Hélène Boelens

Reclassification of Staphylococcus aureus nasal carriage types.

BACKGROUND Persistent nasal carriers have an increased risk of Staphylococcus aureus infection, whereas intermittent carriers and noncarriers share the same low risk. This study was performed to provide additional insight into staphylococcal carriage types. METHODS Fifty-one volunteers who had been decolonized with mupirocin treatment and whose carriage state was known were colonized artificially with a mixture of S. aureus strains, and intranasal survival of S. aureus was compared between carriage groups. Antistaphylococcal antibody levels were also compared among 83 carriage-classified volunteers. RESULTS Persistent carriers preferentially reselected their autologous strain from the inoculum mixture (P=.02). They could be distinguished from intermittent carriers and noncarriers on the basis of the duration of postinoculation carriage (154 vs. 14 and 4 days, respectively; P=.017, by log-rank test). Cultures of swab samples from persistent carriers contained significantly more colony-forming units per sample than did cultures of swab samples from intermittent carriers and noncarriers (P=.004). Analysis of serum samples showed that levels of immunoglobulin G and immunoglobulin A to 17 S. aureus antigens were equal in intermittent carriers and noncarriers but not in persistent carriers. CONCLUSIONS Along with the previously described low risk of infection, intermittent carriers and noncarriers share similar S. aureus nasal elimination kinetics and antistaphylococcal antibody profiles. This implies a paradigm shift; apparently, there are only 2 types of nasal carriers: persistent carriers and others. This knowledge may increase our understanding of susceptibility to S. aureus infection.

Natural population dynamics and expansion of pathogenic clones of Staphylococcus aureus

The population structure of Staphylococcus aureus carried by healthy humans was determined using a large strain collection of nonclinical origin (n = 829). High-throughput amplified fragment length polymorphism (AFLP) analysis revealed 3 major and 2 minor genetic clusters of S. aureus, which were corroborated by multilocus sequence typing. Major AFLP cluster I comprised 44.4% of the carriage isolates and showed additional heterogeneity whereas major AFLP groups II and III presented 2 homogeneous clusters, including 47.3% of all carriage isolates. Coanalysis of invasive S. aureus strains and epidemic methicillin-resistant S. aureus (MRSA) revealed that all major clusters contained invasive and multiresistant isolates. However, clusters and subclusters with overrepresentation of invasive isolates were also identified. Bacteremia in elderly adults, for instance, was caused by a IVa cluster-derived strain significantly more often than by strains from other AFLP clusters. Furthermore, expansion of multiresistant clones or clones associated with skin disease (impetigo) was detected, which suggests that epidemic potential is present in pathogenic strains of S. aureus. In addition, the virulence gene encoding Panton-Valentine leukocidin was significantly enriched in S. aureus strains causing abscesses and arthritis in comparison with the carriage group. We provide evidence that essentially any S. aureus genotype carried by humans can transform into a life-threatening human pathogen but that certain clones are more virulent than others.

Anti-Staphylococcal Humoral Immune Response in Persistent Nasal Carriers and Noncarriers of Staphylococcus aureus

BACKGROUND Persistent carriers have a higher risk of Staphylococcus aureus infections than noncarriers but a lower risk of bacteremia-related death. Here, the role played by anti-staphylococcal antibodies was studied. METHODS Serum samples from 15 persistent carriers and 19 noncarriers were analyzed for immunoglobulin (Ig) G, IgA, and IgM binding to 19 S. aureus antigens, by means of Luminex technology. Nasal secretions and serum samples obtained after 6 months were also analyzed. RESULTS Median serum IgG levels were significantly higher in persistent carriers than in noncarriers for toxic shock syndrome toxin (TSST)-1 (median fluorescence intensity [MFI] value, 11,554 vs. 4291; P < .001) and staphylococcal enterotoxin (SE) A (742 vs. 218; P < .05); median IgA levels were higher for TSST-1 (P < .01), SEA, and clumping factor (Clf) A and B (P < .05). The in vitro neutralizing capacity of anti-TSST-1 antibodies was correlated with the MFI value (R(2) = 0.93) and was higher in persistent carriers (90.6% vs. 70.6%; P < .05). Antibody levels were stable over time and correlated with levels in nasal secretions (for IgG, R(2) = 0.87; for IgA, R(2) = 0.77). CONCLUSIONS Antibodies to TSST-1 have a neutralizing capacity, and median levels of antibodies to TSST-1, SEA, ClfA, and ClfB are higher in persistent carriers than in noncarriers. These antibodies might be associated with the differences in the risk and outcome of S. aureus infections between nasal carriers and noncarriers.

Key role for clumping factor B in Staphylococcus aureus nasal colonization of humans.

Background Staphylococcus aureus permanently colonizes the vestibulum nasi of one-fifth of the human population, which is a risk factor for autoinfection. The precise mechanisms whereby S. aureus colonizes the nose are still unknown. The staphylococcal cell-wall protein clumping factor B (ClfB) promotes adhesion to squamous epithelial cells in vitro and might be a physiologically relevant colonization factor. Methods and Findings We define the role of the staphylococcal cytokeratin-binding protein ClfB in the colonization process by artificial inoculation of human volunteers with a wild-type strain and its single locus ClfB knock-out mutant. The wild-type strain adhered to immobilized recombinant human cytokeratin 10 (CK10) in a dose-dependent manner, whereas the ClfB− mutant did not. The wild-type strain, when grown to the stationary phase in a poor growth medium, adhered better to CK10, than when the same strain was grown in a nutrient-rich environment. Nasal cultures show that the mutant strain is eliminated from the nares significantly faster than the wild-type strain, with a median of 3 ± 1 d versus 7 ± 4 d (p = 0.006). Furthermore, the wild-type strain was still present in the nares of 3/16 volunteers at the end of follow-up, and the mutant strain was not. Conclusions The human colonization model, in combination with in vitro data, shows that the ClfB protein is a major determinant of nasal-persistent S. aureus carriage and is a candidate target molecule for decolonization strategies.

Host- and Tissue-Specific Pathogenic Traits of Staphylococcus aureus

Comparative genomics were used to assess genetic differences between Staphylococcus aureus strains derived from infected animals versus colonized or infected humans. A total of 77 veterinary isolates were genetically characterized by high-throughput amplified fragment length polymorphism (AFLP). Bacterial genotypes were introduced in a large AFLP database containing similar information for 1,056 human S. aureus strains. All S. aureus strains isolated from animals in close contact with humans (e.g., pet animals) were predominantly classified in one of the five main clusters of the AFLP database (cluster I). In essence, mastitis-associated strains from animals were categorized separately (cluster IVa) and cosegregated with bacteremia-associated strains from humans. Distribution of only 2 out of 10 different virulence genes differed across the clusters. The gene encoding the toxic shock syndrome protein (tst) was more often encountered among veterinary strains (P < 0.0001) and even more in the mastitis-related strains (P<0.0001) compared to human isolate results. The gene encoding the collagen binding protein (cna) was rarely detected among invasive human strains. The virulence potential, as indicated by the number of virulence genes per strain, did not differ significantly between the human- and animal-related strains. Our data show that invasive infections in pets and humans are usually due to S. aureus strains with the same genetic background. Mastitis-associated S. aureus isolated in diverse farm animal species form a distinct genetic cluster, characterized by an overrepresentation of the toxic shock syndrome toxin superantigen-encoding gene.

Effect of Mupirocin Treatment on Nasal, Pharyngeal, and Perineal Carriage of Staphylococcus aureus in Healthy Adults

ABSTRACT Nasal carriage of Staphylococcus aureus is an important risk factor for S. aureus infections. Mupirocin nasal ointment is presently the treatment of choice for decolonizing the anterior nares. However, recent clinical trials show limited benefit from mupirocin prophylaxis in preventing nosocomial S. aureus infections, probably due to (re)colonization from extranasal carriage sites. Therefore, we studied the effectiveness of mupirocin nasal ointment treatment on the dynamics of S. aureus nasal and extranasal carriage. Twenty noncarriers, 26 intermittent carriers, and 16 persistent carriers had nasal, throat, and perineum samples taken 1 day before and 5 weeks after mupirocin treatment (twice daily for 5 days) and assessed for growth of S. aureus. The identities of cultured strains were assessed by restriction fragment length polymorphisms of the coagulase and protein A genes. The overall carriage rate (either nasal, pharyngeal, or perineal carrier or a combination) was significantly reduced after mupirocin treatment from 30 to 17 carriers (P = 0.003). Of the 17 carriers, 10 (60%) were still colonized with their old strain, 6 (35%) were colonized with an exogenous strain, and 1 (5%) was colonized with both. Two noncarriers became carriers after treatment. The acquisition of exogenous strains after mupirocin treatment is a common phenomenon. The finding warrants the use of mupirocin only in proven carriers for decolonization purposes. Mupirocin is effective overall in decolonizing nasal carriers but less effective in decolonizing extranasal sites.

Characterization of the humoral immune response during Staphylococcus aureus bacteremia and global gene expression by Staphylococcus aureus in human blood.

Attempts to develop an efficient anti-staphylococcal vaccine in humans have so far been unsuccessful. Therefore, more knowledge of the antigens that are expressed by Staphylococcus aureus in human blood and induce an immune response in patients is required. In this study we further characterize the serial levels of IgG and IgA antibodies against 56 staphylococcal antigens in multiple serum samples of 21 patients with a S. aureus bacteremia, compare peak IgG levels between patients and 30 non-infected controls, and analyze the expression of 3626 genes by two genetically distinct isolates in human blood. The serum antibody levels were measured using a bead-based flow cytometry technique (xMAP®, Luminex corporation). Gene expression levels were analyzed using a microarray (BµG@s microarray). The initial levels and time taken to reach peak IgG and IgA antibody levels were heterogeneous in bacteremia patients. The antigen SA0688 was associated with the highest median initial-to-peak antibody fold-increase for IgG (5.05-fold) and the second highest increase for IgA (2.07-fold). Peak IgG levels against 27 antigens, including the antigen SA0688, were significantly elevated in bacteremia patients versus controls (P≤0.05). Expression of diverse genes, including SA0688, was ubiquitously high in both isolates at all time points during incubation in blood. However, only a limited number of genes were specifically up- or downregulated in both isolates when cultured in blood, compared to the start of incubation in blood or during incubation in BHI broth. In conclusion, most staphylococcal antigens tested in this study, including many known virulence factors, do not induce uniform increases in the antibody levels in bacteremia patients. In addition, the expression of these antigens by S. aureus is not significantly altered by incubation in human blood over time. One immunogenic and ubiquitously expressed antigen is the putative iron-regulated ABC transporter SA0688.

Panton-Valentine Leukocidin Genes in Staphylococcus aureus

The pathogenicity of Staphylococcus aureus depends on various bacterial surface components and extracellular proteins. However, the precise role of single virulence determinants in relation to infection is hard to establish. The frequent recovery of staphylococcal isolates that produce leukocidal toxins from patients with deep skin and soft tissue infections, particularly furunculosis, cutaneous abscesses, and severe necrotizing pneumonia, suggests that the Panton-Valentine leukocidin (PVL) is 1 such virulence factor that has a major role in pathogenicity (1–3). In 1932, Panton and Valentine described PVL as a virulence factor belonging to the family of synergohymenotropic toxins (4). These toxins form pores in the membrane of host defense cells by synergistic action of 2 secretory proteins, designated LukS-PV and LukF-PV, which are encoded by 2 cotranscribed genes of a prophage integrated in the S. aureus chromosome (5). PVL is mostly associated with community-acquired methicillin-resistant S. aureus (MRSA) infections and distinguishable from nosocomial MRSA by nonmultidrug resistance and carriage of the type IV staphylococcal chromosome cassette element (SCCmec type IV) (6,7). Despite the presumed importance of PVL as a virulence factor, few data are available on its prevalence among S. aureus isolates from the nares of healthy persons compared with stains isolated from infections. This lack of data led us to investigate the frequency of PVL gene–positive S. aureus strains obtained from the nares of healthy carriers in the community. For this purpose, a single polymerase chain reaction method was used to detect both lukS-PV and lukF-PV genes (2). In a previous study, the population structure of S. aureus, isolated from the nares of healthy persons in the Rotterdam area, the Netherlands, was elucidated (8). Strains were obtained from healthy children ( 55 years). Invasive strains (blood culture, skin and soft tissue infections, and impetigo isolates) were included in this study (Table). All carriage and clinical isolates (n = 1,033) were mecA negative. We used the same strain collection to study the PVL prevalence in carriage and invasive isolates of S. aureus from a single geographic region. Table Panton-Valentine Leukocidin (PVL) distribution among carriage and invasive isolates per genetic cluster of Staphylococcus aureus Five PVL-positive S. aureus strains (0.6%) were found in the carriage group (n = 829), and 3 (2.1%) of 146 blood-culture isolates carried the PVL gene (Table). This finding is in agreement with previously reported low PVL prevalences by Prevost et al. (0% in 31 carriage isolates and 1.4% in 69 blood-culture isolates) and Von Eiff et al. (1.4% in 210 carriage isolates and 0.9% in 219 blood-culture isolates) (9,10). However, a higher prevalence of PVL (38.9%) was found in S. aureus strains causing abscesses and arthritis (Fisher exact test, p <0.0001) (8). This finding is also in agreement with the proposed involvement of PVL in severe and invasive (soft tissue) staphylococcal infections (1–3). No significant differences were found in the presence of PVL when carriage isolates were compared with invasive blood-culture isolates. PVL was found in each major genomic amplified fragment length polymorphism (AFLP) cluster, indicating that PVL has been introduced in distinct phylogenetic subpopulations of S. aureus (Figure). Multilocus sequence typing analysis of a subset of the strain collection showed that the 15 PVL-positive strains were within clonal complex (CC) 30 (n = 7), CC 121 (n = 3), CC 1 (n = 2), CC 8 (n = 1), CC 22 (n = 1), and CC 45 (n = 1) (Table) (8). Although PVL was found among several staphylococcal genotypes, it was slightly overrepresented in AFLP cluster IVb (CC 121) compared with major clusters I and III. Whether the prevalence of PVL in carriage- and blood-culture isolates is higher and differs among distinct genetic clusters of S. aureus in countries with endemic CA-MRSA has to be investigated further. Figure Principal component analysis of amplified fragment length polymorphism (AFLP) data of 1,033 Staphylococcus aureus strains. The different cubes (plotted in 3-dimensional space) represent every strain in the study. Each axis represents the score calculated ... In conclusion, we have shown that the PVL-encoding phage has entered distinct staphylococcal lineages, although its prevalence differs per clonal group. PVL is associated with skin and soft tissue infections but not with bacteremia, which suggests that PVL is not likely to be involved in the pathogenesis of bacteremia. Infections caused by PVL-positive S. aureus strains have been documented since the 1930s. Expansion and increased incidence of such infections, however, are more recent, and further epidemiologic studies for tracking this phenomenon are still warranted.

Multilocus Variable-Number Tandem-Repeat Polymorphism among Brazilian Enterococcus faecalis Strains

ABSTRACT Multilocus variable-number tandem-repeat analysis (MLVA) for seven genomic loci was developed for Enterococcus faecalis. MLVA and pulsed-field gel electrophoresis (PFGE) resulted in 37 and 31 genotypes among 83 strains, respectively. Both typing schemes were highly concordant (90.4%). MLVA is an excellent alternative to PFGE.

Epidemiology of Nasopharyngeal Carriage of Neisseria meningitidis in Healthy Dutch Children

We investigated the prevalence and determinants of nasopharyngeal carriage of Neisseria meningitidis in 3200 healthy children aged 1-19 years. The incidence of meningococcal carriage was, on average, 1.5%. Peak incidences were seen at age 1 year and after age 15 years. The independent determinants of meningococcal carriage included age, regular visits to youth clubs (odds ratio [OR], 2.2) and discotheques (OR, 4.3), and pneumococcal carriage (OR, 4.1).