transposons.Objectives: We aimed to combine vanB-carrying transposon data with core-genome MLST (cgMLST) typing and epidemiological data to understand the pathways of transmission in nosocomial outbreaks.Methods: Retrospectively, 36 VREfm isolates obtained from 34 patients from seven VREfm outbreak investigations in 2014 were analysed. Isolates were sequenced on a MiSeq and a MinION instrument. De novo assembly was performed in CLC Genomics Workbench and the hybrid assemblies were obtained through Unicycler v0.4.1. Ridom SeqSphere+ was used to extract MLST and cgMLST data. Detailed analysis of each transposon and their integration points was performed using the Artemis Comparison Tool (ACT) and multiple blast analyses.Results: Four different vanB transposons were found among the isolates. cgMLST divided ST80 isolates into three cluster types (CTs); CT16, CT104 and CT106. ST117 isolates were divided into CT24, CT103 and CT105. Within VREfm isolates belonging to CT103, two different vanB transposons were found. In contrast, VREfm isolates belonging to CT104 and CT106 harboured an identical vanB transposon.Conclusions: cgMLST provides a high discriminatory power for the epidemiological analysis of VREfm. However, additional transposon analysis is needed to detect horizontal gene transfer. Combining these two methods allows investigation of both clonal spread as well as the spread of MGEs. This leads to new insights and thereby better understanding of the complex transmission routes in VREfm outbreaks.

Rapid and accurate detection of VRE (vancomycin-resistant enterococci) is required for adequate antimicrobial treatment and infection prevention measures. Previous studies using PCR for the detection of VRE, including Cepheid's Xpert vanA / vanB assay, reported accurate detection of vanA VRE; however, many false-positive results were found for vanB VRE. This is mainly due to nonenterococcal vanB genes, which can be found in the gut flora. Our goal was to optimize the rapid and accurate detection of vanB VRE and to improve the positive predictive value (PPV) by limiting false-positive results. We evaluated the use of the Xpert vanA / vanB assay on rectal swabs and on enriched inoculated broths for the detection of vanB VRE. By adjusting the cycle threshold ( C T ) cutoff value to ≤25 for positivity by PCR on enriched broths, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 96.9%, 100%, 100%, and 99.5% for vanB VRE, respectively. As shown in this study, C T values of ≤25 acquired from enriched broths can be considered true positive. For broths with C T values between 25 and 30, we recommend confirming the results by culture. C T values of >30 appeared to be true negative. In conclusion, this study shows that the Cepheid's Xpert vanA / vanB assay performed on enriched inoculated broths with an adjusted cutoff C T value is a useful and rapid tool for the detection of vanB VRE.

The study describes the transmission of a CTX-M-15-producing ST15 Klebsiella pneumoniae between patients treated in a single center and the subsequent inter-institutional spread by patient referral occurring between May 2012 and September 2013. A suspected epidemiological link between clinical K. pneumoniae isolates was supported by patient contact tracing and genomic phylogenetic analysis from May to November 2012. By May 2013, a patient treated in three institutions in two cities was involved in an expanding cluster caused by this high-risk clone (HiRiC) (local expansion, CTX-M-15 producing, and containing hypervirulence factors). A clone-specific multiplex PCR was developed for patient screening by which another patient was identified in September 2013. Genomic phylogenetic analysis including published ST15 genomes revealed a close homology with isolates previously found in the USA. Environmental contamination and lack of consistent patient screening were identified as being responsible for the clone dissemination. The investigation addresses the advantages of whole-genome sequencing in the early detection of HiRiC with a high propensity of nosocomial transmission and prolonged circulation in the regional patient population. Our study suggests the necessity for inter-institutional/regional collaboration for infection/outbreak management of K. pneumoniae HiRiCs.

Contains fulltext : 172306.pdf (Publisher’s version ) (Closed access) A survey of diagnosis and treatment of invasive aspergillosis was conducted in eight University Medical Centers (UMCs) and eight non-academic teaching hospitals in the Netherlands. Against a background of emerging azole resistance in Aspergillus fumigatus routine resistance screening of clinical isolates was performed primarily in the UMCs. Azole resistance rates at the hospital level varied between 5% and 10%, although rates up to 30% were reported in high-risk wards. Voriconazole remained first choice for invasive aspergillosis in 13 out of 16 hospitals. In documented azole resistance 14 out of 16 centres treated patients with liposomal amphotericin B.

Meningitis in adults due to infection with Mycoplasma hominis is rarely reported. Here, we document the third case of M. hominis meningitis in an adult individual, developed upon neurosurgery following a subarachnoid haemorrhage. Our findings are noteworthy, because the presence of M. hominis in cerebrospinal fluid cannot be identified by standard culturing, Gram-staining, or matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Importantly, however, 16S rDNA sequencing did lead to an unambiguous diagnosis and guided successful antimicrobial therapy. Based on our present findings and a review of the respective literature, we conclude that M. hominis should be considered as a candidate causative agent of infections of the central nervous system following neurosurgical procedures, especially if there is no response to standard antimicrobial therapy, and routine culturing yields negative results. (C) 2012 Elsevier GmbH. All rights reserved.

The detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) infections and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settings as early and swiftly as possible. In this study, we investigated the application potential of a new approach based on multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and microfluidics technology for a rapid discrimination of MRSA lineages in outbreak settings. A total of 206 nonrepetitive MRSA isolates recovered from infected patients at the University Medical Center Groningen between 2000 and 2010 were tested. The results obtained by MLVF using microcapillary electrophoresis with newly designed primers were compared to those obtained by spa typing and multiple-locus variable-number tandem-repeat analysis (MLVA). The discriminatory power was 0.980 (107 patterns), 0.969 (85 allelic profiles), and 0.959 (66 types) for MLVF, MLVA, and spa typing, respectively. All methods tested showed a good concordance of results calculated by the adjusted Rand's coefficient method. Comparisons of data obtained by the three approaches allowed us to propose an 88% cutoff value for the similarity between any two MLVF patterns, which can be used in S. aureus epidemiological studies, including analyses of outbreaks and strain transmission events. Of the three tested methods, MLVF is the cheapest, fastest, and easiest to perform. MLVF applied to microfluidic polymer chips is a rapid, cheap, reproducible, and highly discriminating tool to determine the clonality of MRSA isolates and to trace the spread of MRSA strains over periods of many years. Although spa typing should be used due to its portability of data, MLVF has a high added value because it is more discriminatory.

Sequencing of at least 13 Staphylococcus aureus isolates has shown that genomic plasticity impacts significantly on the repertoire of virulence factors. However, genome sequencing does not reveal which genes are expressed by individual isolates. Here, we have therefore performed a comprehensive survey of the composition and variability of the S. aureus exoproteome. This involved multilocus sequence typing, virulence gene, and prophage profiling by multiplex PCR, and proteomic analyses of secreted proteins using 2-DE. Dissection of the exoproteomes of 25 clinical isolates revealed that only seven out of 63 identified secreted proteins were produced by all isolates, indicating a remarkably high exoproteome heterogeneity within one bacterial species. Most interesting, the observed variations were caused not only by genome plasticity, but also by an unprecedented variation in secretory protein production due to differences in transcriptional and post-transcriptional regulation. Our data imply that genomic studies on virulence gene conservation patterns need to be complemented by analyses of the extracellular protein pattern to assess the full virulence potential of bacterial pathogens like S. aureus. Importantly, the extensive variability of secreted virulence factors in S. aureus also suggests that development of protective vaccines against this pathogen requires a carefully selected combination of invariably produced antigens.

Objective.To reveal the source of a nosocomial outbreak of colonization and infection with a strain ofSerratia marcescenspositive for Guiana extended-spectrumβ-lactamase 1 (GES-1) that occurred among patients in a neurosurgical intensive care unit (ICU) in a Dutch university medical center from May 2002 through March 2003.Methods.Samples from the environment and from the hands of healthcare workers (HCWs) were cultured. A retrospective case-control study was carried out.Results.Fifteen neurosurgical ICU patients who had 1 or more cultures that yielded the epidemic strain ofS. marcescensfrom May 2002 through March 2003 were defined as case patients and matched with 30 control patients. Environmental cultures did not reveal a prominent source ofS. marcescens. Cultures of specimens from the hands of 100 HCWs revealed colonization of a single HCW with the epidemic strain. Although this HCW instantly went on leave, serial cultures detected prolonged carriage of the epidemic strain on the hands of the HCW for 3 months. The skin of the HCW's hands was psoriatic. The epidemic abruptly ended after the colonized HCW went on leave. Retrospective case-control analysis showed that the patients colonized or infected withS. marcescensreceived significantly more nursing care from the colonized HCW than did control patients (PS. marcescens. In October 2004, the formerly colonized HCW appeared to have carriage of the epidemic strain on the hands again.Conclusions.A single HCW with the epidemic strain ofS. marcescenson the hands was considered the source of this outbreak.

The efficacy of anidulafungin is driven by the area under the concentration-time curve (AUC)/MIC ratio. Patients in intensive care may be at risk for underexposure. In critically ill patients with an invasive Candida infection, the anidulafungin exposure and a possible correlation with disease severity or plasma protein levels were explored. Concentration-time curves were therefore obtained at steady state. Anidulafungin concentrations were measured with a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The MIC values of the Candida species were determined with the Etest. The target AUC/MIC ratio was based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) data. Twenty patients were included. The patients received a maintenance dose of 100 mg once daily after a loading dose of 200 mg on the first day. The mean (±standard deviation) AUC, maximum concentration of drug in plasma ( C max ), and minimum concentration of drug in plasma ( C min ) were 69.8 ± 24.1 mg · h/liter, 4.7 ± 1.4 mg/liter, and 2.2 ± 0.8 mg/liter, respectively. The MIC values of all cultured Candida species were below the EUCAST MIC breakpoints. The exposure to anidulafungin in relation to the MIC that was determined appeared sufficient in all patients. The anidulafungin exposure was low in our critically ill patients. However, combined with the low MICs of the isolated Candida strains, the lower exposure observed in comparison to the exposure in the general patient population resulted in favorable AUC/MIC ratios, based on EUCAST data. No correlation was observed between anidulafungin exposure and disease severity or plasma protein concentrations. In patients with less-susceptible Candida albicans or glabrata strains, we recommend considering determining the anidulafungin exposure to ensure adequate exposure. (This trial has been registered at ClinicalTrials.gov under registration no. NCT01047267.)

The remineralization of whole human dentine treated with a neutral EDTA solution was investigated: the treatment periods were 15, 30 and 120 min. From the literature it is known that EDTA removes noncollagenous proteins (NCPs) from dentine powders. In order to extract more phosphoproteins from dentine lesions, in part of this work samples were also treated with 1 M NaCl or 4 M guanidine chloride solutions. All the dentine samples after the treatments mentioned were immersed subsequently in a remineralizing solution without fluoride for 2 weeks and microradiographed. To investigate the effects of fluoride, samples treated with EDTA for 120 min were also remineralized with 2ppm fluoride in solution. The results presented show that: (1) Measurable remineralization did not occur when fluoride was free in the remineralizing solution. Because remineralization occurred neither at the lesion front nor in the nonmineral part of the surface–softened tissue, presumably the NCPs (inhibitors) of underlying dentine diffused into the tissue during the remineralization period. (2) 2ppm fluoride caused remineralization at the lesion front. In this case we assume fluoride either acted as nucleating agent or overcame the NCP effects at the lesion front.

Methicillin resistant S. aureus (MRSA) is a major threat for human health and well-being. In recent years, it has become clear that livestock is a potential reservoir for MRSA, most livestock-associated isolates belonging to the ST398 lineage. Importantly, ST398 strains were also reported as causative agents of severe invasive infections in humans with no evidence for livestock associations. Here we document the sequence of the J1 region of the type V (5C2&5) SCCmec element and its right chromosomal junction in the clinical PVL-positive ST398 MRSA isolate UMCG-M4. Sequence comparisons show that this SCCmec element and related type V elements from other S. aureus isolates share a common core structure, but differ substantially in the so-called J1 region. Additional PCR analyses and typing studies indicate that the J1 region of strain UMCG-M4 is specific for SCCmec elements of PVL-positive ST398 isolates. Lastly, we show that the sequenced right chromosomal junction is invariant in strains of the ST398 lineage. (C) 2013 Elsevier GmbH. All rights reserved.

Fluoridated composites are able to reduce or inhibit secondary caries around fillings. The aims of this study were firstly to investigate the amount of F released in vitro from composites with a F content between 0 and 26 vol% as a function of time over a 1-year period. A second aim was to correlate the in vitro data on F release with enamel demineralisation data in situ after a 1-month period. The results show that all fluoridated composites release sizable amounts of F in solution, the total amounts of F released from three composites were proportional to log t over at least 1 year. In one case proportionality of the total amount of F released with t was observed. Possible mechanisms of F release are considered. The results presented also show a linear relation between the log of in vitro F release data and the effects on enamel demineralisation in situ next to the composite. Extrapolation of the data reveals that a F release of about 200-300 micrograms/cm2 over a 1-month period from a fluoridated composite would completely inhibit secondary caries under plaque conditions.

Contains fulltext : 89507.pdf (Publisher’s version ) (Open Access) We report the use of PCR techniques on a formalin-fixed and paraffin-embedded tissue specimen for direct detection of one dominant azole resistance mechanism in a case of disseminated invasive aspergillosis. Rapid detection of mutations associated with azole resistance directly in tissue significantly reduces diagnostic delay. 01 april 2010

Endoscopes, including duodenoscopes, are medical devices that are frequently associated with outbreaks of nosocomial infections. We investigated an outbreak of multidrug-resistant Pseudomonos aeruginosa sepsis affecting three patients after endoscopic retrograde cholangiopancreaticography (ERCP). Epidemiologic investigation supplemented by molecular typing revealed that one ERCP scope was the source of infection with R aeruginosa. No contamination with this microorganism was found after screening of washer-disinfectors, connecting tubes, and environmental surfaces in the endoscopy center. Pseudomonas isolates from blood and endoscope channels before gas sterilization with ethylene oxide (ETO) were characterized by molecular typing as "linked isolates". Though the current surveillance system did not prevent the infections in three patients, our microbiological surveillance protocol with routine culturing of endoscopes was helpful in detecting the source of contamination and probably avoided numerous cross-contaminations in other patients who underwent ERCP procedures with endoscopes.

Rhodococcus equi, mainly known from veterinary medicine as a pathogen in domestic animals, can also cause infections in immunocompromised humans, especially in those with defects in cellular immunity. Alemtuzumab, an anti-CD52 monoclonal antibody, causes lymphocytopenia by eliminating CD52-positive cells. We report a patient in whom Rhodococcus equi infection developed after alemtuzumab therapy.

Methicillin-resistant Staphylococcus aureus (MRSA) belonging to the multilocus sequence type clonal complex 59 (MLST CC59) is the predominant community-associated MRSA clone in Asia. This clone, which is primarily linked with the spa type t437, has so far only been reported in low numbers among large epidemiological studies in Europe. Nevertheless, the overall numbers identified in some Northern European reference laboratories have increased during the past decade. To determine whether the S. aureus t437 clone is present in other European countries, and to assess its genetic diversity across Europe, we analysed 147 S. aureus t437 isolates from 11 European countries collected over a period of 11 years using multiple locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and MLST. Additionally 16 S. aureus t437 isolates from healthy carriers and patients from China were included. Most isolates were shown to be monophyletic with 98% of the isolates belonging to the single MLVA complex 621, to which nearly all included isolates from China also belonged. More importantly, all MLST-typed isolates belonged to CC59. Our study implies that the European S. aureus t437 population represents a genetically tight cluster, irrespective of the year, country and site of isolation. This underpins the view that S. aureus CC59 has been introduced into several European countries, not being restricted to particular geographical regions or specific host environments. The European S. aureus t437 isolates thus bear the general hallmarks of a high-risk clone.

Background, Gram-negative bacteremia in children, a major cause of morbidity and mortality, may in part be induced by intensive treatment procedures and nonspecific use of antibiotics. Our primary objective was to study the causal relationship between the use of vancomycin and Gram-negative bacteremia, for which this antibiotic is not specifically indicated. Methods. The study was conducted in a 105-bed tertiary care children's hospital in the period of 1994 to 1997, The study pertains to a cohort of children with suspected bacteremia, in whom a blood culture was performed during hospital stay. Using the bacteriologic laboratory registration system, we selected all pediatric cases with bacteriologically proved Gram-negative bacteremia (n = 105) and a random sample of 225 pediatric controls with negative blood cultures. Using logistic regression analysis we examined associations between Gram-negative bacteremia and the following factors: preceding use of antibiotics, antacids, corticosteroids, surgery, mechanical ventilation, parenteral nutrition, and invasive instrumentation; and the intensity of care assessed with the Therapeutic Intensity Scoring System (TISS 28), Results. Gram-negative bacteremia was positively associated with the use of aminoglycosides, cephalosporins, surgical interventions, central venous catheters, parenteral nutrition, antacids and dexamethasone, The strongest association was with the use of vancomycin (odds ratio, 8.1; 95% confidence interval, 3.1 to 20,9), In a multiple logistic regression model containing all above-mentioned variables, the use of vancomycin remained positively and strongly associated with Gram-negative bacteremia (odds ratio, 3.88; 95% confidence interval, 1.34 to 11,21), Further adjustments and restrictions in the analysis did not materially change these findings concerning vancomycin. Conclusions. Among children suspected of bacteremia there are several drugs and clinical procedures influencing the risk for Gram-negative bacteremia. Empiric use of vancomycin is strongly and independently associated with Gram-negative bacteremia, The safety of using vancomycin solely on the basis of suspicion of bacteremia in children may not be warranted.

When lesion formation in dental enamel or dentine is studied in vitro, a well–defined area of the material, a 'window', is exposed to the demineralization medium. In the present experiments, we report the effect of window width on lesion formation in enamel and dentine. Rectangular windows, longest dimension always 5 mm and shortest dimension varying from 0.5 to 5 mm, were exposed to demineralization media. Lesions in dentine were formed by placing the specimens in an acidified unstirred 6% carboxymethyl cellulose (CMC) gel, an unstirred 0.5% CMC gel, a mildly stirred 0.5% CMC gel, or in a mildly stirred demineralization solution. Lesions in enamel were formed only in an unstirred acidified 6% CMC gel. pH in the demineralization media was 5.0 in all experiments. The microradiographic technique was used to assess lesion depth and mineral loss (vol%·μm) of lesions formed. Lesions formed in the unstirred gels were deeper, and there was a larger mineral loss, the narrower the window. There was no effect of window size on lesions formed in stirred systems. These effects can be explained by the strong dependence of concentration gradient in stirred gels just outside the exposed window on the window size. The phenomenon found here in vitro may at least partly explain why secondary caries may lead to localized, but relatively deep, lesions.

Pantoea infections are rare in humans, especially in neonates. Infections are usually associated with plant thom injury or outbreaks traced to contaminated parenteral nutrition, intravenous anesthetics or packed er throcytes. Between I st of January 1994 and Ist of June 2005, 125 cf 6383 patients (2%) in a 24-bed level III NICU became colonized with Pantoea agglomerans. Three new-borns exhibited late-onset Pantoea agglomerans septicemia and died. Sporadic cases of Pantoea agglomerans septicemia have not been reported in neonatal intensive care so far.

A case of bacteremia due to Ochrobactrum intermedium , with concomitant liver abscesses, in an orthotopic liver transplant recipient is presented. Identical microorganisms were isolated from fecal specimens and from an aspirate of a liver abscess that was indicative of invasion of the graft by gastrointestinal spread. 16S DNA sequence analysis of the blood isolate revealed the recovery of the recently proposed new species O. intermedium , closely related to Ochrobactrum anthropi and Brucella spp.

The aim of this investigation was to study the effect of the interproximal use of fluoride (F)–impregnated and non–impregnated birch toothpicks on the degree of de– and remineralization of enamel and dentine in situ. Ten volunteers with complete dentures in the upper jaw participated. Each subject had four specimens: (1) sound enamel, (2) demineralized enamel, (3) sound dentine and (4) demineralized dentine; placed pairwise at two approximal sites (15/16 and 25/26) of the maxillary prosthesis. The study involved three test periods (A, B and C), each lasting 4 weeks. In A, the subjects used F toothpicks (impregnated in 4% NaF) and, in B, nonimpregnated toothpicks 3 times daily. During period C, no toothpicks were used. Dentifrice or other F–containing products were not allowed during the 4–week periods. Transversal microradiography was used to determine lesion depth (ld) and mineral loss (ΔZ). The results revealed that all the sound samples lost mineral during the three experimental periods; ΔZ for both enamel and dentine was less for A and B compared with C (p

The aim was to study the effect of two different water rinsing procedures after tootbrushing with an NaF-containing dentifrice on the degree of de- or remineralization of enamel and dentine at approximal and buccal sites. Seven adults, wearing complete dentures, participated in two experimental periods (A and B) in a randomised order. During period A, they blushed with the dentifrice for 2 min, followed by 1-min active mouthrinse with the toothpaste-foam combined with 10 ml of water. No more water was used after the slurry had been spat out. During period B, the brushing was followed by 3 thorough rinsings of approximately 15 ml water each. These two procedures were carried out twice daily, i.e. in the morning (after breakfast) and in the evening (just before bedtime), during 3 months. Demineralized enamel and dentine samples were mounted at two locations - approximally and buccally - in the first molar region of the upper prostheses. Quantitative microradiography (TMR) was used to assess the lesion depth (Id) and the mineral loss (Delta Z). The results showed that the approximally located samples continued to lose mineral during both periods A and B. However, the Id and Delta Z values for enamel (p

We describe the introduction of various multi-drug resistant bacterial strains, including an NDM-1-producing Klebsiella pneumoniae, through a traveller returning from Egypt, where they had been admitted to a private hospital. All family members of the patient were colonised with one or more extended-spectrum beta-lactamase producing strains. These findings emphasise the importance of adherence to isolation precautions for returning patients and suggest the need for inclusion of Enterobacteriaceae in admission screening. We here report of a patient who had been hospitalised in Egypt for appendicitis in July 2013, and was colonised with various multiresistant Enterobacteriaceae including strains producing NDM-1, oxacillinase-48 (OXA-48) and extended spectrum beta-lactamase (ESBL). Explorative screening for multiresistant microorganisms among the patient's family members also yielded several ESBL-producing microorganisms. This report addresses the need for heightened awareness of patients and family members who have recently been exposed to healthcare environments in countries with high levels of antibiotic resistance. Patients repatriated after hospitalisation abroad are a risk for introducing multiresistant microorganisms into hospitals in their home countries. In 2008, New Delhi metallo-beta-lactamase (NDM), which hydrolyses last-line carbapenem antibiotics, has been for the first time described in a Swedish patient returning from India [1]. Most reports on NDM are related to travellers returning from Pakistan and India. However, the global dispersal of NDM is of growing concern [2]. In the past two years, NDM-producing strains have been reported in patients returning from the African continent without obvious links to the Indian subcontinent [3,4].