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.     

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.     

Ribonucleotide reductase class III, an essential enzyme for the anaerobic growth of Staphylococcus aureus, is a virulence determinant in septic arthritis

Staphylococcus aureus is the most common cause of joint infections. It also contributes to several other diseases such as pneumonia, osteomyelitis, endocarditis, and sepsis. Bearing in mind that S. aureus becomes rapidly resistant to new antibiotics, many studies survey the virulence factors, with the aim to find alternative prophylaxis/treatment regimens. One potential virulence factor is the bacterial ability to survive at different oxygen tensions. S. aureus expresses ribonucleotide reductases (RNRs), which help it to grow under both aerobic and anaerobic conditions, by reducing ribonucleotides to deoxyribonucleotides. In this study, we investigated the role of RNR class III, which is required for anaerobic growth, as a virulence determinant in the pathogenesis of staphylococcal arthritis. The wild-type S. aureus strain and its isogenic mutant nrdDG mutant were inoculated intravenously into mice. Mice inoculated with the wild-type strain displayed significantly more severe arthritis, with significantly more synovitis and destruction of the bone and cartilage versus mutant strain inoculated mice. Further, the persistence of bacteria in the kidneys was significantly more pronounced in the group inoculated with the wild-type strain. Together these results indicate that RNR class III is an important virulence factor for the establishment of septic arthritis.     

The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model.

We have studied the role of the accessory gene regulator (agr) of Staphylococcus aureus as a virulence determinant in the pathogenesis of septic arthritis. At least 15 genes coding for potential virulence factors in Staphylococcus aureus are regulated by a putative multicomponent signal transduction system encoded by the agr/hld locus. agr and hld mutants show a decreased synthesis of extracellular toxins and enzymes, such as alpha-, beta-, and delta-hemolysin, leucocidin, lipase, hyaluronate lyase, and proteases, and at the same time an increased synthesis of coagulase and protein A as compared with the wild-type counterpart. We have used a recently described murine model of S. aureus-induced arthritis to study the virulence of S. aureus 8325-4 and two agr/hld mutants derived from it. Sixty percent of the mice injected with the wild-type strain developed arthritis, whereas agrA and hld mutants displayed joint involvement in only 10 and 30%, respectively. In addition, 40% of the mice inoculated with the wild-type strain displayed an erosive arthropathy; such changes were not detectable at all in mice inoculated with the agrA mutant. Serum levels of interleukin-6, a potent B-cell differentiation factor, were significantly higher (P < 0.001) in the mice inoculated with the wild-type strain than in those inoculated with the agrA mutant counterpart. Overall, our results suggest that the agr system of S. aureus is an important virulence determinant in the induction and progression of septic arthritis in mice.     

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.     

Protein A is a virulence factor in Staphylococcus aureus arthritis and septic death

Staphylococcal protein A (SpA), a cell wall anchored protein of Staphylococcus aureus, has the ability to interact with several host components, possibly indicating a role as a virulence factor in S. aureus infections. In this study, the contribution of SpA to bacterial virulence was investigated in a murine model of S. aureus arthritis. Intravenous inoculation of S. aureus wild-type strain Newman gave rise to more severe arthritis and higher mortality than the isogenic spa mutant strain DU5873. The wild-type strain caused more in vitro spleen cell proliferation than the SpA-deficient strain. However, IL-6 and TNF-alpha levels were higher after stimulation with thespa mutant strain compared to the wild-type strain. To conclude, our results clearly indicate that SpA is a virulence factor of S. aureus in murine septic arthritis.     

Alpha-toxin and gamma-toxin jointly promote Staphylococcus aureus virulence in murine septic arthritis

Septic arthritis is a common and feared complication of staphylococcal infections. Staphylococcus aureus produces a number of potential virulence factors including certain adhesins and enterotoxins. In this study we have assessed the roles of cytolytic toxins in the development of septic arthritis by inoculating mice with S. aureus wild-type strain 8325-4 or isogenic mutants differing in the expression of alpha-, beta-, and gamma-toxin production patterns. Mice inoculated with either an alpha- or beta-toxin mutant showed degrees of inflammation, joint damage, and weight decrease similar to wild-type-inoculated mice. In contrast, mice inoculated with either double (alpha- and gamma-toxin-deficient)- or triple (alpha-, beta-, and gamma-toxin-deficient)-mutant S. aureus strains showed lower frequency and severity of arthritis, measured both clinically and histologically, than mice inoculated with the wild-type strain. We conclude that simultaneous production of alpha- and gamma-toxin is a virulence factor in S. aureus arthritis.     

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.     

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.     

The surface protein Pls of methicillin-resistant Staphylococcus aureus is a virulence factor in septic arthritis

Pls, a surface protein of certain methicillin-resistant Staphylococcus aureus strains, is associated with poor bacterial adherence to solid-phase fibronectin and immunoglobulin G, as well as with reduced invasion of cultured epithelial cells. Here the importance of Pls for the development of septic arthritis and sepsis was investigated by using a mouse model. Mice inoculated with a pls knockout mutant developed a much milder arthritis and showed less grave weight reduction than mice infected with the wild-type Pls(+) clinical isolate. Also, the pls mutant induced a significantly lower frequency of mortality than the wild-type strain. The bacterial load of the kidneys was larger in mice infected with the Pls(+) strain than in animals challenged with the pls mutant. However, there was no evident inflammatory effect due to the Pls molecule alone, as indicated by knee injection of purified Pls. In conclusion, the results show that Pls is a virulence factor for septic arthritis and sepsis.     

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.     

Ribonucleotide reductase class III,an essential enzyme for the anaerobic growth of Staphylococcus aureus,is a virulence determinant in septic arthritis

Staphylococcus aureus is the most common cause of joint infections. It also contributes to several other diseases such as pneumonia, osteomyelitis, endocarditis, and sepsis. Bearing in mind that S. aureus becomes rapidly resistant to new antibiotics, many studies survey the virulence factors, with the aim to find alternative prophylaxis/treatment regimens. One potential virulence factor is the bacterial ability to survive at different oxygen tensions. S. aureus expresses ribonucleotide reductases (RNRs), which help it to grow under both aerobic and anaerobic conditions, by reducing ribonucleotides to deoxyribonucleotides. In this study, we investigated the role of RNR class III, which is required for anaerobic growth, as a virulence determinant in the pathogenesis of staphylococcal arthritis. The wild-type S. aureus strain and its isogenic mutant nrdDG mutant were inoculated intravenously into mice. Mice inoculated with the wild-type strain displayed significantly more severe arthritis, with significantly more synovitis and destruction of the bone and cartilage versus mutant strain inoculated mice. Further, the persistence of bacteria in the kidneys was significantly more pronounced in the group inoculated with the wild-type strain. Together these results indicate that RNR class III is an important virulence factor for the establishment of septic arthritis.     

Diminished platelet binding in vitro by Staphylococcus aureus is associated with reduced virulence in a rabbit model of infective endocarditis.

The direct binding of platelets by bacteria is a postulated central mechanism in the pathogenesis of endocarditis. To address the role of binding more definitively, we employed Tn551 insertional mutagenesis of Staphylococcus aureus parental strain ISP479 to generate an isogenic variant (strain PS12) that bound platelets minimally. As compared with the binding of ISP479, the binding of PS12 to platelet monolayers was reduced by 67.2%. Similarly, the binding of PS12 to platelets in suspension was reduced by 71.3%, as measured by flow cytometry. The low-binding phenotype was transducible into both ISP479 and S. aureus Newman. Southern blotting indicated that a single copy of Tn551 was inserted within the chromosomes of PS12 and the transductants. When tested in a rabbit model, animals inoculated with PS12 were significantly less likely to develop endocarditis and had lower densities of organisms (CFU per gram) within vegetations and a decreased incidence of renal abscess formation, as compared with animals inoculated with the parental strain. The diminished virulence of PS12 was not attributable to a reduction in the initial attachment of organisms to the damaged endocardium, since 30 min after inoculation, PS12-infected animals had microbial densities on the valve surface comparable to those seen with the parental strain. These results indicate that the direct binding of Staphylococcus aureus to platelets is a major determinant of virulence in the pathogenesis of endocarditis. Staphylococcus-platelet binding appears to be critical for pathogenetic events occurring after the initial colonization of the valve surface, such as vegetation formation and septic embolization.     

Sigma factor B and RsbU are required for virulence in Staphylococcus aureus-induced arthritis and sepsis

The prototype Staphylococcus aureus strain 8325-4 produces high levels of hemolysins and proteases. Recently it has been shown that this property depends on a deficiency of sigma factor B (SigB) activity controlling the activation of regulatory genes such as agr and sarA. SigB deficiency is in turn due to a mutation in the rsbU gene, which is required for posttranslational activation of SigB. The rsbU defect of strain 8325-4 has recently been repaired, and we used this strain (SH1000), along with its isogenic sigB-negative mutant, to investigate the contributions of RsbU and SigB in a murine model of septic arthritis. Intravenous inoculation with the rsbU-repaired isogenic strain SH1000 resulted in significantly more severe arthritis, weight decrease, and mortality compared to those of the parental strain 8325-4 (rsbU-negative) or the isogenic sigB-negative mutant (MJH502). SH1000 also persisted more in kidneys and joints of infected mice. Our data strongly suggest that RsbU and SigB regulate important virulence factors, thereby contributing significantly to the outcome of staphylococcal infection.     

The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis.

The importance of a collagen-binding adhesin in the pathogenesis of septic arthritis has been examined by comparing the virulence of two sets of Staphylococcus aureus mutants in an animal model. Collagen adhesin-negative mutant PH100 was constructed by replacing the chromosomal collagen adhesin gene (cna) in a clinical strain, Phillips, with an inactivated copy of the gene. Collagen adhesin-positive mutant S. aureus CYL574 was generated by introducing the cna gene into CYL316, a strain that normally lacks the cna gene. Biochemical, immunological, and functional analyses of the generated mutants and their respective parent strains showed that binding of 125I-labeled collagen, expression of an immunoreactive collagen adhesin, and bacterial adherence to cartilage were directly correlated with the presence of a functional cna gene. Greater than 70% of the mice injected with the Cna+ strains developed clinical signs of arthritis, whereas less than 27% of the animals injected with Cna- strains showed symptoms of disease. Furthermore, mice injected with the Cna+ strain Phillips had remarkably elevated levels of immunoglobulin G1 and interleukin-6 compared with mice injected with the Cna- mutant PH100. Taken together, these results demonstrate that collagen adhesin plays an important role in the pathogenesis of septic arthritis induced by S. aureus.     

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.     

Preferential induction of septic arthritis and mortality by superantigen-producing staphylococci.

Staphylococcal enterotoxins A through D (SEA through SED) and toxic shock syndrome toxin-1 display superantigen properties, i.e., they stimulate a great fraction of T cells expressing certain T-cell receptor V beta sequences. Using a newly established rat model of septic Staphylococcus aureus arthritis, we have recently shown that an S. aureus strain producing SEA showed marked arthritogenic properties. In the present study we decided to employ another five S. aureus strains, each one producing a distinct exotoxin. Almost all rats injected with superantigen-producing strains developed arthritis. In contrast, only 20% of rats injected with an S. aureus strain lacking superantigen production displayed mild and transient arthritis. Mortality was preferentially induced among the rats inoculated with the S. aureus strains producing SEB and SED. This study emphasizes that superantigen production is an important virulence factor in the development of septic S. aureus arthritis. Differences concerning mortality between staphylococcal strains producing different exotoxins may be dependent on the degree of activation of the immune system.     

The role of staphylococcal polysaccharide microcapsule expression in septicemia and septic arthritis.

Staphylococcus aureus arthritis is a rapidly progressive and highly erosive disease of the joints in which both host and bacterial factors are of pathogenic importance. One potential bacterial virulence factor is the ability to express a polysaccharide capsule (CP). Among 11 reported capsular serotypes, CP type 5 (CP5) and CP8 comprise 80 to 85% of all clinical blood isolates. The aim of this study was to assess the role of CP5 as a virulence factor in staphylococcal septicemia and septic arthritis with a recently established murine model of hematogenously spread S. aureus arthritis. NMRI mice were inoculated intravenously with S. aureus strains isogenic for expression of CP5, and clinical, bacteriological, serological, and histopathological progression of disease was studied. Inoculation of 7 x 10(6) CFU of S. aureus per mouse induced 55% mortality in the group inoculated with the CP-expressing bacteria, compared to 18% in the group inoculated with CP- mutants. A lower dose of inoculum (3 x 10[6] per mouse) did not give rise to mortality in mice inoculated with CP mutant strains, whereas 18% of the mice inoculated with the CP5-expressing S. aureus died. Importantly, mice inoculated with S. aureus expressing CP5 had a significantly higher frequency of arthritis and a more severe form of the disease. In vitro assays suggested that macrophages were not able to phagocytize CP5+ staphylococci as efficiently as they were CP5- strains. In addition, once phagocytized, CP5+ bacteria were less efficiently killed than CP- mutants. In summary, CP5 leads to a higher frequency of arthritis and a more severe course of the disease. This seems to be related to the effects of the downregulatory properties of CP on the ingestion and intracellular killing capacity of phagocytes. Our results clearly indicate that the expression of CP5 is a determinant of the virulence of S. aureus in arthritis and septicemia.     

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.