Introduction: 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. We aimed to combine vanB-carrying transposons characteristics 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, 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 were performed using Artemis Comparison Tool (ACT) and multiple blast analyses. Results Four different vanB transposons were found among the isolates. CgMLST divided ST80 isolates into three 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. Conclusion: 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 to investigate 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.

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.

Background: The occurrence of highly resistant microorganisms (HRMOs) is a major threat to critical care patients, leading to worse outcomes, need for isolation measures, and demand for second-line or rescue antibiotics. The aim of this study was to quantify the burden of HRMOs in an intensive care unit (ICU) for adult patients in a university hospital in the Netherlands. We evaluated local distribution of different HRMO categories and proportion of ICU-imported versus ICU-acquired HRMOs. Outcome of HRMO-positive patients versus controls was compared.Methods: In this prospective single-centre study, culture results of all ICU patients during a four-month period were recorded, as well as APACHE scores, ICU mortality and length of stay (LOS) in the ICU.Results: 58 of 962 (6.0%) patients were HRMO positive during ICU stay. The majority (60%) of those patients were HRMO positive on ICU admission. HRMO-positive patients had significantly higher APACHE scores, longer LOS and higher mortality compared with controls.Conclusions: Our study suggests that a large part of antibiotic resistance in the ICU is imported. This underscores the importance of a robust surveillance and infection control program throughout the hospital, and implies that better recognition of those at risk for HRMO carriage before ICU admission may be worthwhile. Only a small minority of patients with HRMO at admission did not have any known risk factors for HRMO.

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.

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 (CT) 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, CT values of ≤ 25 acquired from enriched broths can be considered true positive. For broths with CT values between 25 and 30, we recommend confirming the results by culture. CT 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 CT value is a useful and rapid tool for the detection of vanB VRE.

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.

Anti-platelet drugs have been used to treat inflammatory bowel disease. In this study, we observed the therapeutic effects of magnesium lithospermate B, a main component of salvianolate, on colitis induced by dextran sodiumsulfate (DSS). Colitis was induced by 5% DSS oral administration in BALB/C male mice. Magnesium lithospermate B (60–240 mg/kg) was given by subcutaneous injection for 2 weeks. Then, mice were sacrificed; serum and colon tissues were collected for biomarker assay, histological examination, immunohistochemical study and real-time quantitative polymerase chain reaction. DSS induced gross bleeding, inflammation, crypt damage and mucosal damage in colon. Treatment with magnesium lithospermate B could reduce colon inflammation induced by DSS. Magnesium lithospermate B could reverse the high CD40/CD40L expression and hypercoagulable state induced by DSS in colon. This study showed that magnesium lithospermate B could be used to treat colitis. The protective effects of magnesium lithospermate B may be due to its effects on CD40/CD40L expression and blood clotting status.

Background Selective digestive tract decontamination ( SDD) and selective oropharyngeal decontamination ( SOD) are infection- prevention measures used in the treatment of some patients in intensive care, but reported effects on patient outcome are conflicting.Methods We evaluated the effectiveness of SDD and SOD in a crossover study using cluster randomization in 13 intensive care units ( ICUs), all in the Netherlands. Patients with an expected duration of intubation of more than 48 hours or an expected ICU stay of more than 72 hours were eligible. In each ICU, three regimens ( SDD, SOD, and standard care) were applied in random order over the course of 6 months. Mortality at day 28 was the primary end point. SDD consisted of 4 days of intravenous cefotaxime and topical application of tobramycin, colistin, and amphotericin B in the oropharynx and stomach. SOD consisted of oropharyngeal application only of the same antibiotics. Monthly point- prevalence studies were performed to analyze antibiotic resistance.Results A total of 5939 patients were enrolled in the study, with 1990 assigned to standard care, 1904 to SOD, and 2045 to SDD; crude mortality in the groups at day 28 was 27.5%, 26.6%, and 26.9%, respectively. In a random- effects logistic- regression model with age, sex, Acute Physiology and Chronic Health Evaluation (APACHE II) score, intubation status, and medical specialty used as covariates, odds ratios for death at day 28 in the SOD and SDD groups, as compared with the standard- care group, were 0.86 ( 95% confidence interval [CI], 0.74 to 0.99) and 0.83 ( 95% CI, 0.72 to 0.97), respectively.Conclusions In an ICU population in which the mortality rate associated with standard care was 27.5% at day 28, the rate was reduced by an estimated 3.5 percentage points with SDD and by 2.9 percentage points with SOD. (Controlled Clinical Trials number, ISRCTN35176830.).

Endoscopes, including duodenoscopes, are medical devices that are frequently associated with outbreaks of nosocomial infections. We investigated an outbreak of multidrug-resistant PSEUDOMONAS 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 P. 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.

Infections caused by community-acquired methicillin-resistant Staphylococcus aureus (MRSA) are emerging as a major public health problem. In this study, we describe the distribution of 54 Panton-Valentine leucocidin (PVL)-carrying MRSA isolates in the northern Netherlands between 1998 and 2005, of which 43 (80%) consisted of the European PVL-positive strain multi locus sequence type 80 with staphylococcal cassette chromosome mec type IVc (ST80). Individual cases and small clusters of ST80 predominated in the community (74%), but ST80 was also found in nursing homes (16%) and hospitals (9%). Long-term carriership (months to years) and reinfection of patients with ST80 has probably led to the strain spreading in the community and subsequently to further migration to health care environments.

SUMMARY The gram-positive bacterium Staphylococcus aureus is a frequent component of the human microbial flora that can turn into a dangerous pathogen. As such, this organism is capable of infecting almost every tissue and organ system in the human body. It does so by actively exporting a variety of virulence factors to the cell surface and extracellular milieu. Upon reaching their respective destinations, these virulence factors have pivotal roles in the colonization and subversion of the human host. It is therefore of major importance to obtain a clear understanding of the protein transport pathways that are active in S. aureus . The present review aims to provide a state-of-the-art roadmap of staphylococcal secretomes, which include both protein transport pathways and the extracytoplasmic proteins of these organisms. Specifically, an overview is presented of the exported virulence factors, pathways for protein transport, signals for cellular protein retention or secretion, and the exoproteomes of different S. aureus isolates. The focus is on S. aureus , but comparisons with Staphylococcus epidermidis and other gram-positive bacteria, such as Bacillus subtilis , are included where appropriate. Importantly, the results of genomic and proteomic studies on S. aureus secretomes are integrated through a comparative “secretomics” approach, resulting in the first definition of the core and variant secretomes of this bacterium. While the core secretome seems to be largely employed for general housekeeping functions which are necessary to thrive in particular niches provided by the human host, the variant secretome seems to contain the “gadgets” that S. aureus needs to conquer these well-protected niches.

Department of Medical Microbiology, University Medical Centre Groningen and University of Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB Groningen, The Netherlands; Institut für Mikrobiologie, Ernst-Moritz-Arndt Universität, Greifswald, F.-L.-Jahnstr. 15, D-17487 Greifswald, Germany; Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, 9751 NN Haren, The Netherlands; and Department of Cell Biology and Electron Microscopy, University Medical Centre Groningen and University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands

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.

Enterococci are increasingly recognized as important nosocomial pathogens. Studies in children have shown that neonates and compromised hosts are especially at risk; that bacteremia and sepsis were the most prominent manifestations of infection; that enterococcal bacteremia occurred often in the setting of polymicrobial infections and that the attributable mortality was about 20-25% [1Christie C Hammond J Reising S Patterson JE Clinical and molecular epidemiology of enterococcal bacteremia in a pediatric teaching hospital.J Pediatr. 1994; 125: 392-399Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 2Dobson SRM Baker CJ Enterococcal sepsis in neonates: features by age at onset and occurrence of focal infection.Pediatrics. 1990; 85: 165-171PubMed Google Scholar, 3Bonadio WA Group D streptococcal bacteremia in children.Clin Pediatr. 1993; 32: 20-24PubMed Google Scholar, 4Nourse C Murphy H Byrne C et al.Control of a nosocomial outbreak of vancomycin resistant Enterococcus faecium in a paediatric oncology unit: risk factors for colonization.Eur J Pediatr. 1998; 157: 20-27Crossref PubMed Scopus (57) Google Scholar]. In the present study risk factors for colonization with enterococci in newborns are described. A retrospective case-control study on risk factors for colonization with enterococci was performed in the neonatal intensive care unit (NICU) of the Beatrix Children's Hospital, Groningen, The Netherlands. All newborns admitted to the unit from 1 January 1994 until 31 December 1994 were included. Fecal and throat cultures were taken on admission and twice weekly afterwards. Infants were considered colonized when enterococci were isolated on esculin plates (Merck, Darmstadt, Germany) at least once from throat or faeces. Only faeces cultures in which enterococci were predominant were considered positive. Controls were a random selection of the newborns admitted during the study period and not colonized with enterococci during their stay in the NICU. Resistance to vancomycin was not determined in this study, since it is still a very rare event in the Netherlands [5Endtz HP van den Braak N van Belkum A et al.Fecal carriage of vancomycin-resistant enterococci in hospitalized patients and those living in the community in The Netherlands.J Clin Microbiol. 1997; 35: 3026-3031PubMed Google Scholar]. Between 1 January 1994 and 31 December 1994, a total of 579 newborns were admitted of which 116 (20%) were colonized with enterococci. The site of colonization was the faeces in 54 patients, the throat in 51 patients and both faeces and throat in 11 patients. In an univariate analysis of possible risk factors for colonization with enterococci a shorter gestational age (P < 0.001), a lower birth weight (P = 0.02), an increased number of days of hospitalization, mechanical ventilation, treatment via a central venous line, parenteral nutrition and antibiotic use (all P < 0.001) were associated with enterococcal colonization. Major surgery and episodes of clinical features compatible with infection were also risk factors (P < 0.001). Multiple regression analysis showed that a longer gestational age [odds ratio(OR), 2.12; 95% confidence interval (CI), 1.04-4.34], a longer duration of hospitalization (OR, 1.07; 95% CI, 1.04-1.09), the use of antibiotics other than amoxicillin (OR, 3.32; 95% CI, 1.30-8.50) and the use of a central venous line (OR, 2.51; 95% CI, 1.12-5.63) were the only independent risk factors (Table 1).Table 1Multiple regression analysis of risk factors for colonization with enterococciOR95% CIPGestational age >37 weeks2.121.04-4.340.039Days of hospitalization1.071.04-1.09<0.001Deep venous line in place2.511.12-5.630.026Antibiotics other than amoxicillin3.321.30-8.500.013OR, odds ratio; CI, confidence interval. Open table in a new tab OR, odds ratio; CI, confidence interval. Neonates with these risk factors should be monitored closely for the development of enterococcal bacteremia and sepsis.