Staphylococcal small colony variants have novel mechanisms for antibiotic resistance.

Over the past 4 years, a variant subpopulation of Staphylococcus aureus has been characterized that is defective in electron transport. These organisms grow slowly and are typical of the previously described small colony variants (SCVs). Indeed, many earlier papers included data that are consistent with defective respiratory activity in SCVs. We present a hypothesis that serves as biochemical basis for the development of SCVs. These variants are particularly interesting because they have been associated with very persistent infections, and they are more resistant to many antibiotics than normal S. aureus. Because of their slow growth, atypical colonial morphology, and unusual biochemical profile, they are easily missed or misidentified in the clinical laboratory. This is of some significance, as this subpopulation is more resistant to antibiotics than the parent population from which they arose. When an infection is particularly resistant to therapy, persists for a long period, or fails to respond to apparently adequate antimicrobial therapy, clinicians and clinical laboratory personnel should consider special efforts to search for SCVs.

Introduction of the mec element (methicillin resistance) into Staphylococcus aureus alters in vitro functional activities of fibrinogen and fibronectin adhesins.

Some methicillin-resistant strains of Staphylococcus aureus are defective in the production of major surface components such as protein A, clumping factor, or other important adhesins to extracellular matrix components which may play a role in bacterial colonization and infection. To evaluate the impact of methicillin resistance (mec) determinants on bacterial adhesion mediated by fibrinogen or fibronectin adhesins, we compared the in vitro attachment of two genetically distinct susceptible strains (NCTC8325 and Newman) to protein-coated surfaces with that of isogenic methicillin-resistant derivatives. All strains containing an intact mec element in their chromosomes were found to be defective in adhesion to fibrinogen and fibronectin immobilized on polymethylmethacrylate coverslips, regardless of the presence or absence of additional mutations in the femA, femB, or femC gene, known to decrease expression of methicillin resistance in S. aureus. Western ligand affinity blotting or immunoblotting of cell wall-associated adhesins revealed similar contents of fibrinogen- or fibronectin-binding proteins in methicillin-resistant strains compared to those of their methicillin-susceptible counterparts. In contrast to methicillin-resistant strains carrying a mec element in their genomes, methicillin-resistant strains constructed in vitro, by introducing the mecA gene on a plasmid, retained their adhesion phenotypes. In conclusion, the chromosomal insertion of the mec element into genetically defined strains of S. aureus impairs the in vitro functional activities of fibrinogen or fibronectin adhesins without altering their production. This effect is unrelated to the activity of the mecA gene.

Decreased susceptibility to antibiotic killing of a stable small colony variant of Staphylococcus aureus in fluid phase and on fibronectin-coated surfaces.

The frequency of small colony variants of staphylococci associated with persistent, antibiotic resistant and relapsing infections is probably underestimated. These variants demonstrate decreased metabolism, leading to slow growth, increased resistance to cell-wall-active antibiotics, and decreased uptake of aminoglycoside antibiotics. This altered phenotype arises from defects in menadione and haemin biosynthesis resulting in impaired electron transport and decreased ATP concentrations. The recent acquisition of a stable small colony variant (SCV strain JB1), generated from strain 6850 of Staphylococcus aureus, allowed us to study the susceptibilities to antibiotic killing of parent and variant strains. Because differences in susceptibilities have been found between unattached and surface-adherent organisms, we tested both strains in solid and fluid-phase assays. Suspensions of SCV strain JB1 exposed to 8 x MIC of either oxacillin, vancomycin or fleroxacin, exhibited lower reductions in viable counts than the parent strain 6850, especially when high bacterial concentrations (1-2 x 10(7) cfu/mL) of either strain were tested. Susceptibility to antibiotic killing of bacteria attached to fibronectin-coated coverslips was markedly influenced by their growing or nongrowing state on the surface. In the latter condition, surface-bound SCV organisms were highly resistant to the bactericidal action of oxacillin or vancomycin in contrast to the parental strain which was normally eliminated by each antimicrobial agent. In conclusion, the decreased susceptibility of the stable SCV strain of S. aureus to bactericidal concentrations of antibiotics may help to explain the persistence of such organisms in chronic infections.

Increased expression of fibronectin-binding proteins by fluoroquinolone-resistant Staphylococcus aureus exposed to subinhibitory levels of ciprofloxacin.

Bacterial adhesion, which plays an important role in Staphylococcus aureus colonization and infection, may be altered by the presence of antibiotics or/and antibiotic resistance determinants. This study evaluated the effect of fluoroquinolone resistance determinants on S. aureus adhesion to solid-phase fibronectin, which is specifically mediated by two surface-located fibronectin-binding proteins. Five isogenic mutants, derived from strain NCTC 8325 and expressing various levels of quinolone resistance, were tested in an in vitro bacterial adhesion assay with polymethylmethacrylate coverslips coated with increasing amounts of fibronectin. These strains contained single or combined mutations in the three major loci contributing to fluoroquinolone resistance, namely, grlA, gyrA, and flqB, which code for altered topoisomerase IV, DNA gyrase, and increased norA-mediated efflux of fluoroquinolones, respectively. Adhesion characteristics of the different quinolone-resistant mutants grown in the absence of fluoroquinolone showed only minor differences from those of parental strains. However, more important changes in adhesion were exhibited by mutants highly resistant to quinolones following their exponential growth in the presence of one-quarter MIC of ciprofloxacin. Increased bacterial adhesion of the highly quinolone-resistant mutants, which contained combined mutations in grlA and gyrA, was associated with and explained by the overexpression of their fibronectin-binding proteins as assessed by Western ligand affinity blotting. These findings contradict the notion that subinhibitory concentrations of antibiotics generally decrease the expression of virulence factors by S. aureus. Perhaps the increased adhesion of S. aureus strains highly resistant to fluoroquinolones contributes in part to that emergence in clinical settings.

A low-fibronectin-binding mutant of Staphylococcus aureus 879R4S has Tn918 inserted into its single fnb gene.

A low-fibronectin-binding mutant of Staphylococcus aureus strain 879R4SSp generated by transposon Tn918 mutagenesis is attenuated in a rat endocarditis model (J.M. Kuypers & R.A. Procter, 1989, Infect Immun 57, 2306-2312). PCR and Southern hybridization analysis with primers and probes, respectively, for the fnbA and fnbB genes of strains 8325-4 showed that strain 879R4SSp possesses a single fnb gene which is homologous to fnbA. This was confirmed by sequencing 41 bp of 5' non-coding and 237 bp of 5' coding DNA, which showed 97% base identity to fnbA. Southern hybridization and sequencing showed that Tn918 was inserted 41 bp 5' to fnbA in the mutant 879R4SSp/1536, between the promoter and initiation codon. Reduced adherence of the mutant to surface-bound fibronectin correlated with lower expression of a 180 kDa wall-associated fibronectin-binding protein.

Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes.

Staphylococcus aureus 8325-4 has the potential to express two distinct cell wall-associated fibronectin-binding proteins called FnBPA and FnBPB. In order to test if both proteins are expressed in S. aureus and if both are required for promoting bacterial adhesion to fibronectin-coated surfaces, insertion mutations were isolated in each gene. A DNA fragment encoding tetracycline resistance was inserted into fnbA and a fragment encoding erythromycin resistance was inserted into fnbB. A double fnbAfnbB mutant was also constructed. The fnbA and fnbB single mutants showed no significant reduction in their adhesion to polymethylmethacrylate coverslips that had been coated in vitro with fibronectin. However, the double mutant was completely defective in adhesion. Monospecific antibodies directed against the non-conserved N-terminal regions of both proteins confirmed the lack of expression of FnBPs in the mutant strains. Wild-type fnbA and fnbB genes cloned seperately on a multicopy plasmid were each able to restore fully the adhesion-defective phenotype of the 8325-4 fnbAfnbB mutant. This demonstrates that both fnb genes are expressed in S. aureus and that both contribute to the ability of strain 8325-4 to adhere to fibronectin-coated surfaces. The double mutant was also defective in adhesion to coverslips that had been removed from tissue cages implanted subcutaneously in guinea-pigs, which suggests that fibronectin is important in promoting attachment of S. aureus to biomaterial in vivo.

Comparison of sparfloxacin, temafloxacin, and ciprofloxacin for prophylaxis and treatment of experimental foreign-body infection by methicillin-resistant Staphylococcus aureus.

The prophylactic and therapeutic activities of three broad-spectrum fluoroquinolones were evaluated in two different experimental models of foreign-body infections caused by methicillin-resistant Staphylococcus aureus (MRSA) susceptible to quinolones. In a guinea pig model of prophylaxis, subcutaneously implanted tissue cages were infected at a > 90% rate by 10(2) CFU of MRSA in control animals. A single dose of 50 mg of ciprofloxacin per kg of body weight administered intraperitoneally 3 h before bacterial challenge was less effective than an equivalent regimen of either sparfloxacin or temafloxacin in decreasing the rate of experimental infection in tissue cages challenged with increasing inocula of MRSA. In a rat model evaluating the therapy of chronic tissue cage infection caused by MRSA, the efficacy of a 7-day high-dose (50-mg/kg twice-daily) regimen of sparfloxacin, temafloxacin, or ciprofloxacin was compared to that of vancomycin (50 mg/kg twice daily). Active levels of sparfloxacin, temfloxacin, or ciprofloxacin were continuously present in tissue cage fluid during therapy, exceeding their MBCs for MRSA by 6- to 20-fold. Either temafloxacin, sparfloxacin, or vancomycin was significantly (P < 0.01) more active than ciprofloxacin in decreasing the viable counts of MRSA in tissue cage fluids. The different activities of ciprofloxacin compared with those of the other two quinolones against chronic tissue cage infections caused by MRSA did not involve the selective emergence of quinolone-resistant mutants. Temafloxacin and ciprofloxacin, which showed the most prominent differences in their in vivo activities, however, exhibited similar bactericidal properties and pharmacokinetic parameters in the rat model. In conclusion, both temafloxacin and sparfloxacin were significantly more active than ciprofloxacin for the prophylaxis or treatment of experimental foreign-body infections caused by a quinolone-susceptible strain of MRSA.

Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts.

We used an ex vivo canine arteriovenous shunt model, previously developed to study plasma protein adsorption and thrombogenesis on polymeric biomaterials, to define the role of host proteins in promoting adhesion of Staphylococcus aureus. Either polyethylene or polyvinyl chloride tubings were exposed to canine blood for 5, 15, or 60 min at a flow rate of 300 ml/min and then were flushed in phosphate-buffered saline (PBS), cut into 1.5-cm segments, and stored at -70 degrees C. After thawing, each segment was preincubated in 0.5% albumin in PBS to prevent nonspecific staphylococcal attachment to surfaces that were not exposed to blood. Each segment was then incubated with 4 x 10(6) CFU of [3H]thymidine-labelled S. aureus per ml for 60 min at 37 degrees C in an in vitro adhesion assay. Two site-specific mutants of S. aureus were tested: one specifically defective in adhesion to surface-bound fibronectin (FnAd-def) and the other defective in adhesion to fibrinogen (FgAD-def) [corrected]. Compared with their respective parental strains, the FgAd-def, but not the FnAd-def, mutant of S. aureus showed a strong (> 80%) decrease in attachment to ex vivo tubings. The adhesion of each strain of S. aureus onto polyethylene was consistently more than twofold higher than the adhesion onto polyvinyl chloride segments exposed to flowing blood for 5 or 15 min, but adhesion became similar to that on polyvinyl chloride after 60 min of exposure. In conclusion, the specific adhesion-defective mutants of S. aureus suggested that fibrinogen was the most active adhesion-promoting protein in a short-term blood-material interaction. The experimental approach described in this study should prove useful for screening materials thought to be resistant to protein-mediated staphylococcal adhesion and colonization.

Perioperative antibiotic prophylaxis of wound and foreign body infections: microbial factors affecting efficacy.

Numerous microbial factors are responsible for perioperative infections and influence the efficacy of antibiotic prophylaxis. These factors include the staphylococcal carrier state, bacterial adherence to a number of host proteins, the production of glycocalyx by sessile bacteria, and shifts in antibiotic resistance. A full understanding of the mechanisms involved will lead to further reductions in the number of postoperative infections. Unfortunately, the microbial factors affecting prophylaxis cannot be evaluated separately under clinical conditions; they are easier to study under circumstances whose bacteriologic features are well defined and in which the presence of foreign materials (e.g., sutures) greatly potentiates pathogenic mechanisms. Such circumstances exist, for example, in infections developing after "clean" surgery and in experimental models. Since even clean wounds are found to be contaminated when sampled carefully, the control of infection is more a quantitative than a qualitative problem. The critical period for the development of infection is short: an antibiotic course not exceeding 24 hours seems effective in preventing infection.

Successful single-dose prophylaxis of Staphylococcus aureus foreign body infections in guinea pigs by fleroxacin.

Single-dose administration of fleroxacin was evaluated as a means of preventing foreign body infection due to staphylococci. Tissue cages were implanted into guinea pigs and subsequently infected (100% rate) with 10(2) or more CFU of Staphylococcus aureus Wood 46. When a single dose of 30 mg of fleroxacin or vancomycin per kg of body weight was administered intraperitoneally, bactericidal levels of the antimicrobial agent were found in the tissue cage fluid after 3 h (when guinea pigs were inoculated with S. aureus) and during the next 24 h. Either fleroxacin or vancomycin successfully prevented experimental infection in all tissue cages challenged by 10(2) CFU of S. aureus Wood 46. When tissue cages were challenged with 10(4) CFU of S. aureus Wood 46, however, fleroxacin was more effective than vancomycin (P less than 0.05) in reducing colony counts below the detection limit of 10 CFU/ml in the inflammatory fluid of all tissue cages during the initial 48 h. In contrast to their initially different actions, the effects of the antibiotics were similar after 7 days, mostly because bacterial regrowth occurred more frequently in the fleroxacin-treated than in the vancomycin-treated tissue cages. These data show that experimental infections of subcutaneous tissue cages are a useful model for studying the prophylaxis of foreign body infections with antimicrobial agents.

Inhibition by immunoglobulins of Staphylococcus aureus adherence to fibronectin-coated foreign surfaces.

Recent data suggest that fibronectin may favor Staphylococcus aureus infection by promoting attachment to either injured tissues or implanted foreign bodies. Using a previously described in vitro assay, we show that promotion of S. aureus adherence by surface-bound fibronectin, adsorbed on polymethylmethacrylate (PMMA) coverslips, is antagonized by antistaphylococcal antibodies present in immunoglobulin G (IgG) purified from human plasma. Among the different organisms tested, the protein A-deficient strain Wood 46 of S. aureus was the most strongly inhibited by purified IgG or whole serum dose-dependently. Bacterial adherence was not influenced by preincubating fibronectin-coated PMMA with either purified IgG or whole serum. However, inhibition of bacterial adherence was directly related to the extent of IgG binding to S. aureus Wood 46. When F(ab')2 fragments of purified IgG were tested in the adherence assay, they could also reduce the interaction between S. aureus Wood 46 and fibronectin-coated PMMA. Two other staphylococcal strains were also tested in the adherence inhibition assay: Whereas the protein A-rich strain Cowan I of S. aureus was moderately inhibited by purified IgG or whole serum, S. epidermidis KH 11 was not at all inhibited by IgG which bound poorly to the bacterial cells. This study has demonstrated that bacterial coating by humoral factors, and specifically IgG, may influence significantly subsequent adherence of S. aureus to surface-bound fibronectin.

Fibronectin, fibrinogen, and laminin act as mediators of adherence of clinical staphylococcal isolates to foreign material.

Bacterial adherence to polymer surfaces is a required early step in intravenous (iv) device infection. We collected eight strains of Staphylococcus aureus and 19 of coagulase-negative staphylococci from patients with proven iv device bacteremia and studied the role of plasma or connective-tissue proteins in promoting bacterial adherence to polymethylmethacrylate (PMMA) coverslips. Although only a negligible percentage of organisms adhered to albumin-coated PMMA, surface-bound fibronectin significantly promoted adherence of all isolates. Fibrinogen markedly promoted adherence of all S. aureus strains but of only four coagulase-negative strains. Thus, coagulase-negative staphylococci revealed a marked heterogeneity in adherence to fibrinogen-coated surfaces, a result suggesting the existence of heretofore unknown receptors for fibrinogen. Laminin promoted adherence of staphylococci to a much lower extent. Although strain specific, adherence of clinical staphylococcal isolates to foreign surfaces is significantly increased by fibronectin, fibrinogen, and laminin, an observation suggesting the possible contribution of these proteins to the pathogenesis of iv device infection.

Attachment of Staphylococcus aureus to polymethylmethacrylate increases its resistance to phagocytosis in foreign body infection.

The mechanisms responsible for the development of a pyogenic infection (most commonly due to staphylococci) in the vicinity of an implanted foreign body have been studied recently by several investigators. Thus, we have been able to demonstrate that the phagocytic function of residential polymorphonuclear leukocytes (PMN) is deficient in the presence of a foreign body. Others have shown that in the presence of foreign surfaces, microorganisms produce extracellular amorphous material, the pathogenic role of which is still to be defined. In the present study we use a novel assay system to demonstrate that Staphylococcus aureus Wood 46, after attachment to polymethylmethacrylate (PMMA), shows increased resistance to the phagocytic-bactericidal action of normal PMN. The first step of this assay involves the reproducible attachment of [3H]thymidine-labeled bacteria to PMMA cover slips. During the second step, attached bacteria were exposed to guinea pig peritoneal exudate PMN. In the third and final step, attached S. aureus cells were removed from the cover slips using a procedure harmless to the bacteria. The extent of bacterial detachment was estimated by radioactive counts and their viability by standard colony counts. Whereas bacteria that were attached artificially and rapidly by centrifugation and immediately exposed to PMN were killed in the phagocytic assay, bacteria adhering spontaneously to the cover slips for a prolonged period of time were more resistant to the killing action of the phagocytes. The spontaneous adherence of S. aureus to PMMA renders it poorly susceptible to the killing action of PMN.

Adsorption of fibronectin onto polymethylmethacrylate and promotion of Staphylococcus aureus adherence.

Recent data suggest that fibronectin may favor Staphylococcus aureus infection by promoting attachment to either injured tissues or implanted foreign bodies. We studied the quantitative adsorption of fibronectin onto polymethylmethacrylate (PMMA) cover slips by using a 125I-labeled preparation of the purified plasma glycoprotein. Fibronectin in buffer solutions showed a high affinity to PMMA coverslips. Adherence of S. aureus Wood 46 was studied on PMMA pre-exposed to fibronectin, using an assay specifically adapted to the cover slip model. Whereas S. aureus adherence in an albumin-containing buffer was less than or equal to 10(3) CFU on control uncoated cover slips, adherence in the same medium increased up to maximum of 7.7 X 10(4) CFU on cover slips preincubated in a solution of fibronectin (125-micrograms/ml). At intermediate fibronectin concentrations, bacterial adherence was a linear function of both the quantity in solution and of the quantity adsorbed on the PMMA cover slips. The presence of human serum proteins, as represented by a fibronectin-depleted pool, essentially prevented adsorption of radiolabeled fibronectin on PMMA and subsequent bacterial adherence on the cover slips. Precoating of PMMA with denatured collagen resulted in increased fibronectin adsorption on PMMA, even in the presence of serum proteins, and S. aureus adherence was optimal on such surfaces. Collagen may therefore play a role as a cofactor contributing to S. aureus adherence onto fibronectin-coated substrata or foreign bodies.

Inter-strain variability of structural proteins in Tetrahymena.

The high molecular weight proteins found in isolated pellicles of Tetrahymena have been compared in several individual strains within the genus using SDS polyacrylamide gel electrophoresis. Three variants of the beta-protein of epiplasm (MW 174,000; 155,000; 145,00) and 2 of the C-protein (MW 140,000; 122,000) were found among the strains examined. No variation was observed in the major kinetodesmal fiber protein (MW 250,000). The variation found between strains in the proteins of a structure which is (as far as we know) the same in all strains indicates a disjunction between evolutionary change at the 2 levels of organization. The taxonomic implications of the observed variation in structural proteins in Tetrahymena are discussed.