Antipneumococcal activity of ertapenem compared with gatifloxacin in a temperature-sensitive murine model of acute pneumonia.

Fluoroquinolone-resistance among pneumococci is low; however the number of isolates with a single ParC mutation has increased. Consequently, more potent agents are needed to minimize resistance selection. We investigated the efficacy of ertapenem versus gatifloxacin in a temperature-sensitive mouse model of pneumonia caused by a wildtype Streptococcus pneumoniae strain (A66) and an isogenic mutant with a ParC mutation (R222). Treatment started at 24 h and lasted for 5 days. Temperature was used to assess disease progression before and during treatment. Of mice infected with either strain and treated at an early stage of infection, 79-94% of those given ertapenem survived compared with 56-61% given gatifloxacin. If treated at a later stage, the results were similar for ertapenem (71-84%) but were considerably lower for gatifloxacin (17-33%). Ertapenem was as bactericidal as gatifloxacin against A66 (94-100% vs 92-100%) but was superior to gatifloxacin against R222 (95-100% vs 50-77%). Ertapenem is a promising new treatment for patients with pneumococcal pneumonia, including those at risk of infection with a fluoroquinolone-resistant strain.

Relative potential for selection of quinolone-resistance-determining-region mutations in Streptococcus pneumoniae by gemifloxacin, gatifloxacin and moxifloxacin.

Serial passage of a clinical isolate of Streptococcus pneumoniae, in the presence of moxifloxacin, gatifloxacin or gemifloxacin, gave rise to resistant isolates. Non-susceptibility as defined by Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) breakpoints arose on Days 10, 11, and 12 with gatifloxacin, gemifloxacin, and moxifloxacin respectively. Moxifloxacin and gatifloxacin selected for a single step quinolone-resistant-determining-region (QRDR) mutation in DNA gyrase (GyrA) on Day 4 and 7 respectively, whereas gemifloxacin selected simultaneously for multi-step mutations in gyrase and topoisomerase IV (ParC) on Day 17 and activated a non-reserpine inhibited efflux mechanism by Day 4. As found in clinical isolates, mutations included Ser-81-Phe and Glu-85-Lys in GyrA and Ser-79-Phe or Asp-83-Tyr in ParC. At high MICs, moxifloxacin showed a previously unreported 4 amino-acid deletion in GyrB as well as a more unusual substitution Ser-79-Leu/Ile in ParC. Gemifloxacin showed a 2- to 16-fold greater activity than moxifloxacin or gatifloxacin against strains with two or more QRDR mutations, however, its potency did not translate to nonsusceptibility and gemifloxacin MIC values were either at or well above the CLSI nonsusceptible breakpoint concentration.

Short-course therapy of gemifloxacin effective against pneumococcal pneumonia in mice.

Standard 7-14 day (d) courses of antimicrobial therapy for community-acquired pneumonia (CAP) are thought to have contributed to the emergence of resistant pneumoccoci. Consequently, short-course fluoroquinolone regimens have been proposed to minimize resistance. To test this, we examined 2-day versus 5-day regimens of gemifloxacin and levofloxacin for treatment of pneumonia in a murine model. In doing so, we also investigated whether the enhanced potency of gemifloxacin would influence outcomes. CD1 Swiss mice were infected intratracheally with 10(5)-CFU of a virulent Streptococcus pneumoniae strain. Drugs were administered every 8 h for 2 d and 5 d, starting at 24 h postinfection. Temperature was used to assess disease progression. Gemifloxacin remained effective for 2 d and 5 d, with survival rates of 100%-83% compared with 40%-58% for levofloxacin. Eighty-nine to 100% of gemifloxacin-treated mice were clear of pulmonary bacteria compared with only 0%-20% for levofloxacin. For levofloxacin-treated mice, 2 of 7 (29%) isolates with a levofloxacin minimum inhibitory concentration (MIC) 4 times that of the infecting parent strain had ParC mutations. By contrast, no isolates recovered from gemifloxacin-treated mice were reduced in susceptibility. Gemifloxacin could be effective in shortening duration of therapy for CAP treatment as well as minimize resistance development.

Selection of Bacillus anthracis isolates resistant to antibiotics.

OBJECTIVE: Long-term therapy for anthrax might induce antimicrobial resistance in Bacillus anthracis. The aim of the present study was to investigate the potential of 18 different antibiotics to select resistant isolates of B. anthracis, (ST-1 and Sterne strains). METHODS: Resistant isolates were selected by serial passages on brain heart infusion agar containing increasing concentrations of antibiotics (from the MIC upwards). RESULTS: The MICs of ciprofloxacin, ofloxacin and levofloxacin increased from 0.125-0.25 to 8 mg/L, that of moxifloxacin increased from 0.03-0.06 to 8 mg/L, in both strains, and the MIC of garenoxacin increased from 0.015 to 0.5 mg/L for the ST-1 strain and from 0.03 to 8 mg/L for the Sterne strain. The MICs of tetracycline and minocycline increased from 0.125 to 2-8 mg/L and 0.06 to 1 mg/L, respectively. The MIC of vancomycin increased from 2.5 to 20 mg/L for the ST-1 strain and from 5 to 20 mg/L for the Sterne strain. Linezolid exhibited an MIC increase from 2 to 4 mg/L for both strains. The MIC of quinupristin/dalfopristin increased from 0.125 to 64-128 mg/L. Erythromycin demonstrated an MIC increase from 1 to 128 mg/L, that of clarithromycin increased from 0.125 to 8-64 mg/L and that of telithromycin increased from 0.06-0.125 to 1-4 mg/L. The clindamycin MIC increased from 0.125-0.25 to 8 mg/L. Penicillin G and amoxicillin MICs increased from <1 mg/L to 128-512 mg/L. Isolates made resistant to one fluoroquinolone exhibited cross-resistance to the other quinolones except the ST-1 mutant strain which remained susceptible to garenoxacin. Cross-resistance to fluoroquinolones did not correlate with resistance to other antibiotics. CONCLUSION: The ease with which B. anthracis can be made resistant in vitro suggests that close monitoring of patients treated for anthrax is mandatory.

In vitro post-antibiotic effect of fluoroquinolones, macrolides, beta-lactams, tetracyclines, vancomycin, clindamycin, linezolid, chloramphenicol, quinupristin/dalfopristin and rifampicin on Bacillus anthracis.

OBJECTIVES: The aim of this study was to investigate in vitro the post-antibiotic effect (PAE) of 19 antibacterial agents against two strains of Bacillus anthracis (ST-1 and Sterne strains). METHODS: PAE was determined by calculating the time required for the viable counts of antibiotic-exposed bacteria (at concentrations of 10x MIC and exposure for 2 h) at 37 degrees C to increase by 1 log10 above the counts observed immediately after antibiotic removal compared with the corresponding time for controls not exposed to antibiotics. RESULTS: The PAEs of the fluoroquinolones (ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin and garenoxacin) were 2-5 h. The macrolide (erythromycin, clarithromycin and telithromycin) PAEs were 1-4 h, and that of clindamycin was 2 h. The PAEs induced by tetracycline and minocycline were 1-3 h. The PAEs induced by the beta-lactams (penicillin G, amoxicillin and ceftriaxone), vancomycin, linezolid and chloramphenicol were 1-2 h. The PAE induced by rifampicin was 4-5 h. Quinupristin/dalfopristin had the longest PAE, lasting for 7-8 h. CONCLUSIONS: Our results indicate that the PAE is unrelated to the MIC but may be related to the rapidity of bacterial kill. These observations may bear importance on treatment regimens of human anthrax.

Activity of BMS-284756, a novel des-fluoro(6) quinolone, against Staphylococcus aureus, including contributions of mutations to quinolone resistance.

The in vitro activity of BMS-284756 against 602 Staphylococcus aureus isolates, including 152 that were both methicillin and ciprofloxacin resistant (MIC > or = 4 microg/ml), was determined. For ciprofloxacin-susceptible and nonsusceptible isolates, the MICs at which 50% of organisms were inhibited were 0.015 and 2 microg/ml and the MICs at which 90% of organisms were inhibited were 0.03 and 4 microg/ml, respectively.

Interspecies recombination contributes minimally to fluoroquinolone resistance in Streptococcus pneumoniae.

Analysis of 71 ciprofloxacin-resistant (MIC > or = 4 microg/ml) Streptococcus pneumoniae clinical isolates revealed only 1 for which the quinolone resistance-determining regions of the parC, parE, and gyrB genes were genetically related to those of viridans group streptococci. Our findings support the occurrence of interspecies recombination of type II topoisomerase genes; however, its contribution to the emergence of quinolone resistance among pneumococci appears to have been minimal.

A nosocomial outbreak of fluoroquinolone-resistant Streptococcus pneumoniae.

Over the course of a 20-month period, in a hospital respiratory ward in which ciprofloxacin was often used as empirical antimicrobial therapy for lower respiratory tract infections (LRTIs), 16 patients with chronic bronchitis developed nosocomial LRTIs caused by penicillin- and ciprofloxacin-resistant Streptococcus pneumoniae (serotype 23 F). The minimum inhibitory concentration (MIC) of ciprofloxacin for all isolates from the first 9 patients was 4 microg/mL, in association with a parC mutation. Isolates from the subsequent 7 patients all had a ciprofloxacin MIC of 16 microg/mL, in association with an additional mutation in gyrA. The MICs for this isolate were 8 microg/mL of levofloxacin (resistant), 2 microg/mL of moxifloxacin and gatifloxacin (intermediately resistant), and 0.12 microg/mL of gemifloxacin. This outbreak demonstrates the ability of S. pneumoniae to acquire multiple mutations that result in increasing levels of resistance to the fluoroquinolones and to be transmitted from person to person.

Streptococcus iniae virulence is associated with a distinct genetic profile.

Streptococcus iniae causes meningoencephalitis and death in commercial fish species and has recently been identified as an emerging human pathogen producing fulminant soft tissue infection. As identified by pulsed-field gel electrophoresis (PFGE), strains causing disease in either fish or humans belong to a single clone, whereas isolates from nondiseased fish are genetically diverse. In this study, we used in vivo and in vitro models to examine the pathogenicity of disease-associated isolates. Strains with the clonal (disease-associated) PFGE profile were found to cause significant weight loss and bacteremia in a mouse model of subcutaneous infection. As little as 10(2) CFU of a disease-associated strain was sufficient to establish bacteremia, with higher inocula (10(7)) resulting in increased mortality. In contrast, non-disease-associated (commensal) strains failed to cause bacteremia and weight loss, even at inocula of 10(8) CFU. In addition, disease-associated strains were more resistant to phagocytic clearance in a human whole blood killing assay compared to commensal strains, which were almost entirely eradicated. Disease-associated strains were also cytotoxic to human endothelial cells as measured by lactate dehydrogenase release from host cells. However, both disease-associated and commensal strains adhered to and invaded cultured human epithelial and endothelial cells equally well. While cellular invasion may still contribute to the pathogenesis of invasive S. iniae disease, resistance to phagocytic clearance and direct cytotoxicity appear to be discriminating virulence attributes of the disease-associated clone.

Fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae: contributions of type II topoisomerase mutations and efflux to levels of resistance.

We report on amino acid substitutions in the quinolone resistance-determining region of type II topisomerases and the prevalence of reserpine-inhibited efflux for 70 clinical isolates of S. pneumoniae for which the ciprofloxacin MIC is >/=4 microgram/ml and 28 isolates for which the ciprofloxacin MIC is

Genetic locus for streptolysin S production by group A streptococcus.

Group A streptococcus (GAS) is an important human pathogen that causes pharyngitis and invasive infections, including necrotizing fasciitis. Streptolysin S (SLS) is the cytolytic factor that creates the zone of beta-hemolysis surrounding GAS colonies grown on blood agar. We recently reported the discovery of a potential genetic determinant involved in SLS production, sagA, encoding a small peptide of 53 amino acids (S. D. Betschel, S. M. Borgia, N. L. Barg, D. E. Low, and J. C. De Azavedo, Infect. Immun. 66:1671-1679, 1998). Using transposon mutagenesis, chromosomal walking steps, and data from the GAS genome sequencing project (www.genome.ou.edu/strep. html), we have now identified a contiguous nine-gene locus (sagA to sagI) involved in SLS production. The sag locus is conserved among GAS strains regardless of M protein type. Targeted plasmid integrational mutagenesis of each gene in the sag operon resulted in an SLS-negative phenotype. Targeted integrations (i) upstream of the sagA promoter and (ii) downstream of a terminator sequence after sagI did not affect SLS production, establishing the functional boundaries of the operon. A rho-independent terminator sequence between sagA and sagB appears to regulate the amount of sagA transcript produced versus transcript for the entire operon. Reintroduction of the nine-gene sag locus on a plasmid vector restored SLS activity to the nonhemolytic sagA knockout mutant. Finally, heterologous expression of the intact sag operon conferred the SLS beta-hemolytic phenotype to the nonhemolytic Lactococcus lactis. We conclude that gene products of the GAS sag operon are both necessary and sufficient for SLS production. Sequence homologies of sag operon gene products suggest that SLS is related to the bacteriocin family of microbial toxins.

Prevalence and mechanisms of macrolide resistance in clinical isolates of group A streptococci from Ontario, Canada.

A total of 3,205 group A streptoccal isolates were collected in 1997 through a private laboratory which serves community physicians in southern Ontario and which represents a population base of 6 million people. Nonsusceptibility to erythromycin was detected for 67 (2.1%) isolates both by disk diffusion and by broth microdilution. Of these, 47 (70%) were susceptible to clindamycin and were found by PCR to possess the mef gene. Of the other 20 strains, 18 and 2 showed inducible and constitutive resistance, respectively, to clindamycin. Nineteen of these strains were shown by PCR to possess the ermTR gene, and a single constitutively resistant strain harbored an ermB gene. Sixteen (24%) erythromycin-resistant strains were also resistant to tetracycline. All were susceptible to penicillin and chloramphenicol.

The identification of three biologically relevant globotriaosyl ceramide receptor binding sites on the Verotoxin 1 B subunit.

The Verotoxin 1 (VT1) B subunit binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). Receptor-binding specificity is associated with the terminally linked Galalpha(1-4) Galbeta disaccharide sequence of the receptor. Recently, three globotriose (Galalpha[1-4] Galbeta [1-4] Glcbeta) binding sites per B-subunit monomer were identified by crystallography. Two of these sites (sites I and II) are located adjacent to phenylalanine-30. Site I was originally predicted as a potential Gb3 binding site on the basis of sequence conservation, and site II was additionally predicted based on computer modelling and receptor docking. The third (site III) was also identified by crystallography and is located at the N-terminal end of the alpha-helix. To determine the biological significance of sites II and III, and to support our previous findings of the significance of site I, we examined the binding properties and cytotoxicity of VT1 mutants designed to block Gb3 binding at each site selectively. The Scatchard analysis of saturation-binding data for each mutant revealed that only the amino acid substitutions predicted to affect site I (D-17E) or site II (G-62T) caused reductions in the binding affinity and capacity of VT1 for Gb3. Similarly, those mutations at sites I and II also caused significant reductions in both Vero and MRC-5 cell cytotoxicity (by seven and five logs, respectively, for G-62T and by four and two logs, respectively, for D-17E). In contrast, the substitution of alanine for W-34 at site III did not reduce the high-affinity binding of the B subunit, despite causing a fourfold reduction in the receptor-binding capacity. The corresponding mutant W-34A holotoxin had a two-log reduction in cytotoxicity on Vero cells and no statistically significant reduction on MRC-5 cells. We conclude that the high-affinity receptor binding most relevant for cell cytotoxicity occurs at sites I and II. In contrast, site III appears to mediate the recognition of additional Gb3 receptor epitopes but with lower affinity. Our results support the significance of the indole ring of W-34 for binding at this site.

Murine antibody responses to the verotoxin 1 B subunit: demonstration of major histocompatibility complex dependence and an immunodominant epitope involving phenylalanine 30.

Structurally conserved verotoxin 1 (VT1) mutant derivatives, showing reduced receptor binding and cytotoxicity, may serve as natural toxoids to protect against VT-mediated disease. In this study, the antibody responses to the wild-type VT1 B subunit, a B-subunit mutant (Phe30Ala B), and the corresponding holotoxin (Phe30Ala HT) were examined in three inbred mouse strains. BALB/c (H-2d) and CBA (H-2k) mice produced strong antibody responses to both wild-type and mutant B subunits. VT1 B-raised sera reacted more strongly with VT1 B than with Phe30Ala B in enzyme-linked immunosorbent assays, while Phe30Ala B-raised sera reacted equally with VT1 B and Phe30Ala B. C57BL/6 (H-2b) and congenic BALB/c (BALB x B [H-2b]) mice produced no detectable antibody response to either VT1 B or Phe30Ala B. However, an anti-VT1 B antibody response was detected in H-2b mice immunized with biologically active Phe30Ala HT. Based on these observations, we conclude that the VT1 B subunit possesses a B-cell immunodominant epitope formed partly by phenylalanine 30 and that the B-subunit antibody response is dependent on the H-2 haplotype of the mouse strain. Our results also support a potential role for the A subunit in providing the T-cell help necessary to overcome a deficient B-subunit antibody response in H-2b mice.

Toxicity and immunogenicity of a verotoxin 1 mutant with reduced globotriaosylceramide receptor binding in rabbits.

The verotoxins (VT1 and VT2), produced by strains of enterohemorrhagic Escherichia coli, have been implicated in the pathogenesis of hemorrhagic colitis and the hemolytic uremic syndrome. To better understand the role of globotriaosylceramide (Gb3) receptor binding by the verotoxins in disease production, we examined the clinicopathologic effects of an intravenously (i.v.) administered verotoxin 1 mutant holotoxin (Phe30Ala) in rabbits. The substitution of alanine for phenylalanine 30 in the VT1 B subunit has been shown previously to reduce both Gb3 binding affinity and capacity in vitro. This reduction in receptor binding corresponded to a 10(5)-fold reduction in the toxic activity of VT1 on a Vero cell monolayer. In this study, purified 125I-labeled Phe30Ala was administered i.v. to rabbits to determine its specific distribution in rabbit tissues. In contrast to the rapid elimination of i.v. administered 125I-VT1 from the bloodstream, 125I-Phe30Ala had a 52-fold-longer half-life in serum and failed to localize preferentially in the gastrointestinal tract and central nervous system (CNS). Rabbits challenged with Phe30Ala at a dose equivalent to 10 times the 50% lethal dose (LD50) of VT1 showed no visible clinical symptoms typical of VT effect after 7 days. Administration of Phe30Ala at a dose equivalent to 100 times the LD50 of VT1, however, caused both clinical and histopathologic features indistinguishable from VT1 toxemia in rabbits, although the onset of symptoms was delayed. Rabbits were immunized with Phe30Ala and challenged i.v. with either 125I-VT1 or 125I-VT2. The specific uptake of 125I-VT1 in the gastrointestinal tract and CNS was totally inhibited in Phe30Ala immune rabbits. Only a partial decrease in target organ uptake was observed in Phe30Ala immune rabbits challenged with 125I-VT2. From this study, we conclude that Gb3 binding is responsible for target organ localization of VT1 and disease production in the rabbit. The ability of Phe30Ala to induce both strong antibody and protective responses against VT1 suggests that VT mutants with reduced receptor binding properties may be useful in vaccine strategies. A further reduction in the toxicity of Phe30Ala would be required for its use as a natural toxoid to protect against human verotoxigenic E. coli infections.