Virulence of a hemB mutant displaying the phenotype of a Staphylococcus aureus small colony variant in a murine model of septic arthritis

Persistence of Staphylococcus aureus during invasive infections has been associated with a small-colony variant (SCV) phenotype. SCVs are frequently auxotrophic for menadione or hemin, two compounds involved in the biosynthesis of the electron transport chain. SCVs have been shown to be more resistant to antibiotics such as aminoglycosides, grow slowly and persist intracellularly. The aim of this study was to assess the virulence of an hemB mutant, which has been shown to display the typical characteristics of clinical SCVs, in a murine model of septic arthritis. NMRI mice were inoculated intravenously with either the wild type strain Newman or with its mutant displaying the SCV phenotype. The clinical, bacteriological, and histopathological progression of the disease was studied. Mice inoculated with the hemB mutant displayed a higher frequency and a significantly higher severity of arthritis than mice inoculated with the wild type Newman strain. Despite that, the mutant inoculated mice displayed significantly lower bacterial burden in their kidneys and joints compared with mice exposed to the wild parental strain. Notably, the hemB mutant produced almost 20 times more protease in vitro than the parental strain. We conclude that the small colony variants of S. aureus are more virulent on a per organism basis than its isogenic parental strain in the model of septic arthritis. This can at least in part be explained by the ability of SCV to produce high amounts of destructive proteases.     

Mutations are involved in emergence of aminoglycoside-induced small colony variants of Staphylococcus aureus

Staphylococcus aureus small colony variants (SCVs) occur frequently after local treatment with aminoglycosides and cause persistent as well as recurrent infections. So far, the molecular mechanism of the emergence of SCVs is not understood and regulatory as well as genetic mechanisms seem conceivable. To screen for possible mutations, the hemin biosynthetic gene cluster of a gentamicin-induced SCV was sequenced and was found to contain a deletion in the gene hemH. To further assess the influence of a high mutation rate on the development of SCVs, we tested the emergence of SCVs in a strain that had been inactivated in the DNA proofreading enzyme MutS. In the mutant, spontaneous SCVs emerged 556-fold more frequently than in the parent strain. By incubation in the presence of subinhibitory concentrations of gentamicin, the SCV frequency in the parent strain could be increased to 9.7 x 10(-6), whereas it remained rather stable in the mutant (1.8 x 10(-5)). Eighty percent of the gentamicin-induced SCVs were hemin auxotrophic in contrast to only 20% of the spontaneous SCVs which may explain the large proportion of hemin auxotrophs among clinical SCVs from patients previously treated with aminoglycosides. Additionally, a clinical S. aureus SCV isolate with a mutator phenotype, indicated by the generation of rifampicin-resistant mutants at a 16-fold higher frequency than in the reference strain S. aureus NCTC 8325, was characterized. The results demonstrate that a high mutation rate favours the emergence of SCVs, and suggest that mutations in general play an important role in the development of SCVs.     

Growth control of small-colony variants by genetic regulation of the hemin uptake system

Small-colony variants (SCVs) are slow-growing variants of human bacterial pathogens. They are associated with chronic persistent infections, and their biochemical identification and antimicrobial treatment are impaired by altered metabolic properties. To contribute to the understanding of SCV-mediated infections, we analyzed a clinical SCV isolate derived from a chronic prosthetic hip infection. A sequence analysis of housekeeping genes identified an Enterobacter hormaechei-like organism. The SCV phenotype, with growth as microcolonies, was caused by a block within the heme biosynthesis pathway through deletion of the hemB locus, as shown by hybridization and complementation experiments. At a low frequency, large-colony variants (LCVs) arose that were dependent on exogenous hemin. To investigate this phenomenon, we cloned and sequenced the 5.8-kb hemin uptake system, denoted ehu. Gene expression analysis indicated regulation of this locus in wild-type bacteria by the global iron regulator Fur. Inactivation of Fur in LCVs caused the derepression of ehu expression and facilitated bacterial growth. Genetic alterations of the fur locus in LCVs were identified as insertions of IS1A elements and point mutations. In contrast, SCVs could utilize exogenous hemin only in the absence of iron. Thus, we provide the first molecular characterization of the growth properties of a clinical SCV isolate, which may help to improve the diagnostic and therapeutic management of patients with chronic persistent infections.     

Fourier-transform infrared spectroscopic analysis is a powerful tool for studying the dynamic changes in Staphylococcus aureus small-colony variants

Infections due to small-colony variants (SCVs) of Staphylococcus aureus in patients with chronic and recurrent infections are an emerging problem; however, studies with this subpopulation are hampered by the fact that SCVs may exhibit unstable phenotypes, making them difficult to study, particularly in broth media. In this study, two S. aureus sets comprising the (i) normal and the (ii) SCV phenotype (clonal with normal phenotype) recovered from clinical specimens, as well as (iii) corresponding site-directed mutants displaying the SCV phenotype (knockout of hemB) and (iv) their complemented mutants were examined by Fourier-transform infrared (FTIR) spectroscopy. Phenotypes were defined on solid and in broth media. Using first-derivative infrared spectra to calculate spectral distances, hierarchical clustering based on spectral information resulted in a dendrogram with clear discrimination between SCV and normal phenotypes. The SCVs gave an FTIR fingerprint that was easily recognizable and that was much closer to other SCVs than to their parent strains. This technique offers for the first time a noninvasive approach to investigate dynamic processes of reversion of SCVs to the normal phenotype and vice versa. Thus, FTIR spectroscopy allowed a rapid and reproducible tool for the examination of different subpopulations of S. aureus on solid and in broth media for diagnostic and research purposes.     

Physiology and antibiotic susceptibility of Staphylococcus aureus small colony variants

Small colony variants (SCV) are slow-growing subpopulations with altered metabolism and reduced antibiotic susceptibility which, in the case of Staphylococcus aureus, can cause persisting and recurrent infections. We studied four SCVs and their corresponding parent strains: one clinical strain pair, one menaquinone-deficient spontaneous mutant, and two constructed mutants obtained by inactivation of hemB in S. aureus 8325-4 and COL, respectively. SCVs growing in chemically defined medium (CDM) with glucose limitation and enhanced buffering capacity were found to generate deltapsi of -120 to -140 mV, which is comparable to the parent strains. However, glucose is consumed inefficiently with small growth yields. In contrast to wild-type strains, deltapsi dropped immediately to values below -100 mV when glucose expired and other nutrients such as acetate and lactate did not allow for further growth. Accordingly, the sensitivity of SCVs toward antibiotics known to be taken up through deltapsi, such as aminoglycosides, dropped 10- to 30-fold when compared to the parent strain under routine MIC determination conditions. When growing in CDM, the susceptibility of SCVs varied according to the magnitude of deltapsi.     

Virulence of Staphylococcus aureus small colony variants in the Caenorhabditis elegans infection model

Small colony variants (SCVs) of Staphylococcus aureus are slow-growing morphological variants that have been implicated in persistent, relapsing, and antibiotic-resistant infections. The altered phenotype of SCVs in most strains has been attributed to defects in electron transport due to mutations in hemin or menadione biosynthesis. The pathogenic capacity of SCVs compared to phenotypically normal strains is variable depending on the attribute examined, with some studies showing reduced virulence of SCVs and others demonstrating normal or heightened virulence. Recently, the nematode Caenorhabditis elegans has been successfully employed as an alternative host to investigate virulence mechanisms of a variety of bacterial pathogens, including S. aureus. In this study, we show that clinical SCVs as well as hemB- and menD-deficient mutants of S. aureus are greatly reduced in virulence in the C. elegans infection model.     

The multifunctional Staphylococcus aureus autolysin aaa mediates adherence to immobilized fibrinogen and fibronectin

Staphylococci can cause a wide spectrum of infections, including endocarditis, osteomyelitis, and sepsis, which is reflected by the numerous virulence factors they produce, among them a recently identified new class of adhesins, namely, the multifunctional autolysins/adhesins. Here we report the identification and molecular characterization of Aaa, a novel autolysin/adhesin from Staphylococcus aureus. The gene encoding Aaa was cloned from the clinical isolate Staphylococcus aureus 4074. DNA sequence analysis revealed that aaa encodes a deduced protein of 334 amino acids with a predicted molecular mass of 35.8 kDa. Aaa contains three N-terminal repetitive sequences that comprise features of a peptidoglycan-binding domain, the LysM domain. The expression of aaa by Escherichia coli and its subsequent characterization revealed that Aaa possesses bacteriolytic activity as well as adhesive properties, such as binding to extracellular matrix proteins. Real-time biomolecular interaction analysis demonstrated that the interaction of Aaa with fibrinogen, fibronectin, and vitronectin is dose dependent and saturable and occurs with a high affinity. Furthermore, we demonstrate that Aaa binds to the Aalpha and Bbeta chains of fragment D of fibrinogen. Immunofluorescence microscopy revealed that Aaa is located at the cell surface. Finally, an aaa knockout mutant showed reduced adherence to surface-adsorbed fibrinogen and fibronectin, strongly suggesting a role for Aaa in the colonization of host factor-coated polymer surfaces and/or host tissue.     

Identification of Point Mutations in Clinical Staphylococcus aureus Strains That Produce Small-Colony Variants Auxotrophic for Menadione

Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.     

The Agr Quorum-Sensing System Regulates Fibronectin Binding but Not Hemolysis in the Absence of a Functional Electron Transport Chain

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain.     

The influence of adhesive and invasive properties of Staphylococcus aureus defective in fibronectin-binding proteins on secretion of interleukin-6 by human endothelial cells

Fibronectin-binding proteins (FnBP) are surface adhesins of Staphylococcus aureus documented to be virulence attributes in, for example, endovascular infections. By using mutants of S. aureus defective in the FnBPA and B genes we have investigated whether these adhesins affect cytokine expression in human umbilical vein endothelial cells (HUVEC). S. aureus expressing FnBPA and B adhered to and were internalized into HUVEC to a greater extent compared to mutants defective in expression of FnBP. Production and release of IL-6 was higher from endothelial cells infected with the parent FnBP-expressing strain compared to the FnBP-defective mutants. These results indicate that adhesion to and invasion of S. aureus into endothelial cells are important regulators of cytokine expression.     

Functional blocking of Staphylococcus aureus adhesins following growth in ex vivo media

Defining the role of Staphylococcus aureus adhesins in disease pathogenesis may depend on the use of bacteria grown in culture media that more closely reflect the human milieu than conventional broth. This study examined the functional effect on S. aureus adhesins following growth in an ex vivo medium containing a complex mixture of human proteins (used peritoneal dialysate) relative to growth in Todd-Hewitt broth. The adherence of S. aureus, cultured in dialysate, to fibronectin and fibrinogen was markedly reduced despite the expresion of full-length ClfA, ClfB, and fibronectin-binding proteins. Growth in dialysate resulted in the acquisition of a surface coat, as visualized by transmission electron microscopy, which was shown to contain fibronectin, fibrinogen, and immunoglobulins. Adherence of S. aureus to fibrinogen following growth in dialysate was significantly reduced by expression of protein A but was restored following growth in immunoglobulin-depleted dialysate. We conclude that bacterial adherence to solid-phase protein is critically dependent on the culture medium, that S. aureus adhesins may become saturated with target protein prior to contact with solid surfaces, and that there is an interaction between fibrinogen-binding proteins and immunoglobulin bound to protein A following contact with host proteins. These findings have important implications for future studies of S. aureus adhesins.     

Evaluation of two chromogenic agar media for recovery and identification of Staphylococcus aureus small-colony variants

To identify the most rapid and reliable technique for recovery and identification of Staphylococcus aureus small-colony variants (SCVs), the colonial appearance of 106 isolates representing SCVs and the normal phenotype were evaluated on two newly described chromogenic agar media. Although almost all of the SCVs grew on the chromogenic agar media, they did not exhibit a change of color. In comparison with conventional media, S. aureus ID agar (SAID; bioMerieux, La Balme Les Grottes, France) showed the most reliable results, with 49 of 53 SCVs tested growing either as an SCV colony or with a normal phenotype after only 24 h of incubation. Growth of SCVs was often not detected before 72 h of incubation on some of the media tested. In conclusion, the most accurate and rapid method to detect both the species S. aureus and the SCV phenotype is to inoculate specimens onto both Columbia blood agar and SAID.     

Influence of dTMP on the phenotypic appearance and intracellular persistence of Staphylococcus aureus

Thymidine-dependent small-colony variants (SCVs) of Staphylococcus aureus are frequently associated with persistent and recurrent infections in cystic fibrosis patients. The phenotypic appearance of S. aureus SCVs or normal-colony variants (NCVs) is postulated to be affected by the intracellular amount of dTMP. This hypothesis was proven by metabolic pathway assays revealing altered intracellular dTMP concentrations, followed by investigation of the associated phenotype. Inhibition of the staphylococcal thymidylate synthase, which generated intracellular dTMP from dUMP, using 5-fluorouracil and co-trimoxazole resulted in an SCV phenotype. Inhibition of a nucleoside transporter, which provided the bacterial cell with extracellular thymidine, caused growth inhibition of SCVs. In turn, reversion of SCVs to NCVs was achieved by supplying extracellular dTMP. High-performance liquid chromatography additionally confirmed the intracellular lack of dTMP in SCVs, in contrast to NCVs. Moreover, the dTMP concentration is postulated to influence the intracellular persistence of S. aureus. Cell culture experiments with cystic fibrosis cells revealed that clinical and co-trimoxazole-induced SCVs with a diminished amount of dTMP showed significantly better intracellular persistence than NCVs. In conclusion, these results show that the dTMP concentration plays a key role in both the phenotypic appearance and the intracellular persistence of S. aureus.     

Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of Staphylococcus aureus

Thymidine-auxotrophic small colony variants (SCVs) of Staphylococcus aureus are frequently isolated from the chronically infected airways of patients suffering from cystic fibrosis. To date, little is known regarding the molecular mechanisms leading to the formation of this special phenotype, but the auxotrophism for thymidine suggests that impaired thymidine metabolism might play a major role. Sequence analysis of the thymidylate synthase-encoding thyA gene of six clinical thymidine-auxotrophic S. aureus SCVs revealed that all isolates had mutations within thyA. In five isolates the function of the thymidylate synthase was definitely impaired: three of them showed a truncation of the thyA coding sequence by nonsense or frame-shift mutations, in one further isolate the active site of the enzyme was affected by an internal 12-bp deletion, and another isolate had a 173-bp deletion spanning the 5'-terminal region of thyA and the preceding DNA sequence. The sixth isolate showed two amino acid substitutions within the thyA gene product. To confirm the importance of impaired thymidylate synthase synthesis or activity for the formation of the thymidine-auxotrophic SCV phenotype, we constructed a thyA knock-out mutant of a wild-type S. aureus strain. This mutant showed all characteristics of clinical SCVs, such as slow growth, decreased pigment production, reduced hemolytic activity, auxotrophism for thymidine, resistance to trimethoprim/sulfamethoxazol, and reduced plasma coagulase activity. Complementation of the thyA knock-out mutant with intact thyA in trans nearly restored the normal phenotype. In conclusion, these data confirm at the molecular level that impaired thymidylate synthase function is causative for the formation of the thymidine-auxotrophic SCV phenotype in S. aureus.     

Adherence properties of Staphylococcus aureus under static and flow conditions: roles of agr and sar loci, platelets, and plasma ligands

Global regulatory genes in Staphylococcus aureus, including agr and sar, are known to regulate the expression of multiple virulence factors, including cell wall adhesins. In the present study, the adherence of S. aureus RN6390 (wild type), RN6911 (agr), ALC136 (sar), and ALC135 (agr sar) to immobilized fibrinogen, fibronectin, von Willebrand factor (vWF), extracellular matrix (ECM), and human endothelial cells (EC) EAhy.926 was studied. Bacteria grown to postexponential phase were subjected to light oscillation (static condition) or to shear stress at 200 s(-1) (flow condition) on tissue culture polystyrene plates coated with either protein ligands, ECM, or EC. Adherence of nonlabeled bacteria to immobilized ligands was measured by an image analysis system, while adherence of [(3)H]thymidine-labeled S. aureus to ECM and EC was measured by a beta-scintillation counter. The results showed increased adherence of agr and agr sar mutants to immobilized fibrinogen and higher potential of these mutants to induce platelet aggregation in suspension, decreased adherence of sar and agr sar mutants to immobilized fibronectin and vWF as well as to ECM and EC, increased adherence of both S. aureus wild type and sar mutant to EC treated with platelet-rich plasma (PRP) compared to platelet-poor plasma (PPP) and to EC treated with PPP compared to the control, and increased adherence of S. aureus wild type to EC coated with PRP in which platelets were activated with phorbol 12-myristate 13-acetate compared to intact PRP. This finding paralleled the increased adherence to EC of activated compared to intact platelets. It is suggested that platelet-mediated S. aureus adherence to EC depends on platelet activation and the number of adherent platelets and available receptors on the platelet membrane. In conclusion, the agr locus downregulates S. aureus adherence to fibrinogen, while the sar locus upregulates S. aureus adherence to fibronectin, vWF, ECM, and EC. The effect of both agr and sar on S. aureus adherence properties develops primarily under flow conditions, which suggests different adhesion mechanisms in static and flow conditions.     

Prevalence and clinical significance of Staphylococcus aureus small-colony variants in cystic fibrosis lung disease

Small-colony variants (SCVs) of Staphylococcus aureus can be isolated from the chronically infected airways of patients suffering from cystic fibrosis (CF). These slow-growing morphological variants have been associated with persistent and antibiotic-resistant infections, such as osteomyelitis and device-related infections, but no information is available to date regarding the clinical significance of this special phenotype in CF lung disease. We therefore investigated the prevalence of S. aureus SCVs in CF lung disease in a 12-month prospective study and correlated the microbiological culture results with the patients' clinical data. A total of 252 patients were screened for the presence of SCVs. The prevalence rate was determined to be 17% (95% confidence interval, 10 to 25%) among S. aureus carriers. S. aureus isolates with the SCV phenotype showed significantly higher antibiotic resistance rates than those with the normal phenotype. Patients positive for SCVs were significantly older (P = 0.0099), more commonly cocolonized with Pseudomonas aeruginosa (P = 0.0454), and showed signs of more advanced disease, such as lower forced expiratory volume in 1 s (P = 0.0148) than patients harboring S. aureus with a solely normal phenotype. The logistic regression model determined lower weight (P = 0.016), advanced age (P = 0.000), and prior use of trimethoprim-sulfamethoxazole (P = 0.002) as independent risk factors for S. aureus SCV positivity. The clinical status of CF patients is known to be affected by multiple parameters. Nonetheless, the independent risk factors determined here point to the impact of S. aureus SCVs on chronic and persistent infections in advanced CF lung disease.     

Evaluation of different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureus

To evaluate different methods for their abilities to detect methicillin resistance in small-colony variants (SCVs) of Staphylococcus aureus, 11 different methicillin-resistant S. aureus (MRSA) clones with the SCV phenotype were used in this study. The slow growth of SCVs often makes testing by disk diffusion or by automated methods invalid. Only detection of the mecA gene by PCR and the MRSA-Screen latex agglutination test using a higher colony number were shown to be reliable methods to rapidly detect methicillin resistance in these variants.     

Expression of the biofilm-associated protein interferes with host protein receptors of Staphylococcus aureus and alters the infective process

The adherence of Staphylococcus aureus to soluble proteins and extracellular-matrix components of the host is one of the key steps in the pathogenesis of staphylococcal infections. S. aureus presents a family of adhesins called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that specifically recognize host matrix components. We examined the influence of biofilm-associated protein (Bap) expression on S. aureus adherence to host proteins, epithelial cell cultures, and mammary gland sections and on colonization of the mammary gland in an in vivo infection model. Bap-positive strain V329 showed lower adherence to immobilized fibrinogen and fibronectin than isogenic Bap-deficient strain m556. Bacterial adherence to histological sections of mammary gland and bacterial internalization into 293 cells were significantly lower in the Bap-positive strains. In addition, the Bap-negative strain showed significantly higher colonization in vivo of sheep mammary glands than the Bap-positive strain. Taken together, these results strongly suggest that the expression of the Bap protein interferes with functional properties of the MSCRAMM proteins, preventing initial bacterial attachment to host tissues and cellular internalization.     

Inhibition by heparin and derivatized dextrans of Staphylococcus epidermidis adhesion to in vitro fibronectin-coated or explanted polymer surfaces

The ability of Staphylococcus aureus to recognize several extracellular matrix or plasma proteins (e.g., fibrinogen, fibronectin, and collagen) promotes bacterial attachment to artificial surfaces. Whereas most S. aureus clinical isolates elaborate a wide repertoire of bacterial surface receptors' called adhesins, exhibiting specific binding of individual host proteins, S. epidermidis is lacking most of such protein adhesins. To document the interactions between S. epidermidis and various surface-adsorbed proteins, we first compared promotion of bacterial attachment by seven purified human proteins immobilized onto poly(methyl methacrylate) (PMMA) coverslips. Only two of them, namely fibronectin and fibrinogen, exhibited adhesion-promoting activities. In the presence of native heparin or two functionalized dextrans (CMDBS for Carboxy Methyl, Benzylamide sulfonate/sulfate), a dose-dependent inhibition of S. epidermidis adhesion to fibronectin-coated, but not to fibrinogen-coated surfaces was observed. The inhibitory effects of each CMDBS were much stronger than that of native heparin. In contrast, a control highly negatively charged, dextran exclusively substituted with carboxy methyl groups exerted no inhibition on S. epidermidis adhesion. To evaluate how CMDBS could interfere with S. epidermidis attachment to coverslips coated in vivo with extracellular matrix components, we also tested PMMA surfaces retrieved from tissue cages subcutaneously implanted in guinea pigs. Each CMDBS, but not heparin, strongly inhibited S. epidermidis adhesion to explanted coverslips, even in the presence of tissue cage fluid. In conclusion, fibronectin plays an important role in promoting S. epidermidis attachment to implanted biomaterials. Furthermore, S. epidermidis adhesion to fibronectin-coated or implanted biomaterials can be efficiently blocked in vitro by CMDBS.