Oral quinolones in hospitalized patients: an evaluation of a computerized decision support intervention.

OBJECTIVE: To determine whether a computerized decision support system could increase the proportion of oral quinolone antibiotic orders placed for hospitalized patients. DESIGN: Prospective, interrupted time-series analysis. SETTING: University hospital in the south-eastern United States. SUBJECTS: Inpatient quinolone orders placed from 1 February 2001 to 31 January 2003. INTERVENTION: A web-based intervention was deployed as part of an existing order entry system at a university hospital on 5 February 2002. Based on an automated query of active medication and diet orders, some users ordering intravenous quinolones were presented with a suggestion to consider choosing an oral formulation. MAIN OUTCOME MEASURE: The proportion of inpatient quinolone orders placed for oral formulations before and after deployment of the intervention. RESULTS: There were a total of 15 194 quinolone orders during the study period, of which 8962 (59%) were for oral forms. Orders for oral quinolones increased from 4202 (56%) before the intervention to 4760 (62%) after, without a change in total orders. In the time-series analysis, there was an overall 5.6% increase (95% CI 2.8-8.4%; P < 0.001) in weekly oral quinolone orders due to the intervention, with the greatest effect on nonintensive care medical units. CONCLUSIONS: A web-based intervention was able to increase oral quinolone orders in hospitalized patients. This is one of the first studies to demonstrate a significant effect of a computerized intervention on dosing route within an antibiotic class. This model could be applied to other antibiotics or other drug classes with good oral bioavailability.

Iron-cofactored superoxide dismutase inhibits host responses to Mycobacterium tuberculosis.

Superoxide dismutase (SOD) is a ubiquitous metalloenzyme in aerobic organisms that catalyzes the conversion of superoxide anion to hydrogen peroxide. Mycobacterium tuberculosis is unusual in that it secretes large quantities of iron-cofactored SOD. To determine the role of SOD in pathogenesis, we constructed mutants of M. tuberculosis H37Rv with reduced SOD production. Compared with controls, SOD-diminished isolates were more susceptible to killing by hydrogen peroxide. The isolates were markedly attenuated, exhibiting nearly 100,000-fold fewer bacilli than virulent control strains in the lungs and spleens of C57BL/6 mice 4 wk after intravenous inoculation. In the lung, SOD-attenuated M. tuberculosis induced robust interstitial mononuclear cell infiltration within 24 h and many cells were apoptotic by TUNEL staining, whereas virulent H37Rv exhibited minimal early inflammatory response and only rare interstitial mononuclear cell apoptosis. During prolonged infections, C57BL/6 mice tolerated SOD-attenuated M. tuberculosis better than BCG, exhibiting 68% greater weight gain, quicker eradication of bacilli from the spleen, and less alveolar lung infiltration. These results establish the importance of SOD in the pathogenesis of tuberculosis. Its effect appears to be mediated in part by inhibiting innate host immune responses, including early mononuclear cell infiltration of infected tissues and apoptosis.

Enhanced production of recombinant Mycobacterium tuberculosis antigens in Escherichia coli by replacement of low-usage codons.

A major obstacle to development of subunit vaccines and diagnostic reagents for tuberculosis is the inability to produce large quantities of these proteins. To test the hypothesis that poor expression of some mycobacterial genes in Escherichia coli is due, in part, to the presence of low-usage E. coli codons, we used site-directed mutagenesis to convert low-usage codons to high-usage codons for the same amino acid in the Mycobacterium tuberculosis genes for antigens 85A and 85B and superoxide dismutase. Replacement of five codons in the wild-type gene for antigen 85B increased recombinant protein production in E. coli 54-fold. The recombinant antigen elicited proliferation and gamma interferon production by lymphocytes from healthy tuberculin reactors and was recognized by monoclonal antibodies to native antigen 85, indicating that the recombinant antigen contained T-cell and B-cell epitopes. Northern blotting demonstrated only a 1.7- to 2.5-fold increase in antigen 85B mRNA, suggesting that the enhanced protein production was due primarily to enhanced efficiency of translation. Codon replacement in the genes encoding antigen 85A and superoxide dismutase yielded four- to sixfold increases in recombinant protein production, suggesting that this strategy may be generally applicable to overexpression of mycobacterial genes in E. coli.

Characterization of a chromosomal gene encoding type B beta-lactamase in phage group II isolates of Staphylococcus aureus.

In contrast to most Staphylococcus aureus isolates in which the gene for staphylococcal beta-lactamase (blaZ) is plasmid borne, isolates typeable by group II bacteriophages frequently carry blaZ on the chromosome. Furthermore, the chromosomal gene encodes the type B variant of staphylococcal beta-lactamase for which the nucleotide and deduced amino acid sequences have not yet been reported. To better understand beta-lactamase production among phage group II staphylococci and the nature of the type B beta-lactamase, we determined the type and amount of enzyme produced by 24 phage group II isolates. Of these isolates, 1 did not produce beta-lactamase, 8 produced the type B enzyme, and 15 produced the type C enzyme. In all eight type B beta-lactamase-producing isolates, blaZ was located on the chromosome. This was in contrast to the type C beta-lactamase-producing isolates, in which blaZ was located on a 21-kb plasmid. The nucleotide sequence corresponding to the leader peptide and the N-terminal 85% of the mature exoenzyme form of type B S. aureus was determined. The deduced amino acid sequence revealed 3 residues in the leader peptide and 12 residues in the exoenzyme portion of the beta-lactamase that differ from the prototypic type A beta-lactamase sequence. These include the serine-to-asparagine change at residue 216 found in the kinetically similar type C enzyme, a threonine-to-lysine change at residue 128 close to the SDN loop (residues 130 to 132), and several substitutions not found in any of the other staphylococcal beta-lactamases. In summary, modern isolates of S. aureus typeable by group II phages produce type B or type C staphylococcal beta-lactamase. The type B gene resides on the chromosome and has a sequence that, when compared to the sequences of the other staphylococcal beta-lactamases, corresponds well with its kinetic properties.

Synergism between poly-(1-6)-beta-D-glucopyranosyl-(1-3)-beta-D-glucopyranose glucan and cefazolin in prophylaxis of staphylococcal wound infection in a guinea pig model.

To determine whether the infection-preventing capability of the neutrophil-activating agent poly-(1-6)-beta-D-glucopyranosyl-(1-3)-beta-D-glucopyranose glucan (PGG-glucan) can be enhanced with antibiotic prophylaxis, we administered PGG-glucan and cefazolin, alone and in combination, to guinea pigs inoculated with isolates of staphylococci. Guinea pigs receiving both PGG-glucan and cefazolin had 50% infective doses that were 8- to 20-fold higher than those obtained with cefazolin alone and 100- to 200-fold higher than those obtained with PGG-glucan alone. PGG-glucan and cefazolin are synergistic in their ability to prevent staphylococcal wound infection.

Recombinant expression and characterization of the major beta-lactamase of Mycobacterium tuberculosis.

New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of beta-lactam antibiotics. beta-Lactamase production appears to be the major mechanism by which M. tuberculosis expresses beta-lactam resistance. beta-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of beta-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing beta-lactamase activity, respectively, were identified. The beta-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the beta-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A beta-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major beta-lactamase of M. tuberculosis is a class A beta-lactamase with predominant penicillinase activity. A second, minor beta-lactamase with relatively greater cephalosporinase activity is also present.

Beta-lactamase production diminishes the prophylactic efficacy of ampicillin and cefazolin in a guinea pig model of Staphylococcus aureus wound infection.

Clinical trials in surgery suggest that some failures of antibiotic prophylaxis are related to the in vivo degradation of beta-lactams by Staphylococcus aureus beta-lactamase. To explore this issue further, isogeneic isolates of S. aureus differing only in whether they contained the structural gene for type A staphylococcal beta-lactamase were constructed and compared for their ability to establish an abscess in a guinea pig model. With ampicillin prophylaxis, the ID50 was 870 cfu for the beta-lactamase-negative isolate VK7114 and 240 cfu for the beta-lactamase-producing isolate VK7115 (P < .001). Similarly, the ID50 was greater for the beta-lactamase-negative isolate when cefazolin prophylaxis was administered (599 vs. 128 cfu, VK7114 and VK7115; P < .001). In the setting of prophylaxis with beta-lactamase-susceptible antibiotics, beta-lactamase contributes to the pathogenesis of S. aureus wound infections.

Prophylactic anti-infective activity of poly-[1-6]-beta-D-glucopyranosyl-[1-3]-beta-D-glucopryanose glucan in a guinea pig model of staphylococcal wound infection.

The judicious use of perioperative antibiotic prophylaxis reduces the infectious complications of surgery. However, increased bacterial resistance within hospitals may make antibiotic prophylaxis less effective in the future and alternative strategies are needed. New immunomodulatory agents might prevent wound infections by stimulation of the host immune system. To test this hypothesis, we administered poly-[1-6]-beta-D-glucopyranosyl- [1-3] -beta-D-glucopyranose glucan (PGG glucan), which enhances neutrophil microbicidal activity, intravenously to guinea pigs in doses ranging from 0.015 to 4 mg/kg of body weight on the day before, on the day of, and on the day after intermuscular inoculation with methicillin-resistant strains of Staphylococcus aureus and Staphylococcus epidermidis. Abscesses were identified at 72 h, and median infective doses (ID50) and statistical significance were determined by logistic regression. Guinea pigs receiving PGG glucan and inoculated with methicillin-resistant S. aureus and S. epidermidis exhibited ID50 of as much as 2.5- and 60-fold higher, respectively, than those of control guinea pigs not receiving PGG glucan. Maximal protection was observed with a dose of 1 mg of PGG glucan per kg, and efficacy was reduced at higher as well as at lower PGG glucan doses. Furthermore, a single dose of PGG glucan given 24 h following bacterial inoculation was found to be effective in preventing infection. We conclude that PGG glucan reduces the risk of staphylococcal abscess formation. Neutrophil-activating agents are a novel means of prophylaxis against surgical infection and may be less likely than antibiotics to be affected adversely by the increasing antibiotic resistance of nosocomial pathogens.

Association of borderline oxacillin-susceptible strains of Staphylococcus aureus with surgical wound infections.

Staphylococcus aureus isolates which produce type A staphylococcal beta-lactamase have been associated with wound infections complicating the use of cefazolin prophylaxis in surgery. To further evaluate this finding, 215 wound isolates from 14 cities in the United States were characterized by antimicrobial susceptibility and beta-lactamase type and correlated with the preoperative prophylactic regimen. Borderline-susceptible S. aureus isolates of phage group 5 (BSSA-5), which produce large amounts of type A beta-lactamase and exhibit borderline susceptibility to oxacillin, comprised a greater percentage of the 120 wound isolates associated with cefazolin prophylaxis than they did of the 95 isolates associated with other prophylactic regimens (25% versus 12.6%, respectively; P < 0.05). In contrast, methicillin-resistant S. aureus isolates were distributed evenly between the two groups (8.3% versus 11.6%, respectively). In vitro assays demonstrated that cefazolin was hydrolyzed faster by BSSA-5 strains than by other beta-lactamase-producing, methicillin-susceptible strains (1.54 versus 0.50 microg/min/10(8) CFU, respectively; P < 0.0001). These data demonstrate that BSSA-5 strains are a distinct subpopulation of methicillin-susceptible S. aureus which frequently cause deep surgical wound infections. Cefazolin use in prophylaxis is a risk factor for BSSA-5 infection.

Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model.

Isogeneic bacterial strains that differ only in the production of a single microbial factor have been invaluable in studying the pathogenesis of bacterial infections. The targeted, intentional inactivation of a gene encoding a potential virulence determinant generally requires homologous recombination to replace the gene with an inactivated allele. To determine whether the insertion and expression of a fragment of a bacterial gene in an antisense orientation could be used as a rapid alternative to allelic inactivation for producing paired isogeneic isolates, we inverted a 600-bp fragment of the Staphylococcus aureus gene encoding alpha-toxin, hla, behind its native promoter on an Escherichia coli-S. aureus shuttle vector. A transformant of an S. aureus strain carrying the antisense hla fragment produced antisense hla RNA and made 16-fold less alpha-toxin than either its parent or an isogeneic transformant containing vector DNA without hla. Also, intraperitoneal injection of 1.5 x 10(9) CFU of the antisense hla-containing transformant was significantly less lethal in a murine model than that of the parent (1 of 10 versus 7 of 10 mice expired [P < 0.02]) or the transformant without hla (1 of 10 versus 7 of 7 mice expired [P < 0.001]). We conclude that the expression of a fragment of hla in an antisense orientation in S. aureus on a plasmid vector reduces alpha-toxin production and the lethal activity of the strain in a murine model. The antisense strategy for creating isogeneic strains of bacteria may facilitate molecular investigations into the pathogenesis of infection. It also may be useful in creating novel live-attenuated strains of bacteria for use as vaccine candidates.

Structure-function relationships among wild-type variants of Staphylococcus aureus beta-lactamase: importance of amino acids 128 and 216.

beta-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the beta-lactam ring and are an important mechanism of resistance for many bacterial pathogens. Four wild-type variants of Staphylococcus aureus beta-lactamase, designated A, B, C, and D, have been identified. Although distinguishable kinetically, they differ in primary structure by only a few amino acids. Using the reported sequences of the A, C, and D enzymes along with crystallographic data about the structure of the type A enzyme to identify amino acid differences located close to the active site, we hypothesized that these differences might explain the kinetic heterogeneity of the wild-type beta-lactamases. To test this hypothesis, genes encoding the type A, C, and D beta-lactamases were modified by site-directed mutagenesis, yielding mutant enzymes with single amino acid substitutions. The substitution of asparagine for serine at residue 216 of type A beta-lactamase resulted in a kinetic profile indistinguishable from that of type C beta-lactamase, whereas the substitution of serine for asparagine at the same site in the type C enzyme produced a kinetic type A mutant. Similar bidirectional substitutions identified the threonine-to-alanine difference at residue 128 as being responsible for the kinetic differences between the type A and D enzymes. Neither residue 216 nor 128 has previously been shown to be kinetically important among serine-active-site beta-lactamases.

Nontropical pyomyositis in patients with AIDS.

Nontropical pyomyositis in persons with acquired immunodeficiency syndrome (AIDS) is an unusual entity with only a few cases having been described in the United States. Staphylococcus aureus is the most common organism implicated. The infection usually presents in a subacute indolent fashion with minimal inflammation. Fever and leukocytosis may be absent, and blood cultures are frequently negative. The diagnosis usually can be established by a combination of clinical features, computed tomography or ultrasound, and prompt examination of material obtained by aspiration or debridement. This article describes two cases of S aureus pyomyositis in patients with AIDS and reviews the literature relevant to this infection.

Passive immunization with antiserum to a nontoxic alpha-toxin mutant from Staphylococcus aureus is protective in a murine model.

A nonhemolytic, nonlethal variant of Staphylococcus aureus alpha-toxin constructed via oligonucleotide-directed mutagenesis and containing a single amino acid substitution (H-35 to L) was used to immunize a rabbit. The resulting antiserum was cross-reactive with wild-type alpha-toxin and neutralized its hemolytic activity in vitro. Passive immunization of mice with rabbit antiserum conferred protection against lethal challenge with wild-type alpha-toxin and against acute lethal challenge with a high-alpha-toxin -producing S. aureus strain. H35L alpha-toxin may be useful as a protective immunogen in S. aureus vaccine studies.

Growth of Staphylococcus aureus with nafcillin in vitro induces alpha-toxin production and increases the lethal activity of sterile broth filtrates in a murine model.

The morbidity and mortality of Staphylococcus aureus infections remain high despite antibiotic therapy. To investigate further the observation that penicillins increase the hemolytic activity of staphylococcal cultures, 37 strains were grown in broth with and without subinhibitory nafcillin. Nafcillin stimulated hemolytic activity in nafcillin-susceptible and -resistant isolates. Sterile broth filtrates of nafcillin-associated cultures injected intraperitoneally in mice were more rapidly lethal than filtrates of the same strain grown without nafcillin. Lethality was neutralized by anti-alpha-toxin antisera. DNA-RNA hybridization revealed a nafcillin-associated increase in alpha-toxin mRNA during the postexponential growth phase after the activation of agr. Isolates grown in slightly inhibitory nafcillin concentrations had more alpha-toxin mRNA than did nafcillin-free cultures, whereas agr RNAIII levels were comparable. This suggests that nafcillin-induced alpha-toxin production is not entirely attributable to agr. A supplemental regulatory mechanism may be involved.

Production of A and C variants of staphylococcal beta-lactamase by methicillin-resistant strains of Staphylococcus aureus.

Most methicillin-resistant Staphylococcus aureus (MRSA) strains produce beta-lactamase. To determine whether this enzyme(s) is identical to one or more of the four beta-lactamases produced by methicillin-susceptible strains, the beta-lactamases of 50 MRSA isolates were typed by using substrate profile analysis. Forty type A, no type B, ten type C, and no type D beta-lactamase-producing strains were identified. The beta-lactamase inhibitor sulbactam reduced the MICs of beta-lactamase-labile antibiotics, including ampicillin, penicillin G, and cefazolin, for type A and type C MRSA strains.

Comparative prophylactic efficacies of ciprofloxacin, ofloxacin, cefazolin, and vancomycin in experimental model of staphylococcal wound infection.

Recent shifts in the species and antibiotic resistance patterns of bacteria causing nosocomial infections present new challenges for providing effective prophylaxis in surgery. Traditional regimens lack activity against methicillin-resistant staphylococci and many gram-negative species causing nosocomial infections. The new fluoroquinolones exhibit in vitro activity against many emerging surgical wound pathogens. To determine the potential of this class of antimicrobial agents for use in surgery, we compared the prophylactic efficacies of ciprofloxacin and ofloxacin with those of cefazolin and vancomycin in a guinea pig model of abscess formation. Four Staphylococcus aureus strains, one Staphylococcus epidermidis strain, and one Staphylococcus haemolyticus strain were evaluated. Vancomycin was the most effective prophylactic agent, exhibiting in vivo activity against all strains which was superior or equivalent to those of all other agents tested. Cefazolin was the least effective agent and surpassed the two quinolones in prophylactic efficacy against only one organism, a quinolone- and methicillin-resistant strain of S. aureus. The two quinolones provided excellent protection against infection with all but the quinolone-resistant isolate. The in vivo emergence of quinolone resistance among quinolone-susceptible isolates was not detected. The methicillin-resistant, quinolone-susceptible S. epidermidis and S. haemolyticus isolates were extremely susceptible to prophylaxis, exhibiting 50% infective doses above 4 x 10(6) CFU for seven of the eight antibiotic-strain combinations. We conclude that ciprofloxacin and ofloxacin may be effective antistaphylococcal agents in surgery. The role of these agents remains to be defined, and the definition should include consideration of an adverse effect upon antibiotic resistance patterns of organisms causing nosocomial infections.

Site-directed mutagenesis of the alpha-toxin gene of Staphylococcus aureus: role of histidines in toxin activity in vitro and in a murine model.

Staphylococcus aureus alpha-toxin is a membrane-damaging exoprotein that oligomerizes to form transmembrane pores. Chemical modification of histidines with diethylpyrocarbonate has been shown to reduce the hemolytic activity of alpha-toxin, suggesting that one or more of the histidine residues is important for toxin function. To individually assess the functional importance of each of the four histidine residues (residues 35, 48, 144, and 259), we used oligonucleotide-directed mutagenesis of the cloned alpha-toxin gene to replace each histidine with leucine. The mutant toxins were expressed in S. aureus and evaluated for hemolytic activity in vitro and for lethality in an intraperitoneal murine model. Substitution of histidine 35 with leucine produced a mutant toxin (H35L) without hemolytic or lethal activity. Mutant toxins H48L, H144L, and H259L exhibited 7, 16, and 46%, respectively, of the hemolytic activity of wild-type toxin. Immunoblotting of purified H35L toxin incubated with liposomal membranes demonstrated intact membrane binding and hexamer formation that was clearly detectable but reduced compared with that of the wild-type toxin. This suggests that hexamer formation alone is not sufficient for the expression of alpha-toxin activity. The nature of the defect underlying the lack of activity of the H35L mutant toxin remains to be elucidated but may involve failure of the hexamer to span the lipid bilayer to form a transmembrane pore or a change in the internal surface and permeability characteristics of the pore.

Comparative prophylactic efficacy of cefazolin and vancomycin in a guinea pig model of Staphylococcus aureus wound infection.

The increasing prevalence of methicillin-resistant Staphylococcus aureus as a wound pathogen in some institutions has prompted the use of vancomycin for surgical prophylaxis, although clinical data comparing vancomycin and cephalosporins are not available. A guinea pig model was used to compare the efficacy of vancomycin and cefazolin in preventing intermuscular abscess formation by 7 S. aureus strains. Both antibiotics were administered to achieve peak serum levels at the time of bacterial inoculation, and each remained > 1 micrograms/mL for a comparable duration. Vancomycin provided equivalent protection from infection by 1 methicillin-susceptible strain and significantly better protection against 4 methicillin-susceptible and both methicillin-resistant S. aureus strains. For most strains, the bacterial inoculum with a 50% probability of causing an abscess was 2 to 4 log10-fold higher with vancomycin than cefazolin prophylaxis. Prophylaxis with vancomycin is superior to cefazolin in preventing intermuscular infection by methicillin-susceptible and -resistant S. aureus.