Efficacy of cethromycin, a new ketolide, against Streptococcus pneumoniae susceptible or resistant to erythromycin in a murine pneumonia model.

Cethromycin is a ketolide with in vitro activity against macrolide-sensitive and -resistant strains of Streptococcus pneumoniae. We compared its in vivo efficacy to erythromycin in a mouse model of acute pneumonia induced by two virulent clinical strains: a serotype 3 susceptible strain (P-4241) (MICs: erythromycin, 0.03 microg/ml; cethromycin, 0.015 microg/ml) and a serotype 1 strain resistant to erythromycin (P-6254; phenotypically MLSB constitutive) (MICs: erythromycin, 1,024 microg/ml; cethromycin, 0.03 microg/ml). Immunocompetent mice were infected with 10(5) CFU of each strain. Six treatments given either subcutaneously (s.c.) or per os (p.o.) at 12-h intervals were initiated at 6 or 12 h after infection. Against P-4241, cethromycin given s.c. at 25 or 12.5 mg/kg protected 100% of the animals, with lungs and blood completely cleared of bacteria. Given p.o., cethromycin maintained its efficacy with 100 and 86% survival at 25 and 12.5 mg/kg, respectively. Erythromycin, given s.c. at 50 or 37.5 mg/kg, provided 50 and 38% survival rates, respectively. Against P-6254, cethromycin was effective at 25 mg/kg (100% survival) regardless of the administration route, whereas only 25 and 8% of animals survived after a 75-mg/kg erythromycin treatment given s.c. and p.o., respectively. The serum protein binding levels of cethromycin were 94.8 and 88.5% after doses of 12.5 and 25 mg/kg, respectively. The higher in vivo activity of cethromycin compared to erythromycin could be explained by favorable pharmacokinetic/pharmacodynamic indexes against P-6254 but not against P-4241.

Activity of gemifloxacin against quinolone-resistant Streptococcus pneumoniae strains in vitro and in a mouse pneumonia model.

Gemifloxacin is a novel fluoronaphthyridone quinolone with enhanced in vitro activity against Streptococcus pneumoniae. We investigated the activities of gemifloxacin and trovafloxacin, their abilities to select for resistance in vitro and in vivo, and their efficacies in a mouse model of acute pneumonia. Immunocompetent Swiss mice were infected with 10(5) CFU of a virulent, encapsulated S. pneumoniae strain, P-4241, or its isogenic parC, gyrA, parC gyrA, and efflux mutant derivatives (serotype 3); and leukopenic mice were infected with 10(7) CFU of two poorly virulent clinical strains (serotype 11A) carrying either a parE mutation or a parC, gyrA, and parE triple mutation. The drugs were administered six times every 12 h, starting at either 3 or 18 h postinfection. In vitro, gemifloxacin was the most potent agent against strains with and without acquired resistance to fluoroquinolones. While control mice died within 6 days, gemifloxacin at doses of 25 and 50 mg/kg of body weight was highly effective (survival rates, 90 to 100%) against the wild-type strain and against mutants harboring a single mutation, corresponding to area under the time-versus-serum concentration curve at 24 h (AUC(24))/MIC ratios of 56.5 to 113, and provided a 40% survival rate against a mutant with a double mutation (parC and gyrA). A total AUC(24)/MIC ratio of 28.5 was associated with poor efficacy and the emergence of resistant mutants. Trovafloxacin was as effective as gemifloxacin against mutants with single mutations but did not provide any protection against the mutant with double mutations, despite treatment with a high dose of 200 mg/kg. Gemifloxacin preferentially selected for parC mutants both in vitro and in vivo.

Efficacy of BAL5788, a prodrug of cephalosporin BAL9141, in a mouse model of acute pneumococcal pneumonia.

BAL5788 is a water-soluble prodrug of BAL9141, a new broad-spectrum cephalosporin with high levels of in vitro activity against methicillin- and vancomycin-resistant staphylococci and penicillin-resistant streptococci. In plasma BAL5788 is rapidly converted to BAL9141. We studied the activity of BAL5788 in a mouse model of acute pneumococcal pneumonia. Leukopenic female Swiss albino mice were challenged intratracheally with 10(7) CFU of clinical Streptococcus pneumoniae strains P-52181 (Pen(s) Cro(s) Ctx(s)), P-15986 (Pen(r) Cro(s) Ctx(s)), P-40422 (Pen(r) Cro(r) Ctx(r)), and P-40984 (Pen(r) Cro(r) Ctx(r)). Infected mice received subcutaneous (s.c.) injections of BAL5788 or ceftriaxone starting 3 h after pneumococcal challenge. Uninfected nonleukopenic mice received single s.c. doses of BAL5788 to determine the BAL9141 concentration-time profiles in serum and lungs. Untreated control mice died within 5 days postinfection. Ten-day cumulative survival rates for infected mice receiving BAL5788 (total daily doses of BAL9141 equivalents, 2.1 to 75 mg/kg of body weight) ranged from 57 to 100%, whereas with ceftriaxone (total daily doses, 10 to 400 mg/kg), the survival rates varied between 13 and 100%. In mice infected with P-15986, the survival rates achieved with BAL5788 (BAL9141 equivalent, 8.4 mg/kg) and those achieved with ceftriaxone (50 mg/kg) were significantly different (93 versus 13%; P < 0.0001) in favor of BAL5788; the outcomes of the trials with all other strains were not significantly different between the two antibiotics, but markedly lower doses of BAL5788 than ceftriaxone were required to obtain similar survival rates. Pharmacokinetic data showed that BAL9141 was effective against the four pneumococcal strains tested at very low values of the time above the MIC (T > MIC), which ranged from 9 to 18% of the dosing interval, whereas the values of T > MICs for ceftriaxone ranged from 30 to 50% of the dosing interval.

Activities of garenoxacin against quinolone-resistant Streptococcus pneumoniae strains in vitro and in a mouse pneumonia model.

Garenoxacin is a novel des-F(6) quinolone with enhanced in vitro activities against both gram-positive and gram-negative bacteria. We compared the activity of garenoxacin with that of trovafloxacin (TVA) against Streptococcus pneumoniae, together with their efficacies and their capacities to select for resistant mutants, in a mouse model of acute pneumonia. In vitro, garenoxacin was more potent than TVA against wild-type S. pneumoniae and against a mutant with a single mutation (parC), a mutant with double mutations (gyrA and parC), and a mutant with triple mutations (gyrA, parC, and parE). Swiss mice were infected with 10(5) CFU of virulent, encapsulated S. pneumoniae strain P-4241 or its derived isogenic parC, gyrA, gyrA parC, and efflux mutants and 10(7) CFU of poorly virulent clinical strains carrying a parE mutation or gyrA, parC, and parE mutations. The drugs were administered six times, every 12 h, beginning at either 3 or 18 h postinfection. The pulmonary pharmacokinetic parameters in mice infected with strain P-4241 and treated with garenoxacin or TVA (25 mg/kg of body weight) were as follows: maximum concentration of drug in serum (C(max); 17.3 and 21.2 micro g/ml, respectively), C(max)/MIC ratio (288 and 170, respectively), area under the concentration-time curve (AUC; 48.5 and 250 microg. h/ml, respectively), and AUC/MIC ratio (808 and 2000, respectively). Garenoxacin at 25 and 50 mg/kg was highly effective (survival rates, 85 to 100%) against the wild-type strain and mutants harboring a single mutation. TVA was as effective as garenoxacin against these strains. TVA at 200 mg/kg and garenoxacin at 50 mg/kg were ineffective against the mutant with the parC and gyrA double mutations and the mutant with the gyrA, parC, and parE triple mutations. The efficacy of garenoxacin was reduced only when strains bore several mutations for quinolone resistance.

Efficacy of BB-83698, a novel peptide deformylase inhibitor, in a mouse model of pneumococcal pneumonia.

The efficacy of BB-83698, a novel potent peptide deformylase inhibitor, was evaluated in a mouse model of acute pneumonia. The Streptococcus pneumoniae isolates tested included four virulent strains (one penicillin-susceptible wild-type strain, one macrolide-resistant strain, and two quinolone-resistant mutants [a mutant carrying mutations in ParC and GyrA and an efflux mutant] isogenic to the wild type) and two poorly virulent penicillin-resistant strains. Pneumonia was induced by intratracheal inoculation of 10(5) CFU (virulent strains) into immunocompetent mice or 10(7) CFU (less virulent strains) into leukopenic mice. Animals received three or six subcutaneous injections of antibiotics at 12- or 24-h intervals, with antibiotic treatment initiated at 3, 6, 12, or 18 h postinfection (p.i.). BB-83698 showed potent in vitro activity against all strains (MICs, 0.06 to 0.25 micro g/ml). In the in vivo model, all control animals died within 2 to 5 days of infection. BB-83698 (80 mg/kg of body weight twice daily or 160 mg/kg once daily) protected 70 to 100% of the animals, as measured 10 days p.i., regardless of the preexisting resistance mechanisms. In contrast, the survival rates for animals treated with the comparator antibiotics were 30% for animals treated with erythromycin (100 mg/kg) and infected with the macrolide-resistant strain, 34% for animals treated with amoxicillin (200 mg/kg every 8 h) and infected with the penicillin-resistant strain, and 0 and 78% for animals treated with ciprofloxacin (250 mg/kg) and infected with the ParC and GyrA mutant and the efflux mutant, respectively. At 80 mg/kg, BB-83698 generated a peak concentration in lung tissue of 61.9 micro g/ml within 1 h and areas under the concentration-times curves of 57.4 and 229.4 micro g. h/ml for plasma and lung tissue, respectively. The emergence of S. pneumoniae isolates with reduced susceptibilities to BB-83698 was not observed following treatment with a suboptimal dosing regimen. In conclusion, the potent in vitro activity of BB-83698 against S. pneumoniae, including resistant strains, translates into good in vivo efficacy in a mouse pneumonia model.

Complex relationship between acquisition of beta-lactam resistance and loss of virulence in Streptococcus pneumoniae.

In a previous study of a murine peritonitis model, no Streptococcus pneumoniae strains were found that were both clinically penicillin resistant and virulent. This study assessed the relationship between acquired resistance and virulence in single- and double-isogenic penicillin-resistant (Peni-R) mutants obtained by transformation of a virulent penicillin-susceptible recipient strain with pbp2b and pbpX polymerase chain reaction fragments from a Peni-R donor strain. Sequence analysis results of the pbp2b and pbpX alleles from these strains were in keeping with acquired penicillin resistance. The virulence of these strains was significantly reduced, which shows a relationship between beta-lactam resistance and loss of virulence. The phenotype of the 23.2x mutant remained stable after in vivo passage, which suggests that the pbpX gene is involved in growth, whereas virulent revertants of the 23.2b and 23.2b.2x mutants had no change in MIC. Compensatory mutations are implicated in the revival of virulence.

Nuclear factor-kappaB activation in mouse lung lavage cells in response to Streptococcus pneumoniae pulmonary infection.

OBJECTIVES: To assess the state and activation kinetics of the nuclear transcription regulatory protein nuclear factor-kappB (NF-kappaB) in lung lavage cells in a murine pneumococcal pneumonia model and to determine how the virulence of the infecting organisms altered the activation state of NF-kappaB. DESIGN: Experimental, comparative study of three Streptococcus pneumoniae strains that induced three distinct pulmonary diseases. SETTING: Experimental laboratory in a university-based medical center. SUBJECTS: Female BALB/cby mice, 8-10 wks of age. INTERVENTIONS: We randomly divided the mice into the following five groups: a) the control group; b) animals infected by virulent encapsulated S. pneumoniae P4241 strain; c) animals infected by avirulent encapsulated S. pneumoniae P15986 strain; d) animals infected by avirulent unencapsulated S. pneumoniae R6 strain; e) animals infected by virulent lysed S. pneumoniae P4241 strain. Animals were anesthetized and infected by intratracheal delivery of 4 x 10(5) colony-forming units (CFU) of S. pneumoniae per mouse or bacterial components equivalent to 4 x 10(5) CFU for lysed S. pneumoniae challenge. After intratracheal challenge with virulent encapsulated strain P4241, mice developed acute pneumonia, became bacteremic, and died within 3 to 5 days. None of the mice infected with the avirulent encapsulated strain P15986 or the avirulent unencapsulated strain R6 died. After collection of lung lavage cells and nuclear extraction, NF-kappaB activation was determined 1 hr, 4 hrs, 6 hrs and 24 hrs after pneumococcal infection. At the same time, pulmonary and blood clearance, bronchoalveolar lavage cells population, and tumor necrosis factor-alpha production were assessed (six mice per time point). MEASUREMENTS AND MAIN RESULTS: NF-kappaB was constitutively expressed within nuclear extracts of lung lavage cells from uninfected control mice. A significant increase in NF-kappaB activation was detected within 1 hr after injection of virulent lysed S. pneumoniae P4241 strain (bacterial components equivalent to 4 x 10(5) CFU), and was still present 24 hrs after the injection. After live pneumococcal challenge, significant NF-kappaB activation was detected within 4 hrs with a peak at 24 hrs. Responses to all three strains (P4241, P15986 and R6) were time-dependent (p < .0001), as NF-kappaB activation gradually increased during the first 24 hrs. Moreover, compared with the control uninfected mice, the intensity of the retarded KB oligonucleotide, as determined by densitometry, was increased approximately four- to five-fold and seven-fold in reactions containing nuclear extracts isolated 24 hrs after infection with the avirulent strains P15986 or R6 and the virulent strain P4241, respectively. With the virulent strain P4241, responses were significantly stronger than with the avirulent strains P15986 and R6 (p < .01). Responses were of similar order with avirulent strains P15986 and R6 (p > .05). CONCLUSION: Pulmonary infection by S. pneumoniae induced delayed and time-dependent activation of NF-kappaB in mouse lung lavage cells. The degree of NF-kappaB activation in lung lavage cells correlated with the virulence of the infecting organisms. Our results suggest that the more severe the infection, the higher the rise in NF-kappaB.

Relationship between capsular type, penicillin susceptibility, and virulence of human Streptococcus pneumoniae isolates in mice.

We examined the relationship between penicillin susceptibility, peritoneal virulence in Swiss mice, and capsular type in a selection of 122 clinical Streptococcus pneumoniae isolates belonging to 24 serotypes. Regardless of the serotype, all 32 virulent strains were susceptible to penicillin, and all 41 strains with diminished susceptibility or resistance to penicillin were avirulent. The remaining 49 strains were both susceptible to penicillin and avirulent, irrespective of the serotype. On the basis of their capsular type and pathogenic behavior, strains fell into one of four groups. In the group consisting of serotypes 1, 3, and 4 (n = 16), strains were predominantly virulent (81.3%), and all were penicillin susceptible. In the serotype 6 group (n = 32), the frequency of virulence was significantly lower (34.4 versus 81.3%, P = 0.002), and strains were predominantly penicillin susceptible (71.9%). In the group composed of serotypes 9, 14, 19, and 23 (n = 50), all strains were avirulent, and 56% had decreased susceptibility (n = 12) or resistance to (n = 16) penicillin. The fourth group was heterogenous, as it pooled 24 strains of 15 different serotypes; in this group the frequency of virulence was 33.3%, and strains were predominantly penicillin susceptible (83.3%). These data point to a complex relationship between penicillin susceptibility and virulence in mice but do not entirely separate these characteristics from the role of the capsular type. The possibility that the mechanisms conferring penicillin resistance are related to those leading to a loss of virulence is supported by these findings.

[Selection of virulent mutants of Streptococcus pneumoniae. Utilization of a murine model of septicemia]. / Sélection de mutants virulents de Streptococcus pneumoniae. Utilisation d'un modèle murin de septicémie.

Genetic construction of virulence deficient mutant is a strategy to analyse virulence genes of Streptococcus pneumoniae and was used to virulence factors as capsule, pneumolysin, autolysin and PspA. We perform a model allowing the in vivo positive selection of virulent S. pneumoniae mutants. Mice which are the most susceptible animals to pneumococcal infection, offer the best model for screening virulent S. pneumoniae. Indeed, after intraperitoneal injection of bacterial mix which was composed to a lot of avirulent bacteria (6 log10 CFU per mouse) (V1015 strain, DL50 = 7.05) and few virulent pneumococci (1 to 2 log10 CFU per mouse) (P4241 strain, DL50 < 1), mice cleared all avirulent bacteria but not virulent pneumococci. Thus, mice dead in 3 to 4 days with septicaemia and positive hemoculture contained only virulent strain. This model was validated by in vivo selection of a virulent mutant (V1042, DL50 = 4.1) which was obtained after transformation of avirulent strain V1015 with the genomic fragment of virulent strain P4241. Our model of screening was the only one allowing detection of virulent S. pneumoniae mutants. This new genetic strategy which consisted in gene addition and used mouse as selection agent, could be used to discover new virulence genes required to in vivo bacterial development.

[Current microbiologic problems. Pulmonary infection by Streptococcus pneumoniae. Current physiopathological aspects]. / Problèmes microbiologiques d'actualité. Infection pulmonaire à Streptococcus pneumoniae. Aspects physiopathologiques actuels.

STILL A SERIOUS DISEASE: Despite advances in antibiotic therapy, and despite the emergence of beta-lactam-resistant strains, mortality of pneumococcal pneumonia has remained relatively unchanged. The pathogenicity of a pneumococcal strain results from an interaction between a specific host with its own capacity to "resist or yield" to the multiple virulence factors intrinsic to each S. pneumoniae strain. ROLE OF THE CAPSULE: The capsule is the pneumococci's principal arm of resistance against the host's defense systems. There is a wide variability depending on the capsule serotype. Inversely, the capsule plays no role in triggering the inflammatory reaction which is secondary to substances released from the bacterial wall such as teichoic acid and peptidoglycan. HOST RESPONSE: The molecular mechanisms of cellular activation and induction of proinflammatory cytokines triggered by S. pneumoniae follow similar pathways which are different from those for endotoxin triggered by Gram negative bacteria. PERSPECTIVES: A better understanding of the clinical expression, distinguishing simple nasopharyngeal carriage from bacterial pneumonia should lead to the design of new therapeutic agents which will reduce the capacity of certain S. pneumoniae strains to invade the host and trigger deleterious inflammatory reactions.

Pleiotropic mutations alter the kinetics of calcium transport, competence regulation, autolysis and experimental virulence in Streptococcus pneumoniae.

Streptococcus pneumoniae is a pathogen in which the extracellular calcium concentration plays a major physiological role, in growth as well as in the induction of competence for genetic transformation and activation of autolysis. Both responses are under the control of a protein activator exported in the medium. We have checked the impact of mutations which alter the regulation of competence and autolysis on experimental virulence. Isogenic encapsulated derivatives carrying the relevant mutations were serotype 3 smooth clones, obtained by transformation of the relevant rough strains with DNA from a serotype 3 smooth isolate. Survival kinetics and bacterial clearance from the blood were followed after intraperitoneal infection of Swiss mice with the different bacterial cultures. In this model, mutants showing an attenuation of virulence relative to the wild type fell into two classes. In the first, represented by the lytA::ery mutant V1095 defective for calcium-induced autolysis, attenuated virulence could be correlated with rapid bacterial clearance from the blood. In the second, represented by the dmb mutants V2200 and V3300, attenuation was associated with delayed bacterial clearance from the blood, and correlated with altered kinetics of calcium transport and of regulation of competence and autolysis. It appeared unlikely that attenuation of virulence for strains V2200 and V3300 was a direct consequence of their competence phenotype, since the com::ery mutants V1008 and V1019, defective for the production of the competence activator, were as virulent as the wild-type strain. Autolysis involving an N-acetyl-muramyl-alanine amidase encoded by lytA was also regulated by calcium. The inserted allele lytA0::ery further reduced virulence in the dmb1 background (V2200). This additive effect of lytA- to dmb1 points to different routes of virulence regulation by LYT and DMB1 and suggests that the kinetics of calcium traffic controls several pathways involved in the virulence of pneumococcus.

Pharmacodynamic activities of ciprofloxacin and sparfloxacin in a murine pneumococcal pneumonia model: relevance for drug efficacy.

We looked for associations between pharmacokinetic (Pk) and pharmacodynamic (Pd) parameters of ciprofloxacin (CPFX) and sparfloxacin (SPFX) and the in vivo efficacy of these antimicrobials in an immunocompetent mouse model of severe Streptococcus pneumoniae pneumonia. Bacterial killing curves recorded in the lungs during the 24 h after single subcutaneous injections of the fluoroquinolones (FQs) in doses ranging from 6.25 to 200 mg/kg were compared with mean Pk/Pd parameters in the serum of the same mice. The impact of the dosing interval on the antimicrobial dose response was evaluated based on the survival of mice treated for 3 days with CPFX (25-200 mg/kg) or SPFX (6.25-50 mg/kg) administered at various intervals from 3 to 24 h. Bacterial killing curves showed that the maximal bacterial decrease achieved in the lungs was correlated, similarly for both FQs, with the area under the curve (AUC) above the minimal inhibitory concentration (MIC) (overall correlation: r = 0.968, P < 10(-4)). CPX attained higher maximal bactericidal effect values, a steeper killing slope and a shorter time to maximal bactericidal effect in comparison with SPX for the highest doses tested. The lower MIC of SPFX compared with CPFX (0.25 vs. 0.75 microgram/ml) and its higher AUC/dose ratio (resulting from a lower serum peak but a longer half-life) translated into a greater area under the bactericidal curve. In the dose fractionation experiments, the Pk/Pd parameter most closely correlated with the survival rate for both FQs was the daily AUC/MIC ratio (r = 0.976, P < 10(-4)). When the AUC/MIC ratio was greater than 160, the probability of a clinical cure was 100%, independently of the dosage schedule.

Efficacy of trovafloxacin against penicillin-susceptible and multiresistant strains of Streptococcus pneumoniae in a mouse pneumonia model.

The increasing emergence of penicillin-resistant and multidrug-resistant strains of Streptococcus pneumoniae will create a serious therapeutic problem in coming years. Trovafloxacin is a novel naphthyridone quinolone with promising activity against S. pneumoniae, including penicillin-resistant strains (MIC for 90% of the isolates tested, 0.25 microg/ml). We compared its in vivo efficacy with that of other fluoroquinolones (ciprofloxacin, temafloxacin, and sparfloxacin) and a reference beta-lactam (amoxicillin) in a model of acute experimental pneumonia. Immunocompetent Swiss mice were infected by peroral tracheal delivery of a virulent, penicillin-susceptible strain (MIC, 0.03 microg/ml); leukopenic Swiss mice were infected with three poorly virulent, penicillin-resistant strains (MICs, 4 to 8 microg/ml) and a ciprofloxacin-resistant strain (MIC, 32 microg/ml). Treatments were started 6 h (immunocompetent mice) or 3 h (leukopenic mice) after infection. Doses ranging from 12.5 to 300 mg/kg were given at 12- or 8-h intervals for 3 days. Trovafloxacin (25 mg/kg) was the most effective agent in vivo against penicillin-susceptible and -resistant strains. Corresponding survival rates were 2- to 4-fold higher than with 50-mg/kg sparfloxacin or temafloxacin and 8- to 16-fold higher than with 100-mg/kg ciprofloxacin. The ratios of the area under the concentration-time curve to the MIC in serum and lung tissue were more favorable with trovafloxacin than with the other quinolones. Efficacy in vivo correlated with pharmacokinetic parameters. Trovafloxacin shows potential for the treatment of infections due to penicillin-susceptible and -resistant S. pneumoniae but appears to be ineffective against a ciprofloxacin-resistant strain.

Correlation between in vitro and in vivo activity of amoxicillin against Streptococcus pneumoniae in a murine pneumonia model.

We studied the relationship between in vitro bacteriological parameters [minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and killing rate, defined as the reduction in the inoculum within 1, 3 or 6 hr] and in vivo activity of amoxicillin against 12 strains of Streptococcus pneumoniae, with penicillin MICs of < 0.01 to 16 micrograms/ml, in a cyclophosphamide-induced neutropenic murine pneumonia model. Dose-response curves were determined for amoxicillin against each strain, and three quantitative parameters of in vivo amoxicillin activity were defined, i.e., maximal attainable antimicrobial effect attributable to the drug [i.e., reduction in log colony-forming units (CFU) per lung, compared with untreated controls], dose required to reach 50% of maximal effect and dose required to achieve a reduction of 1 log CFU/lung. We demonstrated a highly significant correlation between the dose required to reach 50% of maximal effect and MIC (Spearman r = 0.98, P < .0001) or MBC (Spearman r = 0.95, P < .0001) for amoxicillin against strains of S. pneumoniae with a wide range of amoxicillin MICs (0.01-8 micrograms/ml). Significant correlations between the dose required to achieve a reduction of 1 log CFU/lung and MIC (Spearman r = 0.98, P < .0001) or MBC (Spearman r = 0.95, P < .0001) were also observed. In contrast, there were no significant correlations between the maximal attainable antimicrobial effect attributable to the drug and MIC, MBC or killing rate or between killing rate and the dose required to reach 50% of maximal effect or the dose required to achieve a reduction of 1 log CFU/lung. We conclude that in vitro susceptibility test results (MICs and MBCs) correlated well with in vivo amoxicillin activity against pneumococcal strains, including highly penicillin-resistant strains, in this animal model. Furthermore, these data suggest that the estimated MIC breakpoints for amoxicillin against S. pneumoniae would be 2 micrograms/ml for intermediate-resistant and 4 micrograms/ml for resistant, although this remains to be confirmed in clinical studies.

Western blot analysis of antibody response to pneumococcal protein antigens in a murine model of pneumonia.

To detect new antigen candidates for serological tests, we studied the antibody response to pneumococcal protein antigens in mice infected intratracheally with various Streptococcus pneumoniae strains. Sera were tested by Western blotting against whole-cell protein extracts. Mice developed a detectable immunoglobulin G-type response against a small number of polypeptides. The antibody response was strain dependent: sera from individuals infected with the same strain gave similar banding patterns on immunoblots. The banding patterns varied with the strain used for infection. However, a band at 36 to 38 kDa was recognized by all reactive sera. This band appeared to correspond to a polypeptide that was antigenically well conserved among the different S. pneumoniae serotypes. An antibody response to this antigen developed in mice irrespective of the capsular type, the virulence, and the susceptibility to penicillin G of the infecting strain. Thus, this 36- to 38-kDa protein antigen may be of value for the development of a serological test for humans.

Efficacies of cefotaxime and ceftriaxone in a mouse model of pneumonia induced by two penicillin- and cephalosporin-resistant strains of Streptococcus pneumoniae.

We previously demonstrated the efficacy of ceftriaxone (CRO), at 50 mg/kg of body weight every 12 h, against a highly penicillin-resistant (MIC, 4 micrograms/ml) Streptococcus pneumoniae strain with low-level resistance to CRO (MIC, 0.5 microgram/ml) in a leukopenic-mouse pneumonia model (P. Moine, E. Vallée, E. Azoulay-Dupuis, P. Bourget, J.-P. Bédos, J. Bauchet, and J.-J. Pocidalo, Antimicrob. Agents Chemother. 38:1953-1958, 1994). In the present study, we assessed the activity of CRO versus those of cefotaxime (CTX) and amoxicillin (AMO) against two highly penicillin- and cephalosporin-resistant S. pneumoniae strains (P40422 and P40984) (MICs of 2 and 8 for penicillin, 2 and 4 for AMO, and 4 and 8 for CRO or CTX, respectively). Against both strains, a greater than an 80% cumulative survival rate was observed with CRO at a dose of 100 or 200 mg/kg every 12 h (dose/MIC ratio, 25). With CTX, a high dosage of 400 mg/kg (dose/MIC ratio, 100 or 50) administered every 8 h (TID) was needed to protect 66 and 75% of the animals, respectively, with no statistically significant differences versus CRO. Against the P40422 strain, CRO (100 mg/kg) produced the greatest bactericidal effect, from the 8th to the 24th hour after a single injection (1.8-log-unit reduction over 24 h), and the fastest bacterial pulmonary clearance during treatment; with CTX, only multiple injections at a high dosage, i.e., 400 mg/kg TID, demonstrated a significant bactericidal effect. AMO in a high dosage, 400 mg/kg (dose/MIC ratio, 200) TID, showed good activity only against the P40422 strain. Despite the identical MICs of CTX and CRO, the longer time (3.6 to 4.6 h) that serum CRO concentrations remained above the MICs for the pathogens at a dose of 100 mg/kg resulted in greater efficacy versus CTX against highly penicillin- and cephalosporin-resistant S. pneumoniae strains.

Synergy between amoxicillin and gentamicin in combination against a highly penicillin-resistant and -tolerant strain of Streptococcus pneumoniae in a mouse pneumonia model.

In vivo synergy with beta-lactam antibiotics and aminoglycosides has been studied only with penicillin-susceptible Streptococcus pneumoniae strains. We evaluated the interaction between amoxicillin (AMX) and gentamicin (GEN) on the basis of in vitro checkerboard and time-kill curves and of findings in a mouse model of acute bacteremic pneumonia due to a highly penicillin-resistant and -tolerant S. pneumoniae strain of serotype 19 (penicillin and AMX MICs of 4 micrograms/ml; gentamicin MIC of 16 micrograms/ml). Checkerboard results at 18 h of incubation showed indifference. With regard to AMX alone, in vitro time-kill curves demonstrated synergy between AMX (1 microgram/ml) and GEN (16 micrograms/ml) at 5 and 8 h of incubation and for AMX (16 micrograms/ml) in combination with GEN (16 micrograms/ml) at 3, 5, and 8 h of incubation. In leukopenic mice, pulmonary killing curves after a single drug injection demonstrated that AMX (100 mg/kg of body weight) with GEN (16 mg/kg) was more effective than AMX alone (P = 10(-4). With repeated-dose treatment, a synergy was apparent at 8 h after four injections with AMX (100 mg/kg) in combination with GEN (8 or 16 mg/kg) (P < or = 0.05). The cumulative survival rate with AMX (100 mg/kg) every 8 h, combined with GEN (4 or 8 mg/kg) every 8, 12, or 24 h, was better than with AMX alone. Combined use of AMX and GEN may be a valuable therapeutic alternative for pneumococcal pneumonia due to highly penicillin-resistant S. pneumoniae strains.

Amoxicillin dose-effect relationship with Streptococcus pneumoniae in a mouse pneumonia model and roles of in vitro penicillin susceptibilities, autolysis, and tolerance properties of the strains.

We used a mouse model of pneumococcal pneumonia to assess the bactericidal effect of increasing doses of amoxicillin (AMX) against clinical strains with various susceptibilities to penicillin. Twelve strains that exhibited similar virulence in mice were selected. Three were penicillin susceptible (PS) (penicillin and AMX MICs = 0.01 to 0.03 microgram/ml), three were intermediately resistant (PIR) (penicillin and AMX MICs = 0.5 to 1 microgram/ml), and six were penicillin resistant (PR) (penicillin and AMX MICs = 1 to 8 micrograms/ml). Leukopenic Swiss mice were infected intratracheally with 10(7) CFU of each strain. Treatment was initiated 3 h after infection and consisted of a single subcutaneous injection of AMX at doses ranging from 2.5 to 10 mg/kg (PS strains), 5 to 100 (PIR strains), and 25 to 3,000 (PR strains). Bacterial killing kinetics were recorded in the lungs over 9 h. The maximal log CFU reduction (Emax) was observed 3 h postinjection. The relation between Emax and log10(dose/MIC) showed two populations. With seven strains (the three PS, the three PIR, and one of the six PR [MICs, penicillin/AMX = 4/1]) a good correlation was observed between Emax and log10(dose/MIC) (r = 0.772; P < 0.02). A bactericidal effect equal to 3.5 log10 CFU was observed at a log10(dose/MIC) = 2. At this ratio, with the five other PR strains, Emax varied from 0.4 to 1.6 log10 CFU. In brain heart infusion medium containing AMX at 50 times the relevant MIC, these five PR strains were tolerant in vitro. Treatment failure with AMX was found in vivo, with tolerant, highly resistant strains.

In vivo efficacy of a broad-spectrum cephalosporin, ceftriaxone, against penicillin-susceptible and -resistant strains of Streptococcus pneumoniae in a mouse pneumonia model.

The increasing emergence of penicillin-resistant (Pr) strains of Streptococcus pneumoniae could pose a therapeutic problem in the next few years. Ceftriaxone (CRO), a broad-spectrum cephalosporin, exhibits a smaller increase in MICs against Pr S. pneumoniae strains than amoxicillin (AMO) (usually referred as to the "gold standard" therapy for pneumococcal infections). Therefore, we compared their respective efficacies in a leukopenic Swiss mouse model of pneumococcal pneumonia. Infection was induced with two serotype 19 strains: a penicillin-susceptible (Ps) strain (MICs of < 0.01 for penicillin, 0.03 for AMO, and 0.03 for CRO) and a Pr strain (MICs of 4 for penicillin, 2 for AMO, and 0.5 for CRO). Untreated mice died within 2 or 3 days. Against the Ps strain, the minimal protective dose (two subcutaneous injections at 12-h intervals for 3 days) for both CRO and AMO was 5 mg/kg of body weight (87% survivors). Ten-fold-increased doses of CRO (50 mg/kg) gave similar protection (75% survivors) against the Pr strain, whereas 20- and 40-fold-increased doses of AMO protected 0 and 34% of the animals, respectively, against the Ps strain. CRO had a marked and prolonged antibacterial effect in the lungs (2.7-log-unit reduction of CFU in 24 h after a single 50-mg/kg injection) against the Pr strain in comparison with AMO. A standard dosage of 50 mg of CRO per kg in mice resulted in peak levels in serum and protein binding comparable to those observed with 1 g given intravenously in humans. This dosage remained effective against a highly Pr S. pneumoniae strain in this model. The microbiological activity and pharmacodynamic and pharmacokinetic properties of CRO (time during which concentrations exceed the MIC for the test pathogen [delta t MIC], > or less than 8 h; and peak/MIC ratio, >90 for free active drug) accounted for its efficacy relative to AMO (50 mg/kg: delta t MIC, <2; peak/MIC ratio, <25) against the highly Pr S. pneumoniae strain used in this study.

Correlation between macrolide lung pharmacokinetics and therapeutic efficacy in a mouse model of pneumococcal pneumonia.

The correlation between the pharmacokinetics of erythromycin, roxithromycin, clarithromycin, spiramycin and azithromycin and their efficacy was investigated in two pneumococcal pneumonia models. Female Swiss and C57B1/6 mice were infected with Streptococcus pneumoniae strain P4241 by the intratracheal per oral route. This virulent strain produces acute pneumonia with death within 3-4 days (Swiss mice), or subacute pneumonia with death within 10 days (C57B1/6 mice) in untreated mice and the outcome of the disease is closely related to progressive weight loss. Swiss mice received three doses of each macrolide 50 mg/kg bd beginning 18 h post-infection. C57B1/6 mice received three doses of each macrolide 25 mg/kg, bd (except azithromycin was 12.5 mg/kg bd) beginning 48 h post-infection. Cure rates were evaluated on the basis of body weight variations recorded daily after the end of treatment. Pharmacokinetic parameters were determined in infected and non-infected mice after a single dose of each macrolide 50 mg/kg sc. The pharmacokinetics of azithromycin was also determined in leucopenic Swiss mice. We observed a hierarchy of in-vivo efficacy as follows: azithromycin > spiramycin = clarithromycin > roxithromycin = erythromycin which did not correlate with in-vitro MIC or MBC. The same hierarchy was found in terms of the lung T1/2. Lung T1/2s of macrolides could thus be predictive of their efficacy in respiratory tract infections. A reduced tissue AUC of azithromycin was seen in leucopenic mice suggesting leucocytes may help transport macrolides to sites of infection.