[Antibiotic therapy in dental practice]. / Antibiotische therapie in de tandheelkundige praktijk.

Dentists and dental surgeons very frequently prescribe antibiotics to their patients. In a small percentage of cases, that is appropriate; however, patients can often also heal without antibiotic therapy. Microbiological analysis is only carried out in a very limited number of cases, and is complex and time-consuming. A small assortment of oral antibiotics is usually sufficient. Antibiotics are indicated when dental infection is accompanied by fever or indications of infection of a more systemic nature, such as trismus or lymphadenopathy. A patient with cellulitis of the head and neck area, with or without swallowing difficulties, should be treated with antibiotics in any case. In addition, antibiotics have a place in the treatment of periodontitis.

Nucleic Acid Amplification Test Quantitation as Predictor of Toxin Presence in Clostridium difficile Infection.

Multistep algorithmic testing in which a sensitive nucleic acid amplification test (NAAT) is followed by a specific toxin A and toxin B enzyme immunoassay (EIA) is among the most accurate methods for Clostridium difficile infection (CDI) diagnosis. The obvious shortcoming of this approach is that multiple tests must be performed to establish a CDI diagnosis, which may delay treatment. Therefore, we sought to determine whether a preliminary diagnosis could be made on the basis of the quantitative results of the first test in algorithmic testing, which provide a measure of organism burden. To do so, we retrospectively analyzed two large collections of samples (n = 2,669 and n = 1,718) that were submitted to the laboratories of two Dutch hospitals for CDI testing. Both hospitals apply a two-step testing algorithm in which a NAAT is followed by a toxin A/B EIA. Of all samples, 208 and 113 samples, respectively, tested positive by NAAT. Among these NAAT-positive samples, significantly lower mean quantification cycle (Cq ) values were found for patients whose stool eventually tested positive for toxin, compared with patients who tested negative for toxin (mean Cq values of 24.4 versus 30.4 and 26.8 versus 32.2; P < 0.001 for both cohorts). Receiver operating characteristic curve analysis was performed to investigate the ability of Cq values to predict toxin status and yielded areas under the curve of 0.826 and 0.854. Using the optimal Cq cutoff values, prediction of the eventual toxin A/B EIA results was accurate for 78.9% and 80.5% of samples, respectively. In conclusion, Cq values can serve as predictors of toxin status but, due to the suboptimal correlation between the two tests, additional toxin testing is still needed.

Infection and colonization with methicillin resistant Staphylococcus aureus ST398 versus other MRSA in an area with a high density of pig farms.

The purpose of this study was to evaluate the impact of the emergence of animal related methicillin resistant Staphylococcus aureus ST398 in an area with a high density of pig farms. A retrospective analysis was performed of all MRSA isolates in the laboratory database from 2002 till 2008 including typing results and clinical data from infection control archives and patient charts. The implementation of the screening of people in contact with pigs and veal calves for MRSA led to an increase in the average number of newly identified carriers from 16 per year between July 2002 and July 2006 to 148 between July 2006 and December 2008. This is a 925% increase of which 82% (108/132) was due to ST398. The majority (74%) came from targeted screening but 7% was due to unexpected findings. A wide range of infections with ST398 occurred in patients with and without contact with livestock varying from post-operative wound infections to sepsis and post-trauma osteomyelitis with an overrepresentation of spa type t567 among the clinical isolates. ST398 isolates were more often multi-resistant than isolates of other spa-types. The emergence of MRSA ST398 led to an increase in both MRSA carriers and MRSA infections.

[How to diagnose infective endocarditis?] / Hoe herken je infectieuze endocarditis?

We report on three patients with infective endocarditis, which differ greatly in clinical manifestations. Infective endocarditis (IE) is defined by, a mostly bacterial, infection of a native or prosthetic heart valve, the endocardial surface or a cardiac device. It is a rare condition, but it's incidence is increasing because of an increased incidence of elderly patients with chronic disease and cardiac devices. IE is heterogeneous in aetiology, clinical manifestations, and course. It can involve almost any organ system. The presentation often remains subtle and varies with nonspecific symptoms ranging from a mild infection to septic shock and multiorgan failure. IE remains a highly mortal disease, since the diagnosis is missed often. A thorough anamnesis and physical examination can be helpful. Blood cultures prior to antibiotics and echocardiography are key diagnostic steps if there's a clinical suspicion of IE.

Human resources required for antimicrobial stewardship teams: a Dutch consensus report.

SCOPE: Antimicrobial stewardship teams are responsible for implementing antimicrobial stewardship programmes (ASP). However, in many countries, lack of funding challenges this obligation. A consensus procedure was performed to investigate which structural activities need to be performed by Dutch stewardship teams and how much time (and thus full-time equivalent (FTE) labor) is needed to perform these activities. METHODS: In 2015, an electronic survey, based on a nonsystematic literature search and interviews with seven experienced stewardship teams, was sent to 21 stewardship teams that performed an ASP. This was followed by a semistructured face-to-face consensus meeting. Fourteen stewardship teams completed the survey (18% of Dutch acute-care hospitals), and 13 participated in the consensus meeting. RECOMMENDATIONS: The hours needed each year are dependent on hospital size and number of stewardship objectives monitored. If all activities are performed at a minimal base (one stewardship objective; minimal staffing standard), time investment was estimated to be 1393 to 2680 hours annually in the early phase, corresponding with 0.87 (300 beds) to 1.68 FTE (1200 beds), with a further increase to minimally 1.25 to 3.18 FTE in the following years with three stewardship objectives monitored (optimal staffing standards during the first few years of implementing an ASP). This consensus on required human resources provides a directive for structural financial support of stewardship teams in the Dutch context. Some stewardship activities (and related time investments) might be specific to the Dutch context and hospital setting. To develop standards for other settings, our methodology could be applied.

High-throughput amplification fragment length polymorphism (htAFLP) analysis identifies genetic lineage markers but not complement phenotype-specific markers in Moraxella catarrhalis.

Comparative high-throughput amplified fragment length polymorphism (htAFLP) analysis was performed on a set of 25 complement-resistant and 23 complement-sensitive isolates of Moraxella catarrhalis in order to determine whether there were complement phenotype-specific markers within this species. The htAFLP analysis used 21 primer-pair combinations, generating 41 364 individual fragments and 2273 fragment length polymorphisms, with an average of 862 polymorphisms per isolate. Analysis of polymorphism data clearly indicated the presence of two phylogenetic lineages and 40 (2%) lineage-specific polymorphisms. However, despite the presence of 361 (16%) statistically significant complement phenotype-associated polymorphisms, no single marker was 100% complement phenotype-specific. Furthermore, no complement phenotype-specific marker was found within different phylogenetic lineages. These findings agree with previous results indicating that the complement resistance phenotype within M. catarrhalis is probably defined by multiple genes, although not all of these genes may be present within all M. catarrhalis isolates.

[Invasive pneumococcal infection in 5 newborns, 1996-2004]. / Invasieve pneumokokkeninfectie bij 5 pasgeborenen, 1996-2004.

At the Máxima Medisch Centrum in Veldhoven, The Netherlands, neonatal sepsis caused by invasive Streptococcus pneumoniae infection was diagnosed in 5 neonates between 1996 and 2004. This infection is relatively rare and its clinical features are variable, but often particularly severe and fulminant as was the case in 2 of the 5 children, one of whom died and the other was left with serious psychomotor retardation. The other 3 recovered fully. The child who died and one of the children who recovered are described in some detail. They were both prematurely born neonates, a girl and a boy, who presented almost immediately after birth with an early-onset sepsis caused by S. pneumoniae. In both cases neonatal cultures as well as maternal vaginal swabs were positive for S. pneumoniae growth. 2 different patients had other risk factors for peripartal infection. Neonatal pneumococcal infections are most likely transmitted trough the maternal vaginal tract. Maternal vaginal colonization is rare (0.11%), but associated with a high risk of transmission to the newborn. Asymptomatic neonatal colonization was not observed. In light of the likelihood of a high rate of transmission and subsequent infection, peripartal prophylactic antibiotic treatment is advised for all mothers with proven vaginal S. pneumoniae colonization. If this is not given or is not effective, then in contrast with the policy on patients with group B streptococcal colonization, prophylactic antibiotic treatment is advocated for all neonates born to colonized mothers. Amoxicillin is the preferred treatment. In areas of increasing macrolide resistance, erythromycin should only be advised in cases of penicillin allergy.

[A blood culture containing coagulase-negative staphylococci: not always due to contamination]. / Coagulasenegatieve stafylokokken in een bloedkweek.

Staphylococcus lugdunensis (SL) is a species belonging to the group of coagulase-negative staphylococci (CNS). It can cause severe infections such as endocarditis. Three cases of endocarditis caused by SL are presented. The first case describes a 71-year-old man with a fever and endogenous endophthalmitis. The second case describes delirium in an 87-year-old woman, thought to be due to pneumonia. The third case describes a 76-year-old man with an infection of unknown origin. In all cases, the first blood cultures drawn were positive for CNS and considered to be contaminated. However, all three patients were finally diagnosed as having severe endocarditis caused by SL. Two patients underwent valve replacement, one patient died due to ongoing sepsis. The first CNS-positive blood cultures drawn were wrongly denoted as being contaminated. Physicians should be aware of the pathogenic potential of SL and rule out contamination.

Complement-resistant Moraxella catarrhalis forms a genetically distinct lineage within the species.

Moraxella catarrhalis is a bacterial species that has been implicated in 15-20% of all cases of otitis media in the USA and the complement-resistant variant of M. catarrhalis has been considered particularly pathogenic. A collection of geographically diverse, complement-sensitive (n=28) and -resistant strains (n=47) of M. catarrhalis was assembled in order to analyse the bacterial population structure. All strains were identified as M. catarrhalis by conventional microbiological and biochemical methods. Amplification of the small subunit (ssu) ribosomal RNA gene followed by restriction fragment length polymorphism (RFLP) analysis did not reveal consistent differences between serum-susceptible and -resistant M. catarrhalis isolates. Interestingly, upon automated ribotyping using the Qualicon RiboPrinter(R) microbial characterisation system, the complement-sensitive and -resistant strains segregated into two groups. This suggested the existence of two clearly distinguishable lineages within the species M. catarrhalis. This observation was corroborated by pulsed field gel electrophoresis (PFGE) of DNA macro-restriction fragments, a non-ribosomal PCR RFLP procedure and random amplification of polymorphic DNA (RAPD) analysis. All procedures grouped the two variants similarly. Redefinition of the taxonomic status of complement-resistant M. catarrhalis or even the definition of a new species may be opportune.

Changes in genetic types and population dynamics of Moraxella catarrhalis in hospitalized children are not associated with an exacerbation of existing disease.

Pulsed-field gel electrophoresis typing was performed on a retrospective set of 129 Moraxella catarrhalis isolates obtained over a 20 month period from 70 children admitted to, or presenting at, the Erasmus University Medical Center, Rotterdam, The Netherlands. The mean age of the children (at the end of the study) was 2.5 years, with a range of 6 months to 15 years. Fifty-one different M. catarrhalis types were isolated from the hospitalized children, with 31 % (22/70) being infected with two particularly prevalent M. catarrhalis types. These two prevalent types also exhibited different protein profiles. The majority (72%; 16/22) of the children infected with these two predominant types had spent at least 1 week on two paediatric intensive care wards. No exacerbation of existing disease or new disease was observed in children who experienced M. catarrhalis type changes.

Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis.

The purpose of this study was to investigate complement resistance in Branhamella (Moraxella) catarrhalis isolated from healthy schoolchildren or sputum-producing adult patients. Two techniques were used: a serum bactericidal assay as the gold standard and an easier 'culture and spot' test. Children (age 4-13; n = 303) and patients (n = 1047) showed high colonization/infection rates with B. catarrhalis (31% and 19%, respectively). Complement resistance or intermediate sensitivity occurred frequently in patient isolates (62% and 27%, respectively) and less often in children (33% and 8.5%, respectively; P << 0.0001). In young children (age 4-5 years), the proportion of complement-resistant strains was around 50%. Complement resistance in B. catarrhalis is associated with illness and may hence be considered a virulence factor.

The use of Raman spectroscopy in the epidemiology of methicillin-resistant Staphylococcus aureus of human- and animal-related clonal lineages.

In order to perform a cost-effective search and destroy policy for methicillin-resistant Staphylococcus aureus (MRSA), a quick and reliable typing method is essential. In an area with a high level of animal-related MRSA ST398, pulsed field gel electrophoresis (PFGE) typing and spa-typing are not sufficient to discriminate between co-incidental findings and true transmission of MRSA. This study is the first to retrospectively show the performance of Raman spectroscopy in 16 well-documented outbreaks. We analysed 525 isolates, 286 MRSA ST398 and 239 from other PFGE clusters with Raman spectroscopy. When epidemiologically linked isolates from the outbreaks were analysed with PFGE as the reference standard, Raman spectroscopy correctly identified 97% of cases that were indistinguishable from the index case. With Raman cluster analysis, the most dominant distinction was between MRSA ST398 and other MRSA of human clonal lineages. Within MRSA ST398, 22 different Raman clusters were identified. Raman typing correctly identified an ST398 (spa type t567) outbreak in a hospital setting. No direct correlation was observed between Raman clusters and spa types. We conclude that Raman spectroscopy is a quick and reliable method of MRSA typing, which can be used in outbreak settings and it is comparable to PFGE, with the added advantage that PFGE non-typeable isolates can also be readily typed using the same sample preparation protocol.

Experimental evidence for Moraxella-induced penicillin neutralization in pneumococcal pneumonia.

Resistance of microorganisms to antimicrobial agents is an increasing problem in the treatment of infectious diseases. In mixed infections, an interesting development can arise when one organism protects another from being killed by an antibiotic. Unfortunately, in the case of respiratory tract infections, experimental evidence of this development is poor. In this study, mice intranasally infected with a lethal number of pneumococci and treated with a curative dose of penicillin or amoxicillin died from pneumococcal pneumonia when they were coinoculated with beta-lactamase-producing Moraxella catarrhalis. beta-lactamase-negative M. catarrhalis did not show a similar indirect pathogenic effect. Treatment with a combination of amoxicillin and the beta-lactamase inhibitor clavulanic acid was not affected by beta-lactamase-producing M. catarrhalis. These findings help explain antibiotic failure in respiratory tract infections, even though the causative microorganism is sensitive to the antibiotic in vitro.

Differences in complement activation between complement-resistant and complement-sensitive Moraxella (Branhamella) catarrhalis strains occur at the level of membrane attack complex formation.

The mechanism of resistance to human complement-mediated killing in Moraxella catarrhalis was studied by comparing different complement-sensitive and complement-resistant M. catarrhalis strains in a functional bystander hemolysis assay and an enzyme-linked immunosorbent assay (ELISA) for soluble terminal complement complexes. Complement-resistant stains appeared to activate complement to the same extent as, or even slightly better than, complement-sensitive strains. This indicates that complement-resistant strains do not inhibit classical or alternative pathway activation but interfere with complement at the level of membrane attack complex formation. A clear difference in dose-response curves for resistant and sensitive strains was observed both in the bystander hemolysis assay and in the ELISA. Complement-resistant strains showed optimum curves, whereas complement-sensitive strains gave almost linear curves. We conclude that resistant strains bind and/or inactivate one of the terminal complement components or intermediates involved in membrane attack complex formation. Trypsin, known to abolish complement resistance, changed the optimum dose-response curve of a resistant strain to a linear one, which strongly suggests that complement resistance is mediated by an M. catarrhalis-associated protein. This protein acts directly or through the binding of a terminal complement inhibitor present in serum.

Cellular membrane associated mucins in artificial urine as mediators of crystal adhesion: an in vitro enterocystoplasty model.

PURPOSE: Cells are a major component of mucus in enterocystoplasties. We evaluate the role of secreted mucins on cells and cellular membranes as crystal adhesion and agglomeration mediators in artificial urine infected with Proteus mirabilis. MATERIALS AND METHODS: Five human intestinal cell lines, HT29, HT29-18N2, HT29-FU, HT29-MTX, Caco-2 and 1 ureter cell line, SV-HUC-1, were incubated for 3 hours in artificial urine with P. mirabilis (ATCC49565) in monolayer and scraped conditions. We isolated Triton X-100 soluble membrane proteins from cells to evaluate the effect of MUC2 and MUC 5AC as membrane associated proteins on crystal formation and crystal adherence. Scanning electron microscopy, light microscopy, Coulter Counter measurements and x-ray microanalysis were used to evaluate crystal formation. RESULTS: Brushite crystals were adhered to cellular surface sites rich in sulfur as crystal agglomerates. Smaller and more numerous crystals were observed in the presence of scraped cells. Crystal growth and agglomeration was inhibited by the presence of MUC 5AC, whereas MUC2 had the opposite effect. Both are present on cellular membranes and are rich in sulfur. Cellular invasion by bacteria occurred in all cell lines. CONCLUSIONS: Membrane associated cellular secretions such as MUC2 and 5AC are important crystal adhesion molecules on cells and have a clear effect on urease induced crystallization in vitro. MUC2 and MUC 5AC induce crystal adhesion and mucin type dependent effects on crystal agglomeration. The effects of MUC2 and MUC 5AC may explain the high incidence of bladder calculi in enterocystoplasties and emphasize the role of cellular surfaces in urine.

Assessment of complement-mediated killing of Moraxella (Branhamella) catarrhalis isolates by a simple method.

Recently, we showed that complement resistance is an important virulence factor of Moraxella (Branhamella) catarrhalis. Our study used a serum bactericidal assay to determine complement resistance in M. catarrhalis. Although the serum bactericidal assay is considered the "gold standard" for determining complement resistance, it is laborious and time-consuming and therefore not well suited for large-scale studies. Using a large number (n = 324) of M. catarrhalis isolates obtained from the sputa of patients with lower respiratory tract infections (n = 200) and young carriers (n = 124), we assessed the value of a simple "culture-and-spot" test as an alternative to the serum bactericidal assay. For both groups of isolates, the degree of concordance between the two tests used was very significant (P < 0.0001). The agreement between the two assays was estimated to be "excellent beyond chance" (as determined by Cohen's kappa test). The culture-and-spot assay is a valuable alternative to the serum bactericidal assay, not only for screening purposes as shown here but also for studying the mechanism of complement resistance in M. catarrhalis at the molecular level.

Moraxella catarrhalis in acute laryngitis: infection or colonization?

The complement phenotypes of Moraxella catarrhalis isolates obtained from adult patients with acute laryngitis were investigated using a microliter serum bactericidal assay and compared with those of other donor groups. Laryngitis isolates had a higher proportion (57%) of complement-resistant strains than did carrier strains from healthy 8- to 13-year-old schoolchildren (16%). The difference between these groups was statistically significant (chi2 [3 x 2 table] = 21.55; P < .001). The relatively frequent occurrence of the complement-resistant (virulence-associated) phenotype in adults with acute laryngitis supports the theory of an active role of M. catarrhalis in the pathogenesis of acute laryngitis.

Distinguishing species of the Burkholderia cepacia complex and Burkholderia gladioli by automated ribotyping.

Several species belonging to the genus Burkholderia are clinically relevant, opportunistic pathogens that inhabit major environmental reservoirs. Consequently, the availability of means for adequate identification and epidemiological characterization of individual environmental or clinical isolates is mandatory. In the present communication we describe the use of the Riboprinter microbial characterization system (Qualicon, Warwick, United Kingdom) for automated ribotyping of 104 strains of Burkholderia species from diverse sources, including several publicly accessible collections. The main outcome of this analysis was that all strains were typeable and that strains of Burkholderia gladioli and of each species of the B. cepacia complex, including B. multivorans, B. stabilis, and B. vietnamiensis, were effectively discriminated. Furthermore, different ribotypes were discerned within each species. Ribotyping results were in general agreement with strain classification based on restriction fragment analysis of 16S ribosomal amplicons, but the resolution of ribotyping was much higher. This enabled automated molecular typing below the species level. Cluster analysis of the patterns obtained by ribotyping (riboprints) showed that within B. gladioli, B. multivorans, and B. cepacia genomovar VI, the different riboprints identified always clustered together. Riboprints of B. cepacia genomovars I and III, B. stabilis, and B. vietnamiensis did not show distinct clustering but rather exhibited the formation of loose assemblages within which several smaller, genomovar-specific clusters were delineated. Therefore, ribotyping proved useful for genomovar identification. Analysis of serial isolates from individual patients demonstrated that infection with a single ribotype had occurred, despite minor genetic differences that were detected by pulsed-field gel electrophoresis of DNA macrorestriction fragments. The automated approach allows very rapid and reliable identification and epidemiological characterization of strains and generates an easily manageable database suited for expansion with information on additional bacterial isolates.

Identification of Burkholderia spp. in the clinical microbiology laboratory: comparison of conventional and molecular methods.

Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated. This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required. A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B. gladioli (n = 14), Ralstonia pickettii (n = 6), B. multivorans (n = 2), Stenotrophomonas maltophilia (n = 3), and Pseudomonas aeruginosa (n = 11), were collected from environmental, clinical, and reference sources. In addition, 27 clinical isolates putatively identified as Burkholderia spp. were recovered from the sputum of Dutch CF patients. All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism [RFLP] analysis). The best system for the biochemical identification of B. cepacia appeared to be the API 20NE test. None of the biochemical assays successfully grouped the B. gladioli strains. The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different Burkholderia spp. With this method, B. gladioli was also reliably classified in a separate group. For the laboratory diagnosis of B. cepacia, we recommend parallel cultures on blood agar medium and selective agar plates. Further identification of colonies with a Burkholderia phenotype should be performed with the API 20NE test. For final confirmation of species identities, PCR amplification of the small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended.