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blaI and blaR1 regulate beta-lactamase and PBP 2a production in methicillin-resistant Staphylococcus aureus.

For Staphylococcus aureus, it is hypothesized that two genes located upstream of the beta-lactamase gene, blaZ, are required for the inducible expression of beta-lactamase. blaR1 is predicted to encode a signal-transducing membrane protein, and blaI is predicted to encode a repressor protein. These same two genes may also regulate the production of penicillin-binding protein 2a (PBP 2a), a protein essential for expression of methicillin resistance. To confirm that these two genes encode products that can control both beta-lactamase and PBP 2a production, blaI, blaR1, and blaZ with a 150-nucleotide deletion at the 3' end were subcloned from a 30-kb staphylococcal beta-lactamase plasmid and three beta-lactamase-negative strains of methicillin-resistant S. aureus were transformed with the recombinant plasmid containing that insert. The production of PBP 2a and a nonfunctional beta-lactamase was detected by fluorography and by immunoblots with polyclonal antisera directed against each of the proteins. Whereas the parent strains did not produce beta-lactamase and constitutively produced PBP 2a, PBP 2a and a truncated beta-lactamase were now inducible in the transformants. Therefore, two plasmid-derived genes regulate the production of both PBP 2a and beta-lactamase.

Bacterial Proteins↗

Structure and biosynthesis of staphyloxanthin from Staphylococcus aureus.

Most Staphylococcus aureus strains produce the orange carotenoid staphyloxanthin. The staphyloxanthin biosynthesis genes are organized in an operon, crtOPQMN, with a sigma(B)-dependent promoter upstream of crtO and a termination region downstream of crtN. The functions of the five encoded enzymes were predicted on the basis of their sequence similarity to known enzymes and by product analysis of gene deletion mutants. The first step in staphyloxanthin biosynthesis is the head-to-head condensation of two molecules of farnesyl diphosphate to form dehydrosqualene (4,4'-diapophytoene), catalyzed by the dehydrosqualene synthase CrtM. The dehydrosqualene desaturase CrtN dehydrogenates dehydrosqualene to form the yellow, main intermediate 4,4'-diaponeurosporene. CrtP, very likely a mixed function oxidase, oxidizes the terminal methyl group of 4,4'-diaponeurosporene to form 4,4'-diaponeurosporenic acid. CrtQ, a glycosyltransferase, esterifies glucose at the C(1)'' position with the carboxyl group of 4,4'-diaponeurosporenic acid to yield glycosyl 4,4'-diaponeurosporenoate; this compound was the major product in the clone expressing crtPQMN. In the final step, the acyltransferase CrtO esterifies glucose at the C(6)'' position with the carboxyl group of 12-methyltetradecanoic acid to yield staphyloxanthin. Staphyloxanthin overexpressed in Staphylococcus carnosus (pTX-crtOPQMN) and purified was analyzed by high pressure liquid chromatography-mass spectroscopy and NMR spectroscopy. Staphyloxanthin was identified as beta-D-glucopyranosyl 1-O-(4,4'-diaponeurosporen-4-oate)-6-O-(12-methyltetradecanoate).

Acyltransferases↗

Formation of intraperitoneal abscesses by Staphylococcus aureus.

Certain Staphylococcus aureus strains, when inoculated into the peritoneal cavity of mice, were clumped and surrounded by a thick layer of leukocytes. After being enclosed with a connective tissue capsule, the structures histologically resembled staphylococcal abscesses. Of four strains examined, all were destroyed within abscesses, although at different rates. Abscess homogenates possessed bactericidal activity toward staphylococci, and this activity was associated with the sedimentable fraction of the homogenates. Leukocytes did not appear to be responsible for the bactericidal activity. Appreciable quantities of alpha toxin accumulated in these abscesses even without multiplication of the organisms. This model infection offers opportunities for studying some aspects of staphylococcal host-parasite interactions occurring in localized lesions.

Abscess↗

Community-acquired methicillin-resistant Staphylococcus aureus infections.

Staphylococcus aureus causes a variety of minor diseases but also is responsible for staphylococcal pneumonia and sepsis, both of which can be fatal. It is thought to be responsible for many of the pneumonia deaths associated with the influenza pandemics of the 20th century. The introduction of penicillin in the 1940s greatly improved the prognosis for patients with severe staphylococcal infections. However, after a few years of clinical use, most staphylococcal strains were able to hydrolyze penicillin by producing b-lactamases, making penicillin a useless antibiotic to treat staphylococcal infections caused by b-lactamase-producing S aureus. Methicillin, a semisynthetic penicillin introduced in 1959, was specifically designed to be resistant to b-lactamase degradation, but resistance developed soon after its introduction into clinical practice. Methicillin-resistant S aureus (MRSA) was first reported in the United Kingdom in 1961, followed by reports from other European countries, Japan, and Australia. The first reported case of MRSA in the United States was in 1968. Currently, MRSA is an important pathogen in nosocomial infections and is a problem in hospitals worldwide, and it is increasingly recovered from nursing home residents with established risk factors. More recently, community acquired MRSA infections have been documented among healthy individuals with no recognizable risk factors, and it seems clear that community-acquired MRSA (CA-MRSA) strains are epidemiologically and clonally unrelated to hospital-acquired strains. This review focuses on the epidemiology, clinical significance, and virulence markers of CA-MRSA infections.

Anti-Bacterial Agents↗

Use of antimicrobial susceptibility testing for epidemiology and the selection of oral, parenteral and topical regimens for control of CAPD-associated Staphylococcus aureus infection.

Staphylococcus aureus is an important cause of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Using standard broth microdilution and disk diffusion methodology, we evaluated the in vitro activity of selected antimicrobial agents against S. aureus strains isolated from CAPD patients to assess candidate regimens for 1) topical agent control of colonization, 2) oral chemotherapy of CAPD infectious complications, and 3) parenteral treatment of serious CAPD-associated staphylococcal infections. A total of 34 isolates (31 patients) of S. aureus were available for testing, including 29 isolates (29 patients) from pericatheter skin, four isolates (four patients) from the nares, and one isolate from an episode of peritonitis. Six of the isolates were oxacillin-resistant (ORSA). The antimicrobial agents tested by broth microdilution included 17 different quinolones, 10 cephalosporins, six glycopeptides, two aminoglycosides, and imipenem. A total of eight potential topical agents, including the antistaphylococcal agent mupirocin, were tested by disk diffusion. All of the quinolones, with the exception of nalidixic acid (MIC90 greater than 16 micrograms/ml), had excellent activity against both ORSA and oxacillin-susceptible S. aureus (OSSA) with the most active agent being WIN57273 (MIC90 less than or equal to 0.015 microgram/ml). Imipenem and the cephalosporins, with the exception of cefixime, ceftazidime, and E-1040, possessed good activity against OSSA. None of the beta-lactam agents tested were active against ORSA. Likewise, the aminoglycosides, amikacin and gentamicin, exhibited good activity against OSSA strains but no activity against ORSA strains. All glycopeptides tested demonstrated excellent activity against ORSA strains. Of the topical antimicrobial agents tested only bacitracin, mupirocin, and nitrofurantoin were active against all OSSA and ORSA strains tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

The effect of reserpine, an inhibitor of multidrug efflux pumps, on the in-vitro activities of ciprofloxacin, sparfloxacin and moxifloxacin against clinical isolates of Staphylococcus aureus.

In Staphylococcus aureus, in addition to mutations in the grl and gyr gene loci, multidrug efflux pumps like NorA contribute to decreased fluoroquinolone susceptibility. Efflux pumps can be inhibited by the plant alkaloid reserpine, which, at 20 mg/L, reduced sparfloxacin, moxifloxacin and ciprofloxacin IC50s and MICs by up to four-fold in 11, 21 and 48 of the 102 unrelated clinical isolates tested, respectively. The effect was less pronounced with the hydrophobic drugs sparfloxacin and moxifloxacin than with the hydrophilic drug ciprofloxacin and was stable in all 25 clonally related isolates tested.

Anti-Infective Agents↗

Structure of the type 5 capsular polysaccharide of Staphylococcus aureus.

The Staphylococcus aureus type 5 capsular polysaccharide is composed of 2-acetamido-2-deoxy-L-fucose (1 part), 2-acetamido-2-deoxy-D-fucose (1 part), and 2-acetamido-2-deoxy-D-mannuronic acid (1 part). On the basis of methylation analysis, optical rotation, high-field one- and two-dimensional 1H- and 13C-n.m.r. experiments, and selective cleavage with 70% aqueous hydrogen fluoride, the polysaccharide was found to be a partially O-acetylated (50%) polymer of the repeating trisaccharide unit, [----4)-3-O-Ac-beta-D-ManpNAcA-(1----4)-a-L-FucpNAc-(1----3) -beta-D-FucpNAc-(1----]n.

Carbohydrate Sequence↗

Staphylococcus aureus bacteremia.

Staphylococcus aureus bacteremia (SAB) is still associated with a high mortality, and knowledge on risk factors and the clinical and the therapeutic aspects of SAB is still limited. This thesis focuses on the clinical aspects of SAB and its metastatic infections. In a study of all patients with bacteremia in Copenhagen County October 1992 through April 1993 (study I) we emphasized previous findings, that S. aureus is one of the most frequent pathogens in bacteremia, and in a case control study also in Copenhagen County 1994-95 (study II) we demonstrated, that not only an inserted central venous catheter and nasal S. aureus carriage but also hyponatremia and anemia are important risk factors for hospital-acquired SAB (study II). Studies on the treatment of SAB have pointed out, that the eradication of a primary is important, but there are only limited clinical studies dealing with antibiotic treatment. By logistic regression analysis, we were able to demonstrate that focus eradication is essential, but also that treatment with dicloxacillin 1 g x 4 or 2 g x 3 are superior to 1 g x 3 (studie III), indicating that the time for serum concentration above the Minimal Inhibitory Concentration (MIC) for the bacteria plays a role in the outcome of SAB treatment. S. aureus osteomyelitis secondary to SAB is frequently observed. No other countries, however, have a centralized registration, which make it possible to evaluate a large number of these patients. Since 1960, The Staphylococcal Laboratory, Statens Serum Institut in Copenhagen, has registrated selected clinical informations from nearly all patients with positive blood cultures of S. aureus. Based on this registration, we were able to show an increased number of S. aureus osteomyelitis among older patients and a decreased number of S. aureus osteomyelitis of femur and tibia among younger infants in the period 1980-90 (study IV). By reviewing the records of a large number of patients with vertebral S. aureus osteomyelitis, we could evaluate important aspects in the diagnosis and treatment of these patients (study V). We illustrated, that symptoms and laboratory findings were relatively unspecific, and CT-scanning or bone scintigraphy were absolutely necessary for the diagnosis (study V). The relatively high number of patients in the study allowed us to evaluate different treatment regimens, and we found, that treatment with penicillinase-stable penicillins four grams daily for at least eight weeks was necessary (study V). S. aureus meningitis is relatively uncommon and most often a neurosurgical infection based on the presence of a catheter. Meningitis secondary to SAB is relatively rare. The nationwide registration on Statens Serum Institut enabled us to study a large number of patients with special emphasis on clinical, outcome and treatment (study VI). We found, that these patients often were older people with chronic underlying diseases, the infection developed as a community-acquired infection, and the patients also had an unknown focus of infection. Furthermore, these patients often had other secondary manifestations such as endocarditis or osteomyelitis and an extremely high mortality (study VI). Finally, I believe that our studies will contribute to reduce the incidence of SAB and improve the diagnosis and treatment of SAB in the future.

Bacteremia↗

Evidence for the existence of a multidrug efflux transporter distinct from NorA in Staphylococcus aureus.

A Staphylococcus aureus norA disruption mutant was created by allelic replacement. Exposure of this mutant to norfloxacin produced SA K1748, a derivative with raised fluoroquinolone MICs, found to be the result of a grlA mutation, and raised organic cation MICs. Ethidium and enoxacin uptake was identical in SA K1748 and its parent, but pre-exposure of SA K1748 to organic cations caused a reduction in ethidium uptake as a result of increased efflux. Altered ethidium uptake and efflux, as well as increased MICs of other organic cations, suggest that SA K1748 possesses a non-NorA multidrug efflux transporter that is inducible by its substrates.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Molecular genetics of SaPI1--a mobile pathogenicity island in Staphylococcus aureus.

The Staphylococcus aureus gene for toxic shock toxin (tst) is carried by a 15 kb mobile pathogenicity island, SaPI1, that has an intimate relationship with temperate staphylococcal phage 80alpha. During phage growth, SaPI1 is excised from its unique chromosomal site, attC, replicates autonomously, interferes with phage growth, and is efficiently encapsidated into special small phage heads commensurate with its size. Upon transfer to a recipient organism, SaPI1 integrates at attC by means of a self-coded integrase. One or more phage functions are required for excision, autonomous replication and encapsidation of the element and, thus, the overall relationship between SaPI1 and 80alpha is similar to that between coliphages P4 and P2. Among other staphylococcal phages tested, only phi13 interacts with SaPI1, inducing excision but not replication or transfer of the element.

Attachment Sites, Microbiological↗

Outcome following haemodialysis catheter-related Staphylococcus aureus bacteraemia.

Staphylococcus aureus is a frequent cause of haemodialysis access-related bacteraemia. The propensity for this organism to seed from the bloodstream to distant sites is well recognized, but the rate at which this occurs is poorly defined in patients with removable haemodialysis catheters. This retrospective study identified 47 patients with 50 episodes of S. aureus haemodialysis catheter-related bacteraemia between August 1993 and December 1995. Adverse events were recorded until February 1996. Thirty of 50 episodes (60%) were apparently uncomplicated. Bacterial seeding to heart valves or distant sites was documented in eight episodes (16%), of which six occurred during antibiotic therapy. A further 12 patients had persistent bacteraemia in the absence of a defined focus of infection, the last positive blood culture ranging from 2-19 days (mean 6.6, median 5) after removal of the haemodialysis catheter and commencing appropriate antibiotic treatment. The serious nature of this infection confirms the need for prevention, together with effective strategies for investigation and treatment in this patient population.

Adult↗

Penicillin-binding protein 4 overproduction increases beta-lactam resistance in Staphylococcus aureus.

The Staphylococcus aureus mutant strain PVI selected in vitro for methicillin resistance overexpressed penicillin-binding protein (PBP) 4. In the wild-type parent strain the pbp4 gene was separated by 419 nucleotides from a divergently transcribed abcA locus coding for an ATP-binding cassette transporter. The mutant PVI was shown to have a deletion in the pbp4-abcA promoter region that affected pbp4 transcription but not expression of abcA. Introduction of the pbp4 gene plus the mutant promoter region into different genetic backgrounds revealed that PBP 4 overproduction was sufficient to increase in vitro-acquired methicillin resistance independently of other chromosomal genes. The role of the AbcA transporter in methicillin resistance remained unknown.

ATP-Binding Cassette Transporters↗

Low molecular weight heparin aggravates infectious arthritis triggered by Staphylococcus aureus.

PURPOSE: Staphylococcus aureus is responsible for the majority of wound infections in clean surgical procedures that involve implantation of foreign material, grafts or prosthetic devices. The aim of the study was to assess the effect of low molecular weight heparin on the development and progression of S. aureus arthritis. MATERIALS AND METHODS: The murine model of hematogenously acquired septic arthritis was used injecting intravenously toxic shock syndrome toxin-1 (TSST-1) producing S. aureus of LS-1 strain. Mice lacking prosthetic implants were treated with intraperitoneal injections of low molecular weight heparin, used routinely as anti-thrombotic prophylaxis following joint prosthetic surgery. Evaluation of arthritis was performed clinically and histopathologically. In addition, the effect of low molecular weight heparin on T cell dependent and independent inflammation was assessed. RESULTS: Seven days after inoculation with bacteria 18 out of 19 low molecular weight heparin treated mice displayed clinical symptoms of arthritis as compared to 9 out of 23 control animals (p < 0.05), and the severity of arthritis, expressed as arthritic index, was 2.6+/-0.5 versus 1.6+/-0.5 (p = 0.05). The histopathological examination confirmed the clinical findings showing that both inflammation and joint destruction were more substantial in heparin treated animals. CONCLUSION: Our findings indicate that the routine anti-coagulation treatment with heparin contributes to more severe course of joint infection.

Animals↗

Subcellular localization of the major autolysin, ATL and its processed proteins in Staphylococcus aureus.

The Staphylococcus aureus autolysin gene, atl, encodes a unique 138-kDa protein (ATL) with amidase and glucosaminidase domains. ATL has been suggested to undergo proteolytic processing to generate two extracellular peptidoglycan hydrolases, 51-kDa endo-beta-N-acetylglucosaminidase (51-kDa GL) and 62-kDa N-acetylmuramyl-L-alanine amidase (62-kDa AM). To investigate cell-associated bacteriolytic enzymes for atl gene products, proteins were extracted from the cells as follows. The cells were exposed to 3 M LiCl followed by 4% SDS. Thereafter, the cells were disrupted and again extracted with 4% SDS. Whole SDS-stable cell-associated bacteriolytic proteins were extracted without disrupting the cells. Exposure to 3 M LiCl released major 138-, 115-, 85-, 62- and 51-kDa bacteriolytic proteins, and subsequent 4% SDS extraction released major 138- and 115-kDa bacteriolytic proteins. These bacteriolytic proteins were missing in extracts of atl mutant RUSAL2 (S. aureus RN450 atl::Tn551). Immunoblotting studies suggest that these are all atl gene products: the 138-kDa protein is an ATL with a cleaved signal sequence; the 115- and 85-kDa proteins are intermediates; and the 51- and 62-kDa proteins are cell-associated 51-kDa GL and 62-kDa AM, respectively. The trypsin susceptibility of these proteins suggests that they are located outside the cell membrane. Differences in extractability and immunoelectron microscopic studies suggest that atl gene products are associated with cells in two different ways, LiCl extractable and non extractable. We suggest that the 138-kDa ATL undergoes processing through intermediate proteins (115- and 85-kDa proteins) to mature as the active cell cluster-dispersing enzymes 51-kDa GL and 62-kDa AM on the cell surface.

Amino Acid Sequence↗

Protective effect of furocoumarins against 254-nm ultraviolet in Staphylococcus aureus.

For Staphylococcus aureus, pretreatment with furocoumarins (FCs) protect cells against killing by far ultraviolet light (FUV; approximately 254 nm). This protective effect was evident in the repair-proficient, parental strain as well as in the repair-deficient variants in the following order of efficacy: 4,5'',8-trimethylpsoralen << 8-methoxypsoralen congruent with angelicin < 3-carbethoxypsoralen. The extent of protection was greater in the parental strain, indicating that despite the protective effect, a certain number of lethal lesions are nevertheless produced, which would be repaired with greater efficiency in such a strain than in the repair-deficient ones. This protective effect could be attribute to the inhibition of the formation of cyclobutyl pyrimidine dimers. Although the energy-transfer concept could explain the inhibition of pyrimidine dimer formation, and thus the protective effect of FC against FUV, we cannot rule out the possibility that the differences in degree of protection afforded by the FC employed here are related to a subtle and complex combination of effects.

Colony Count, Microbial↗

Contribution of a thickened cell wall and its glutamine nonamidated component to the vancomycin resistance expressed by Staphylococcus aureus Mu50.

Staphylococcus aureus Mu50, which has reduced susceptibility to vancomycin, has a remarkably thickened cell wall with an increased proportion of glutamine nonamidated muropeptides. In addition, Mu50 had enhanced glutamine synthetase and L-glutamine D-fructose-6-phosphate aminotransferase activities, which are involved in the cell-wall peptidoglycan synthesis pathway. Furthermore, significantly increased levels of incorporation of (14)C-labeled D-glucose into the cell wall was observed in Mu50. Unlike a femC mutant S. aureus strain, increased levels of production of nonamidated muropeptides in Mu50 was not caused by lower levels of glutamine synthetase activity but was considered to be due to the glutamine depletion caused by increased glucose utilization by the cell to biosynthesize increased amounts of peptidoglycan. After the cells were allowed to synthesize cell wall in the absence or presence of glucose and glutamine, cells with different cell-wall thicknesses and with cell walls with different levels of cross-linking were prepared, and susceptibility testing of these cells demonstrated a strong correlation between the cell-wall thickness and the degree of vancomycin resistance. Affinity trapping of vancomycin molecules by the cell wall and clogging of the outer layers of peptidoglycan by bound vancomycin molecules were considered to be the mechanism of vancomycin resistance of Mu50. The reduced cross-linking and the increased affinity of binding to vancomycin of the Mu50 cell wall presumably caused by the increased proportion of nonamidated muropeptides may also contribute to the resistance to some extent.

Bacterial Proteins↗

Suppression of DNA synthesis in mitogen stimulated leukocytes with partial trypsin digests of Staphylococcus aureus protein A.

Staphylococcus aureus protein A (SPA) acts as a mitogen stimulating DNA synthesis and is not antagonistic to the mitogenic activity of other mitogens. The mitogenicity of SPA is due to the protein's multivalent Fc antibody receptors. Partial trypsin digestion of SPA yields monovalent Fc antibody receptors which are not mitogenic. Using a thymidine incorporation assay, results indicated that tryptic digests of SPA added to mouse splenic leukocytes 12 hours after stimulation with either concanavalin A, lipopolysaccaride or SPA suppressed the DNA synthesis normally elicited by these mitogens. SPA was digested with 1% trypsin at pH 8 for varying periods of time. As the digestion time increased from 0 to 60 minutes the mitogenicity of the SPA-trypsin digests decreased indicating cleavage from multivalent to monovalent SPA Fc antibody receptors. The decrease in mitogenicity of the SPA digests was directly associated with increased ability to inhibit DNA synthesis in mitogen stimulated leukocytes. A proposed mechanism for the inhibition of DNA synthesis suggests that SPA-monovalent Fc antibody receptors mimic cellular Fc receptor function thereby influencing cellular gene expression.

Animals↗

Cross-linking and O-acetylation of newly synthesized peptidoglycan in Staphylococcus aureus H.

Staphylococcus aureus H growing exponentially was labelled with N-acetyl[14C]glucosamine, which became incorporated into the peptidoglycan. The portion of peptidoglycan not linked to teichoic acid (60-75% of the whole) was degraded with Chalaropsis muramidase to yield disaccharide-peptide monomers and dimers, trimers and oligomers formed by biosynthetic cross-linking of the monomers. The degree of O-acetylation of these fragments was also examined. Pulse-chase experiments showed that the proportion of label initially in the monomer fraction immediately after the 1 min pulse declined rapidly during a 3 min chase, while the oligomer fraction (fragments greater than trimer) gained the radioactivity proportionately. The radioactivity of the dimer and trimer fractions remained virtually unchanged. At 4 min after the commencement of labelling (i.e. approx. one-tenth of a generation time) final values had been reached. The O-acetylation of all fragments had achieved final values even at 1 min, except for the monomer fraction, which showed an increase from 40% to 60% during the first 3 min of chase. Although O-acetylation was clearly a very rapid process, no O-acetylated peptidoglycan lipid-intermediates could be detected.

Acetylation↗