Whole-genome sequencing analysis reveals the spread of a vanB-carrying transposon among different vancomycin-resistant Enterococcus faecium clinical isolates in a non-endemic setting.

BACKGROUND: Vancomycin-resistant enterococci (VRE), particularly Enterococcus faecium (VREfm), can cause serious nosocomial infections, and have been responsible for healthcare-associated outbreaks. Spreading of VREfm can occur both clonally and by the dissemination of mobile genetic elements. AIM: To report prospective analysis of whole-genome sequencing (WGS) data, including both core-genome multi-locus sequence typing (cgMLST) and transposon analysis, during a vanB VREfm outbreak. METHODS: Screening for vanB-positive VREfm isolates was performed by real-time polymerase chain reaction (PCR) on an overnight enriched broth and, if positive, subculture was performed. vanB-positive VREfm isolates underwent WGS. Generated data were used for molecular typing that was performed by cgMLST using SeqSphere. For transposon characterization, sequence data were mapped against the reference sequence of transposon Tn1549 using CLC Genomics Workbench, or de-novo assemblies were used for BLASTN comparisons. RESULTS: In total, 1358 real-time PCRs were performed. Two hundred and fifty-one specimens from 207 patients tested positive on PCR for vanB, of which 13 specimens obtained from six patients were identified as vanB VREfm positive on culture. These six patients harboured seven unique isolates belonging to four cluster types: CT118 (N=2), CT2483 (N=3), CT2500 (N=1) and CT2501 (N=1). Transposon analysis revealed the presence of an identical vanB-carrying transposon in the isolates cultured from all six patients that could be linked based on epidemiological data. CONCLUSION: A vanB VREfm outbreak occurred in the study hospital, including six patients with isolates belonging to four cluster types. In-depth transposon analysis revealed that dissemination of transposon Tn1549 rather than clonal spread was the cause of the outbreak.

Short-course aminoglycosides as adjunctive empirical therapy in patients with Gram-negative bloodstream infection, a cohort study.

OBJECTIVE: Short-course aminoglycosides as adjunctive empirical therapy to ß-lactams in patients with a clinical suspicion of sepsis are used to broaden antibiotic susceptibility coverage and to enhance bacterial killing. We quantified the impact of this approach on 30-day mortality in a subset of sepsis patients with a Gram-negative bloodstream infection. METHODS: From a prospective cohort study conducted in seven hospitals in the Netherlands between June 2013 and November 2015, we selected all patients with Gram-negative bloodstream infection (GN-BSI). Short-course aminoglycoside therapy was defined as tobramycin, gentamicin or amikacin initiated within a 48-hour time window around blood-culture obtainment, and prescribed for a maximum of 2 days. The outcome of interest was 30-day all-cause mortality. Confounders were selected a priori for adjustment using a propensity score analysis with inverse probability weighting. RESULTS: A total of 626 individuals with GN-BSI who received ß-lactams were included; 156 (24.9%) also received aminoglycosides for a median of 1 day. Patients receiving aminoglycosides more often had septic shock (31/156, 19.9% versus 34/470, 7.2%) and had an eight-fold lower risk of inappropriate treatment (3/156, 1.9% versus 69/470, 14.7%). Thirty-day mortality was 17.3% (27/156) and 13.6% (64/470) for patients receiving and not receiving aminoglycosides, respectively; yielding crude and adjusted odds ratios for 30-day mortality for patients treated with aminoglycosides of 1.33 (95% CI 0.80-2.15) and 1.57 (0.84-2.93), respectively. CONCLUSIONS: Short-course adjunctive aminoglycoside treatment as part of empirical therapy with ß-lactam antibiotics in patients with GN-BSI did not result in improved outcomes, despite better antibiotic coverage of pathogens.

First report of a brain abscess caused by Nocardia veterana.

Among Nocardia species causing infections, Nocardia veterana is rarely isolated and is mostly described as causing pulmonary infections. This is the first presentation of a case of brain abscess attributable to an N. veterana infection in a patient with type 2 diabetes. Prolonged antibiotic therapy with trimethoprim-sulfamethoxazole led to successful clinical recovery.

[Relationship between rubella virus and Fuchs heterochromic uveitis; 2 patients]. / Samenhang tussen rubellavirus en fuchs-heterochromie-uveïtis; 2 patiënten.

Two otherwise healthy men, aged 26 and 29 years, were diagnosed with Fuchs heterochromic uveitis (FHU) on the basis of the presence of iris heterochromia or iris atrophy, stellate corneal precipitates, and/or cataract. Microbiological investigation of aqueous humour demonstrated intraocular antibody production against rubella virus, but not against Toxoplasma gondii, herpes simplex virus or varicella zoster virus. Microbial nucleic acid detection was negative for all pathogens. Some time later, both patients underwent cataract surgery, which improved their vision considerably. FHU is a chronic, generally unilateral iridocyclitis, accompanied by the above-mentioned ophthalmologic manifestations in the absence of systemic disease. Little is known about the pathogenesis ofFHU, but recent publications have provided evidence for the possible involvement of the rubella virus.

[Patients co-infected with HIV and hepatitis-B virus (HBV): the favourable effect of lamivudine, as part of combined antiretroviral therapy, on HBV may be dependent upon the number of CD4-cells]. / Patiënten met een coïnfectie van HIV en hepatitis-B-virus (HBV): gunstig effect van lamivudine, als onderdeel van antiretrovirale combinatiebehandeling, op HBV mogelijk afhankelijk van het CD4-celaantal.

OBJECTIVE: To determine the effect of lamivudine on HBV co-infection in HIV-infected patients. DESIGN: Retrospective METHOD: The HBsAg status and the use of lamivudine were determined retrospectively in a cohort of 800 HIV-infected patients under treatment at the Infectious Diseases outpatient clinic of the University Medical Centre in Utrecht, The Netherlands. In the group of HBsAg-positive patients using lamivudine 150 mg twice daily as part of highly active antiretroviral therapy (HAART), the HBV-DNA was measured quantitatively in the remaining plasma. In addition, the HBsAg, HBeAg, activity of alanineaminotransferase (ALAT) and CD4-count were obtained from the patient records. RESULTS: The study identified 29 (3.6%) HIV-infected patients to be HBsAg-positive. Plasma samples of 14 of these 29 patients were positive for HBV-DNA before the start of the therapy. Ten of these 14 patients had CD4 counts of at least 200 x 10(6) cells/l, while four patients had less than 200 x 10(6) cells/l. In contrast to the group with less than 200 x 10(6) cells/l, a significant decrease in HBV-DNA load was seen after six months of therapy in the patients with at least 200 x 10(6) CD4-cells/l (t-test for repeated measurements; p = o.oo1). The difference between the two groups in the effect of lamivudine was statistically significant (p = 0.021). At final evaluation after a mean follow-up of 32 and 13 months, respectively, HBV-DNA could no longer be detected in 7 patients; ALAT normalised in 9 patients (64%). CONCLUSION: In this retrospective study, lamivudine was effective in the therapy of HIV-infected patients with a HBV co-infection. The decrease in the amount of circulating HBV was associated with the number of CD4 cells.

Control of nosocomial multiresistant Enterobacteriaceae using a temporary restrictive antibiotic agent policy.

An observational study on the epidemiology of multiresistant Enterobacteriaceae was conducted in the neurology and neurosurgery wards of a university hospital to determine the impact of hospital hygiene measures and an additional temporary restrictive antibiotic agent policy on the sudden rise in incidence of these bacteria. The incidence and prevalence of patients with multiresistant Enterobacteriaceae were assessed, and patient isolates were typed phenotypically and by random amplified polymorphic DNA analysis. All hospital hygiene measures implemented were recorded, and the influence of the restrictive policy on antibiotic use was analyzed. This policy consisted of a prior authorization requirement and the withdrawal of all antibiotics with a possible selective pressure on multiresistant strains (gentamicin, tobramycin, quinolones, cotrimoxazole, broad-spectrum penicillins, and cephalosporins). This ban left only carbapenems and amikacin for treatment. Typing showed that 17 of the 61 (28%) patients involved were infected or colonized with a single multiresistant strain of Klebsiella oxytoca, for which an environmental source was identified. The isolates recovered from the other patients comprised eight different species, and subsequent genotyping yielded a great variety of strains. The increased incidence could not be controlled with hospital hygiene measures alone. Only after implementation of the restrictive antibiotic policy did the epidemic strain vanish and the endemic incidence of multiresistant Enterobacteriaceae decrease to <50% of the level before intervention. In the years since, the incidence has remained at this low level, and the antibiotic costs have decreased to a level lower than before intervention.

Prevalence and determinants of fecal colonization with vancomycin-resistant Enterococcus in hospitalized patients in The Netherlands.

OBJECTIVE: To determine the prevalence and determinants of fecal carriage of vancomycin-resistant enterococci (VRE) in intensive care unit (ICU), hematology-oncology, and hemodialysis patients in The Netherlands. DESIGN: Descriptive, multicenter study, with yearly 1-week point-prevalence assessments between 1995 and 1998. POPULATION: All patients hospitalized on the testing days in ICUs and hematology-oncology wards in nine hospitals in The Netherlands were included. METHODS: Rectal swabs obtained from 1,112 patients were screened for enterococci in a selective broth and subcultured on selective media with and without 6 mg/L vancomycin. Resistance genotypes were determined by polymerase chain reaction. Further characterization of VRE strains was done by pulsed-field gel electrophoresis (PFGE). We studied possible determinants of VRE colonization with a logistic regression analysis model. Determinants analyzed included gender, age, and log-transformed length of prior hospital stay. RESULTS: The results showed that 614 (55%) of 1,112 patients were colonized with vancomycin-sensitive enterococci, and 15 (1.4%) of 1,112 carried VRE. No increase in VRE colonization was observed from 1995 to 1998. Eleven strains were identified as Enterococcus faecium and four as Enterococcus faecalis. All E faecium and one E faecalis carried the vanA gene; the other E faecalis strains harbored the vanB gene. PFGE revealed that three vanB VRE isolated from patients hospitalized in one single ICU were related, suggesting nosocomial transmission. Though higher age seemed associated with VRE colonization, exclusion of patients with the nosocomial strain from the regression analysis decreased this relation to nonsignificant. Duration of hospital stay was not associated with VRE colonization. CONCLUSION: VRE colonization in Dutch hospitals is an infrequent phenomenon. Although nosocomial spread occurs, most observed cases were unrelated, which suggests the possibility of VRE acquisition from outside the hospital. Prolonged hospital stay, age, and gender proved unrelated to VRE colonization.

A pilot study on infection control in 10 randomly selected European hospitals: results of a questionnaire survey.

We describe and compare the organization of infection control and some infection control practices in 10 hospitals in seven different European countries. Great differences were observed. By evaluating infection control and hygiene practices in different European centers, areas of prime importance for the development of a European infection control standard may be defined.

[Epidemiologic increase of various genotypes of vancomycin-resistant Enterococcus faecium in a university hospital]. / Epidemische verheffing van verscheidene genotypen van vancomycineresistente Enterococcus faecium in een academisch ziekenhuis.

After a report of a possible relationship between an outbreak of vancomycin-resistant enterococci (VRE) in a nearby hospital and earlier admission of two of the patients with this VRE in the University Medical Centre of Utrecht (UMCU), the Netherlands, an extensive search for VRE carriers was started in the UMCU. In the study period of two months, VRE carriership was diagnosed in 51 patients in nine of the 11 wards investigated. Twenty-six patients in eight wards were colonized with the same VRE genotype as in the nearby hospital; spread was demonstrated in three wards. In addition, six patients of one ward were colonized with a second genotype and seven other patients with a third genotype, while 12 patients were carriers of a unique genotype. Most carriers were found in the internal medicine/nephrology and dialysis ward. Far-reaching measures (such as cohort nursing, admission stops, use of gowns and gloves, disinfection and restriction of use of vancomycin) taken in the four wards where spread was demonstrated, appeared effective but in three wards, spread was again demonstrated later. Frequent readmissions and transfers of patients appear to play an important part in this matter. None of the 51 colonized patients developed a serious VRE infection.

Determining risk factors for methicillin-resistant Staphylococcus aureus carriage after discharge from hospital.

At the University Medical Center, Utrecht, methicillin-resistant Staphylococcus aureus (MRSA) patients are considered lifelong MRSA carriers and potentially contagious when readmitted. The purpose of this study was to determine whether patients who become MRSA carriers while in hospital remain colonized after discharge, and whether risk factors for prolonged carriage exist. Thirty-six patients colonized with MRSA during three outbreaks at University Medical Center, Utrecht (group I: 1986-1989), and twenty patients already colonized with MRSA on, or during, admission to the hospital (group II: 1990-1995) were screened for MRSA in two studies. The patients had been discharged from the hospital for periods varying from 15 days to 4.6 years. MRSA was found in five (9%). Four of these patients had skin lesions (wounds), one with an external fixture. The presence of skin- and underlying diseases differed significantly between carriers and non-carriers, supporting the hypothesis that wounds are a major risk factor for long-term MRSA carriage. This study led us to revise our policy concerning readmission of former MRSA patients. We now consider that patients who contracted MRSA in the past no longer need isolation if the following two criteria are met. Absence for at least six months of open wounds, skin lesions, tracheostomy, infections and sources of infection such as abscesses and furuncles, orthopaedic implants, drains, catheters, or tubes. Three MRSA-negative sets of swabs from nose, throat, perineum, urine, and sputum taken at least one hour apart after this six-month period.

Fecal carriage of vancomycin-resistant enterococci in hospitalized patients and those living in the community in The Netherlands.

In order to determine the prevalence of vancomycin-resistant enterococci (VRE) in The Netherlands, 624 hospitalized patients from intensive care units or hemato-oncology wards in nine hospitals and 200 patients living in the community were screened for VRE colonization. Enterococci were found in 49% of the hospitalized patients and in 80% of the patients living in the community. Of these strains, 43 and 32%, respectively, were Enterococcus faecium. VRE were isolated from 12 of 624 (2%) and 4 of 200 (2%) hospitalized patients and patients living in the community, respectively. PCR analysis of these 16 strains and 11 additional clinical VRE isolates from one of the participating hospitals revealed 24 vanA gene-containing, 1 vanB gene-containing, and 2 vanC1 gene-containing strains. All strains were cross-resistant to avoparcin but were sensitive to the novel glycopeptide antibiotic LY333328. Genotyping of the strains by arbitrarily primed PCR and pulsed-field gel electrophoresis revealed a high degree of genetic heterogeneity. This underscores a lack of hospital-driven endemicity of VRE clones. It is suggested that the VRE in hospitalized patients have originated from unknown sources in the community.

Surveillance of nosocomial infections in geriatric patients.

Prospective surveillance of hospital-acquired infections was undertaken in the geriatric ward of the University Hospital, Utrecht, the Netherlands. The medical records of 300 patients were studied for the presence of nosocomial infections using the criteria defined by the Centers for Disease Control (CDC), Atlanta, Georgia, USA. Data were collected from patients with and without infection, which allowed for the analysis of risk factors for nosocomial infection. In 100 out of 300 patients (33.3%), a total of 126 infections was diagnosed. The incidence of nosocomial infections was 16.9 per 1000 days of stay in the hospital. The mean length of stay of patients with infection was 39 days, while that of patients without infection was 17.8 days. Infections developed after an average stay of 13.3 days in the hospital. Patients with infections were 2.6 years older than patients without infections (P = 0.005). Dehydration was shown to be a major risk factor for infection (RR = 2.1, 95% CI: 1.4-3.2). Of the infections, 58.7% were urinary tract infections (UTIs, asymptomatic and symptomatic). The most important risk factor for an asymptomatic UTI was an indwelling urinary catheter (RR = 7.3, 95% CI: 3.1-17.1). The duration of use of the indwelling urinary catheter was of significant influence in the development of a UTI. Seventy percent of the patients with an asymptomatic UTI were treated with antibiotics. Infections of the gastrointestinal tract accounted for 19.8% of all nosocomial infections. The majority of these infections were due to an outbreak of Clostridium difficile. In conclusion, the length of stay may be prolonged by a nosocomial infection. In this study, the main risk factors for developing a nosocomial infection were age, dehydration and the presence of an urinary catheter. Our observations showed that age is a predisposing factor for nosocomial infection and that the risk increases with each year, even for geriatric patients.

Saturable CD14-dependent binding of fluorescein-labeled lipopolysaccharide to human monocytes.

We used rough lipopolysaccharide (ReLPS) to construct a fluorescein-labeled LPS (FITC-LPS) with a very high labeling efficiency that bound to isolated human monocytes in a CD14-dependent fashion and that in this respect behaved indistinctively from native LPS. The CD14-dependent binding could be inhibited either by a 1,000-fold excess of unlabeled LPS or by polymyxin B, bactericidal/permeability-increasing protein, cationic protein 18, or soluble CD14. Although this FITC-LPS preparation no longer possessed the ability to prime neutrophils for the production of reactive oxygen species or to stimulate human monocytes to produce tumor necrosis factor, activation of the Limulus amoebocyte lysate cascade was comparable to activation by native LPS. Binding to monocytes was enhanced by human pooled serum (HPS) or LPS-binding protein (LBP) for LPS concentrations up to 100 ng/ml and was completely CD14 dependent. For LPS concentrations exceeding 100 ng/ml, binding was still partially CD14 dependent, but not HPS or LBP dependent. CD14-dependent association of LPS with monocytes was shown to be totally saturable. In conclusion, we found an HPS- or LBP-dependent binding of FITC-LPS to monocytes that was CD14 dependent at up to 100 ng of LPS per ml, and saturation of binding was shown.

Quantitation of surface CD14 on human monocytes and neutrophils.

The absolute number of membrane-expressed CD14, the most important endotoxin receptor, on human monocytes and neutrophils shows remarkable variation in the literature. To quantify these numbers two fluorescence methods using fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (mAb) were applied. A commercially available set of standard beads was used in flow cytometry to quantitate CD14 with eight different mAbs. Independent from their isotype the various mAbs showed minor differences and indicated that peripheral blood monocytes expressed 99,500-134,600 (115,400 +/- 10,600) and neutrophils 1,900-4,400 (3,300 +/- 800) CD14 receptors. There was no significant difference in CD14 expression on leukocytes in unprocessed freshly obtained whole blood and after a Ficoll isolation procedure. However, a short temperature shift resulted in a 1.3- to 1.6-fold up-regulation of CD14. The results obtained with the reference beads were verified with fluorescence Scatchard analysis and spectrofluorometry using mAb 26ic-FITC and showed 109,500 CD14 per monocyte and 6,700 CD14 per neutrophil. For comparison the number of CD14 on the monocytic THP-1 cells and Fc gamma-receptors on human leukocytes were determined using the reference beads and flow cytometry and gave results comparable to published data. Our data indicate that resting human monocytes express roughly 110,000 CD14 molecules on their surface using a simple fluorometric assay. Correct determination of the number of CD14 and other cell surface receptors is of importance in the monitoring of septic patients.

Modulation of lipopolysaccharide binding to human granulocytes.

Using flow cytometry and fluorescein-labelled lipopolysaccharide (LPS) from Salmonella minnesota R595 (FITC-ReLPS), we studied the role of membrane proteins in the recognition of LPS by human polymorphonuclear granulocytes (PMN) in the absence of serum. Treatment of PMN with trypsin, pronase E or proteinase K reduced both the binding of FITC-ReLPS to PMN at 4 degrees and the response of PMN to LPS at 37 degrees, as measured by luminol-enhanced chemiluminescence. Neuraminidase treatment enhanced both activities. Trypsin treatment of PMN after the binding of FITC-ReLPS effectively reduced fluorescence when cells were kept at 4 degrees, while further incubation of FITC-ReLPS-labelled PMN at 37 degrees rendered fluorescence insensible to trypsin. These results indicate a protein structure of the LPS binding site, association of FITC-ReLPS with the cell membrane at 4 degrees and subsequent internalization at 37 degrees. The binding of FITC-ReLPS was not inhibited by the anti-CD14 monoclonal antibody (mAb) 3C10, which recognizes a functional epitope of CD14. Furthermore, binding of FITC-ReLPS was observed to PMN obtained from a patient with paroxysmal nocturnal haemoglobinuria who lacked membrane-bound CD14. Stimulation of PMN with tumour necrosis factor (TNF) or LPS enhanced the binding of FITC-ReLPS at 4 degrees. This was not observed after activation of PMN devoid of granules (cytoplasts), indicating that the binding of LPS at the cell surface is enhanced by mobilization of LPS-binding proteins from intracellular granules. These studies provide evidence that LPS binding and activation of PMN involves protein structures at the cell surface different from CD14, and that granules constitute a pool of LPS-binding proteins that can be translocated to the cell surface upon stimulation.