Elucidating vancomycin-resistant Enterococcus faecium outbreaks: the role of clonal spread and movement of mobile genetic elements.

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.

Burden of highly resistant microorganisms in a Dutch intensive care unit.

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 versuscontrols 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.

Staphylococcus aureus spa type t437: identification of the most dominant community-associated clone from Asia across Europe.

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.

Evaluation of the Xpert vanA/vanB assay using enriched inoculated broths for direct detection of vanB vancomycin-resistant Enterococci.

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.

Latent introduction to the Netherlands of multiple antibiotic resistance including NDM-1 after hospitalisation in Egypt, August 2013.

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.

Is bacteriologic surveillance in endoscope reprocessing stringent enough?

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.

Decontamination of the digestive tract and oropharynx in ICU patients.

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.)

Spread of a methicillin-resistant Staphylococcus aureus ST80 strain in the community of the northern Netherlands.

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.

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics.

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.

[Three infants with constipation and muscular weakness: infantile botulism]. / 3 zuigelingen met obstipatie en hypotonie: infantiel botulisme.

Two previously healthy infants, a boy of 10 weeks and a girl of 4 months presented with apathy and muscle weakness. A third previously healthy child, a girl of 6 weeks old was admitted with respiratory insufficiency. None of the three had had a bowel movement for a number of days. After extensive investigations which revealed few abnormalities Clostridium botulinum toxin was obtained in serum from all three children. Type-B-toxin was shown in the faeces of the older girl and boy; both recovered quickly. The other girl had type-A toxin; she died. Two of the three children were given honey to comfort them. Infantile botulism must be considered in every infant with symptoms of constipation and hypotonia. The diagnosis can quickly be confirmed by electromyography with repetitive 50-Hz-stimulation. Honey is a well-known source of the C. botulinum spore and should not be given to children under the age of 12 months. These three children are the first cases to be described in the Netherlands.

Does the empiric use of vancomycin in pediatrics increase the risk for Gram-negative bacteremia?

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.

Ochrobactrum intermedium infection after liver transplantation.

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.

Applicability of random primer R143 for determination of Aspergillus fumigatus DNA.

The specificity of random primer R143 for Aspergillus fumigatus DNA was determined in order to test its usefulness in establishing the presence of A. fumigatus DNA in fungal cultures. When PCR reaction products of these cultures were compared with those of 21 other bacterial and fungal DNA samples, R143 proved to produce a 1346 bp band with only A. fumigatus. This band has been sequenced completely and the EcoRI restriction site was used for subsequent confirmation of PCR products. The specificity for A. fumigatus DNA was also confirmed by Southern blotting. Comparison of morphological typing of Aspergillus species in cultures with PCR using R143 on DNA isolated from these cultures showed concordance in 22 of 24 cases. In two cases there was discordance: both times PCR results showed correctly the presence of A. fumigatus, initially not detected by culture. R143 is an A. fumigatus specific random primer, with potential for use in detection of A. fumigatus DNA in clinical specimens.

Clinical use of selective decontamination: the concept.

Infections can be classified according to: (1) the type of offending microorganism (virus, bacteria, fungi, parasites), (2) according to the clearance by the defence system (T cell dependent/independent) and (3) in case bacteria are the causative agents in Gram-positive and Gram-negative infections. The latter classification in Gram-positive and Gram-negative infections has appeared to have a practical consequence. Gram-negative bacteria, often involved in major infections and yeasts, appear to play practically no role in the intestinal ecological system. Consequently, it is nowadays increasingly attempted to eliminate Gram-negative bacteria and yeasts selectively from the digestive tract with antimicrobial agents. Selective suppression of Gram-positive bacteria may severely affect the ecosystem of the digestive tract. This selective suppression of Gram-negatives must be continued as long as patients are immunocompromised (locally or systemically) and is called selective decontamination of the digestive tract.

Differences in virulence between two strains of Streptococcus suis type II after experimentally induced infection of newborn germ-free pigs.

Fifteen newborn germ-free pigs were inoculated with 2 strains, D-282 and T-15, of Streptococcus suis type II. Some pigs also were preinoculated with Bordetella bronchiseptica, which successfully predisposed them to S suis infection. The 2 streptococcal strains were differentiated by muramidase treatment, which released certain high molecular-weight proteins, termed muramidase-released proteins (MRP), from the cell wall of strain D-282, but not from the cell wall of strain T-15. Only strain D-282 (MRP-positive) induced clinical signs of disease and markedly increased neutrophil numbers in pigs. Streptococci were more frequently isolated from fecal swab specimens obtained from pigs inoculated with strain D-282 (MRP-positive) than from specimens obtained from pigs inoculated with strain T-15 (MRP-negative). Both strains were isolated from nasal swab specimens obtained from all infected pigs. Postmortem examination revealed fibrinopurulent meningitis, polyserositis, and polyarthritis in pigs inoculated with strain D-282; this strain was isolated from the CNS, serosae, visceral organs, heart, and joints. Whereas strains D-282 caused several pathologic changes, strain T-15, isolated from the lungs, caused only pneumonia. Both strains were isolated from the tonsils of all pigs. Virulence differed distinctly between the MRP-positive and the MRP-negative strains.

Meningitis caused by Streptococcus suis in humans.

Between 1968 and 1984, 30 strains of Streptococcus suis causing meningitis were isolated in the Netherlands. Twenty-eight strains were type 2, one was type 4, and one was untypable. The average age of the patients infected with these strains was 49 years (range, 21-76 years); the male-to-female ratio was 6.5. Twenty-five patients (83%) were employed in the pork industry. Two patients (7%) died. In seven cases (23%), predisposing factors were identified. The most frequent sequela was hearing loss (54% of surviving patients). The data for these 30 patients were compared with those for 30 patients from outside the Netherlands whose cases of meningitis due to S. suis type 2 were described between 1968 and 1985. No differences were found. The estimated annual risk of developing S. suis meningitis among Dutch abattoir workers and pig breeders was approximately 3.0/100,000--a rate 1,500 times higher than that among persons not working in the pork industry.

Carrier rate of Streptococcus suis capsular type 2 in palatine tonsils of slaughtered pigs.

Palatine tonsils of 143 slaughtered pigs aged 4 to 6 months were investigated for the presence of Streptococcus suis type 2. Slices (50 micron) of frozen tonsils were cultured on a selective agar medium containing antibodies against S. suis type 2 in which colonies of this bacterium showed a halo of immunoprecipitation. When tonsils were sectioned in one plane S. suis type 2 was found in 45 of 143 pigs (32%). This percentage increased to 50% when tonsils were sectioned in more then one plane, which was done on 55 tonsils. The first 45 strains showing a ring of immunoprecipitation were studied and found to be biochemically identical to our reference strain 735 (de Moor) and to 23 isolates from human patients with meningitis. In slices incubated for 24 h at 37 degrees C on selective agar plates and stained with hematoxylin and eosin after fixation, it could be demonstrated that S. suis type 2 was confined to the crypt lumen. The same was true in sections fixed directly (without incubation) that were stained by an indirect immunoperoxidase method with a rabbit anti-S. suis type 2 serum.