Diverse Enterotoxin Gene Profiles among Clonal Complexes of Staphylococcus aureus Isolates from the Bronx,New York

Staphylococcal enterotoxins (SE) can cause toxin-mediated disease, and those that function as superantigens are implicated in the pathogenesis of allergic diseases. The prevalence of 19 enterotoxin genes was determined by PCR in clinical S. aureus strains derived from wounds (108) and blood (99). We performed spa typing and multilocus sequence typing (MLST) to determine clonal origin, and for selected strains staphylococcal enterotoxin B (SEB) production was measured by enzyme-linked immunosorbent assay. Strains carried a median of five SE genes. For most SE genes, the prevalence rates among methicillin-resistant and methicillin-sensitive S. aureus isolates, as well as wound- and blood-derived isolates, did not differ. At least one SE gene was detected in all except two S. aureus isolates (>99%). Complete egc clusters were found in only 11% of S. aureus isolates, whereas the combination of sed, sej, and ser was detected in 24% of clinical strains. S. aureus strains exhibited distinct combinations of SE genes, even if their pulsed-field gel electrophoresis and MLST patterns demonstrated clonality. USA300 strains also showed considerable variability in SE content, although they contained a lower number of SE genes (mean, 3). By contrast, SE content was unchanged in five pairs of serial isolates. SEB production by individual strains varied up to 200-fold, and even up to 15-fold in a pair of serial isolates. In conclusion, our results illustrate the genetic diversity of S. aureus strains with respect to enterotoxin genes and suggest that horizontal transfer of mobile genetic elements encoding virulence genes occurs frequently.As a commensal, Staphylococcus aureus colonizes the nasal mucosa of 20 to 40% of humans (54), and as a pathogen it causes pyogenic diseases and toxin-mediated diseases (38). S. aureus produces many different virulence factors, including enterotoxins (SEs), which can cause defined toxic shock syndromes (4). The characterization of some of these toxins led to the discovery of superantigens (41), which bind to major histocompatibility complex class II molecules and Vβ chains of T-cell receptors, resulting in the activation of large numbers of T cells (20 to 30%) and massive cytokine production (10, 18). These superantigen-induced “cytokine storms” are responsible for the toxic effects seen in staphylococcal entertoxin B (SEB)- and toxic shock syndrome toxin (TSST)-associated shock syndromes in S. aureus infections (13, 40, 47). To date, 19 SEs have been identified based on sequence homologies, and studies have reported enterotoxin genes in up to 80% of all S. aureus strains (4, 21). Although many new enterotoxins have been identified, i.e., seg ser and seu (33, 37, 44, 49), their precise functions have not been characterized yet. The majority of experimental work with SEs is still done with SEB, toxic shock syndrome toxin 1, and SEA (27, 31), because these toxins are commercially available. Most SEs are located on mobile elements in bacterial genomes such as plasmids or pathogenicity islands and can thus be easily transferred horizontally between strains (5, 34, 35). Certain SE genes are grouped together. For instance seg, sei, sem, sen, and seo are commonly found in a gene cluster (egc) on genomic island νSAβ (34), and sel and sek are often found together with seb or sec on S. aureus pathogenicity islands. Other staphylococcal superantigen genes are encoded on plasmids (sed, sej, and ser) or are linked to the antibiotic resistance cassette SCCmec (seh) (44, 55). Phage φ3 carries either sea (strain Mu50), sep (N315), or sea sek seq (MW2) (1, 29).Although a few clinical studies have attempted to correlate shock and outcome with the presence of certain SEs in patients with S. aureus infections (17, 28), the contribution of these toxins to outcome is still unclear. Recent papers have proposed the SEs are immunomodulators and that colonization with S. aureus strains that produce SEB may contribute to the pathogenesis of asthma, chronic rhinitis, and dermatitis (2, 36, 46, 48, 56). The superantigen function of SEs in supernatants of S. aureus cultures can be neutralized by serum of colonized patients (21, 23). With new data emerging implicating SEs in the pathogenesis of chronic allergic syndromes, production of monoclonal antibodies and or vaccine strategies targeting SEs may be considered (6, 24, 26, 30) in the future. It is therefore important to characterize the prevalence of SE genes in clinical S. aureus strains.In this study, we analyzed SE content in both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains that were cultured from wounds (including USA300) and bloodstream infections of patients from a defined geographical area. In addition, SEB production was quantified by enzyme-linked immunosorbent assay (ELISA) in S. aureus strains carrying the seb gene, and spa typing confirmed clonal diversity among S. aureus isolates from different patients, as well as clonal stability in serial isolates, and multilocus sequence typing (MLST) done on a subset of less common spa types. We conclude that SE genes are abundant in S. aureus strains, albeit less abundant in USA300. SE content and combination are highly diverse and therefore more discriminatory than pulsed-field gel electrophoresis (PFGE) and MLST typing, albeit stable in serial isolates. Quantification of SEB production demonstrates that enterotoxin secretion can vary greatly among strains, even if they belong to the same S. aureus lineage. Given the complexities of SE prevalence, regulation, and possible function, we propose that the association of these toxins with chronic allergic diseases or outcome may be oversimplified at present. Precise characterizations of SE function and secretion patterns in individual S. aureus clones are warranted.     

Contribution of Panton-Valentine leukocidin in community-associated methicillin-resistant Staphylococcus aureus pathogenesis

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains typically carry genes encoding Panton-Valentine leukocidin (PVL). We used wild-type parental and isogenic PVL-deletion (Delta pvl) strains of USA300 (LAC and SF8300) and USA400 (MW2) to test whether PVL alters global gene regulatory networks and contributes to pathogenesis of bacteremia, a hallmark feature of invasive staphylococcal disease. Microarray and proteomic analyses revealed that PVL does not alter gene or protein expression, thereby demonstrating that any contribution of PVL to CA-MRSA pathogenesis is not mediated through interference of global gene regulatory networks. Inasmuch as a direct role for PVL in CA-MRSA pathogenesis remains to be determined, we developed a rabbit bacteremia model of CA-MRSA infection to evaluate the effects of PVL. Following experimental infection of rabbits, an animal species whose granulocytes are more sensitive to the effects of PVL compared with the mouse, we found a contribution of PVL to pathogenesis over the time course of bacteremia. At 24 and 48 hours post infection, PVL appears to play a modest, but measurable role in pathogenesis during the early stages of bacteremic seeding of the kidney, the target organ from which bacteria were not cleared. However, the early survival advantage of this USA300 strain conferred by PVL was lost by 72 hours post infection. These data are consistent with the clinical presentation of rapid-onset, fulminant infection that has been associated with PVL-positive CA-MRSA strains. Taken together, our data indicate a modest and transient positive effect of PVL in the acute phase of bacteremia, thereby providing evidence that PVL contributes to CA-MRSA pathogenesis.     

Determination of water quality,and trace metals in endemic Sarotherodon linellii,Pungu maclareni and Clarias maclareni,in crater Lake Barombi Mbo,Cameroon

Considering water pollution as a potential threat to some endemic cichlids of Lake Barombi Mbo, Cameroon, an investigation was done in 2011 to determine trace metals in its water, linking their uptake in gills and liver of fish to water chemistry. ICP-MS and ICP-OES analyses of trace metals based on total concentration of unfiltered lake water samples showed the presence of trace metals. All fish species accumulated Al, Mn and Sr in the highest concentrations in their gills, with Cu, Cd, Co, Cr, Pb and U highest in the liver. Pungu maclareni accumulated Al, Cr, Co, Sr and Pb in the highest concentrations. The highest mean gill Al concentration of 140 µg g^?1 dry weight was measured in P. maclareni gills, this being one of the critically endangered cichlids of the lake. Stable isotope analyses of carbon δ¹³C and nitrogen δ¹⁵N showed that P. maclareni had the highest mean δ¹³C (?30.2‰) and highest concentrations of Cr, Co, Pb and U in liver, probably linking the carbon source to the accumulation of metals. Though trace metal levels in the lake water were low, their presence in fish tissues suggest they are bioavailable, bioaccumulate and may pose a threat to the aquatic biota, and therefore should be monitored.     

Mutations in the CCGTTCACA DnaA box of Mycobacterium tuberculosis oriC that abolish replication of oriC plasmids are tolerated on the chromosome

The origin of replication (oriC) region in some clinical strains of Mycobacterium tuberculosis is a hot spot for IS6110 elements. To understand how clinical strains with insertions in oriC can replicate their DNA, we characterized the oriC regions of some clinical strains. Using a plasmid-based oriC-dependent replication assay, we showed that IS6110 insertions that disrupted the DnaA box sequence CCGTTCACA abolished oriC activity in M. tuberculosis. Furthermore, by using a surface plasmon resonance technique we showed that purified M. tuberculosis DnaA protein binds native but not mutant DnaA box sequence, suggesting that stable interactions of the DnaA protein with the CCGTTCACA DnaA box are crucial for replication of oriC plasmids in vivo. Replacement by homologous recombination of the CCGTTCACA DnaA box sequence of the laboratory strain M. tuberculosis H37Ra with a mutant sequence did not result in nonviability. Together, these results suggest that M. tuberculosis strains have evolved mechanisms to tolerate mutations in the oriC region and that functional requirements for M. tuberculosis oriC replication are different for chromosomes and plasmids.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Typing Staphylococcus aureus using the spa gene and novel distance measures

We developed an approach for identifying groups or families of Staphylococcus aureus bacteria based on genotype data. With the emergence of drug resistant strains, S. aureus represents a significant human health threat. Identifying the family types efficiently and quickly is crucial in community settings. Here, we develop a hybrid sequence algorithm approach to type this bacterium using only its spa gene. Two of the sequence algorithms we used are well established, while the third, the Best Common Gap-Weighted Sequence (BCGS), is novel. We combined the sequence algorithms with a weighted match/mismatch algorithm for the spa sequence ends. Normalized similarity scores and distances between the sequences were derived and used within unsupervised clustering methods. The resulting spa groupings correlated strongly with the groups defined by the well-established Multi locus sequence typing (MLST) method. Spa typing is preferable to MLST typing which types seven genes instead of just one. Furthermore, our spa clustering methods can be fine-tuned to be more discriminative than MLST, identifying new strains that the MLST method may not. Finally, we performed a multidimensional scaling of our distance matrices to visualize the relationship between isolates. The proposed methodology provides a promising new approach to molecular epidemiology.