Damian C. Melles

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.

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.

Staphylococcus aureus Nasal Carriage Is Associated with Glucocorticoid Receptor Gene Polymorphisms

The aim of this study was to determine whether polymorphisms of the glucocorticoid receptor gene, influencing glucocorticoid sensitivity, are associated with persistent nasal carriage of Staphylococcus aureus. Two nasal swab cultures were obtained from each of 2,929 participants. Subjects were classified as persistent carriers (n=563) if both cultures were positive. GG homozygotes of the exon 9beta polymorphism were associated with a 68% reduced risk of persistent S. aureus nasal carriage, whereas carriers of the codon 23 lysine allele displayed an 80% increased risk. Thus, genotype-dependent variation in the sensitivity to glucocorticoids is associated with tolerance toward staphylococcal nasal colonization.

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.

Import and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae by international travellers (COMBAT study): a prospective, multicentre cohort study

BACKGROUND International travel contributes to the dissemination of antimicrobial resistance. We investigated the acquisition of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) during international travel, with a focus on predictive factors for acquisition, duration of colonisation, and probability of onward transmission. METHODS Within the prospective, multicentre COMBAT study, 2001 Dutch travellers and 215 non-travelling household members were enrolled. Faecal samples and questionnaires on demographics, illnesses, and behaviour were collected before travel and immediately and 1, 3, 6, and 12 months after return. Samples were screened for the presence of ESBL-E. In post-travel samples, ESBL genes were sequenced and PCR with specific primers for plasmid-encoded β-lactamase enzymes TEM, SHV, and CTX-M group 1, 2, 8, 9, and 25 was used to confirm the presence of ESBL genes in follow-up samples. Multivariable regression analyses and mathematical modelling were used to identify predictors for acquisition and sustained carriage, and to determine household transmission rates. This study is registered with ClinicalTrials.gov, number NCT01676974. FINDINGS 633 (34·3%) of 1847 travellers who were ESBL negative before travel and had available samples after return had acquired ESBL-E during international travel (95% CI 32·1-36·5), with the highest number of acquisitions being among those who travelled to southern Asia in 136 of 181 (75·1%, 95% CI 68·4-80·9). Important predictors for acquisition of ESBL-E were antibiotic use during travel (adjusted odds ratio 2·69, 95% CI 1·79-4·05), travellers diarrhoea that persisted after return (2·31, 1·42-3·76), and pre-existing chronic bowel disease (2·10, 1·13-3·90). The median duration of colonisation after travel was 30 days (95% CI 29-33). 65 (11·3%) of 577 remained colonised at 12 months. CTX-M enzyme group 9 ESBLs were associated with a significantly increased risk of sustained carriage (median duration 75 days, 95% CI 48-102, p=0·0001). Onward transmission was found in 13 (7·7%) of 168 household members. The probability of transmitting ESBL-E to another household member was 12% (95% CI 5-18). INTERPRETATION Acquisition and spread of ESBL-E during and after international travel was substantial and worrisome. Travellers to areas with a high risk of ESBL-E acquisition should be viewed as potential carriers of ESBL-E for up to 12 months after return. FUNDING Netherlands Organisation for Health Research and Development (ZonMw).

Host polymorphisms in interleukin 4, complement factor H, and C-reactive protein associated with nasal carriage of Staphylococcus aureus and occurrence of boils

BACKGROUND Staphylococcus aureus is capable of persistently colonizing the vestibulum nasi. We hypothesized that polymorphisms in host inflammatory response genes and genetic variation in S. aureus contribute to susceptibility to S. aureus carriage and infection. METHODS The prevalence of persistent nasal carriage of S. aureus in 3851 participants aged 61-101 years was 18% (678 of 3851 participants), whereas 73% of volunteers (2804 of 3851) were not colonized. A total of 1270 individuals had boils. Polymorphisms in TNFA (C -863T), IL4 (C -542T), CFH (Tyr402His), and CRP (C1184T, C2042T, and C2911G) were determined. Genetic similarity among 428 S. aureus isolates was determined by use of amplified fragment length polymorphism analysis (AFLP)-mediated genotyping. RESULTS The IL4 -524 C/C host genotype was associated with an increased risk of persistent S. aureus carriage, irrespective of S. aureus AFLP genotype. The CRP haplotype 1184C; 2042C; 2911C was overrepresented in individuals who were not colonized . In individuals with boils, carriers of the CFH Tyr402 variant, and the CRP 2911 C/C genotype were overrepresented. CONCLUSION Persistent carriage of S. aureus is influenced by genetic variation in host inflammatory response genes. As would be expected in multifactorial host-microbe interactions, these effects are limited. Interestingly, host genotype was associated with the carriage of certain S. aureus genotypes. Apparently, a close interaction between host and bacterial determinants are prerequisites for long-term colonization.

Clonal Distribution and Differential Occurrence of the Enterotoxin Gene Cluster, egc, in Carriage- versus Bacteremia-Associated Isolates of Staphylococcus aureus

ABSTRACT The Staphylococcus aureus enterotoxin gene cluster, egc, was detected in isolates from healthy individuals and in those from patients with bacteremia. The egc genes cooccur and are slightly enriched in strains from healthy carriers (present in 63.7% of carriage-associated isolates versus 52.9% of invasive isolates; P = 0.03). Multilocus sequence typing revealed that successful staphylococcal clones usually harbor the egc locus.

Distribution of the Serine-Aspartate Repeat Protein-Encoding sdr Genes among Nasal-Carriage and Invasive Staphylococcus aureus Strains

ABSTRACT The sdr locus was found in all 497 investigated Staphylococcus aureus strains, although in 29 strains it contained only the sdrC gene (sdrD negative, sdrE negative). The sdrC-positive, sdrD-negative, sdrE-negative gene profile was exclusive to methicillin-sensitive S. aureus (MSSA) strains (Fishers exact test; P = 0.0005) and was not found in the strains collected from bone infections (P = 0.0019). We also found a strong association between the presence of the sdrD gene and methicillin-resistant S. aureus strains (P < 0.0001). Our findings suggest that MSSA strains with the newly uncovered sdrC-positive, sdrD-negative, sdrE-negative gene profile have a substantially decreased potential to establish bone infection.

Overlapping Population Structures of Nasal Isolates of Staphylococcus aureus from Healthy Dutch and American Individuals

ABSTRACT To understand Staphylococcus aureus nasal carriage and its relationship with subsequent disease, insight into the natural (nonclinical) bacterial population structure is essential. This study investigated whether the distributions of S. aureus genotypes that cause colonization differ by geographic locales. High-throughput amplified fragment length polymorphism (AFLP) analysis was performed on nasal isolates of S. aureus from healthy American (n = 391) and Dutch (n = 829) volunteers. In total, 164,970 binary outcomes, covering 135 different markers per isolate, were scored. Methicillin resistance was defined for all strains; pulsed-field gel electrophoresis typing was performed for the American isolates. The overall population structures of the American and Dutch S. aureus isolates were comparable. The same four major AFLP clusters (I to IV) and subclusters were identified for both collections. However, the Dutch methicillin-susceptible S. aureus (MSSA) isolates were overrepresented in AFLP cluster III (P = 0.0016). Furthermore, the majority of the American methicillin-resistant S. aureus isolates (90.5%) were located in AFLP cluster I (P < 0.0001). This result identifies differences in the local prevalence of certain S. aureus genotypes. AFLP clusters II and III, which represent multilocus sequence typing clonal complexes 30 and 45, respectively, account for 46.4% of all MSSA isolates in the study, suggesting that these two lineages have evolved as extremely successful pandemic colonizers of humans. In conclusion, the overall population structures of American and Dutch nasal carriage isolates of S. aureus are surprisingly similar, despite subtle geographic differences in the prevalence of certain S. aureus genotypes.

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.