Review of the in vitro activity and potential clinical efficacy of levofloxacin in the treatment of anaerobic infections

The activity of levofloxacin against aerobic bacteria has been well documented both in vitro and clinically, but its anaerobic activity has been infrequently studied. This new fluoroquinolone exhibits good in vitro activity (MIC(S) < or =2.0 microg/mL) against many anaerobic pathogens associated with acute sinusitis, bite wounds, and other soft-tissue infections. It is less active against Bacteroides fragilis (MIC (90)=2-4 microg/mL ) and has poor inhibitory activity against non-fragilis B. fragilis group species that are associated with gastrointestinal and genitourinary tract infections. Levofloxacin does not antagonize the in vitro activity of clindamycin and metronidazole and often provides additive or synergistic activity against anaerobic bacteria with these agents. In pharmacodynamic models, levofloxacin exhibits rapid bactericidal activity at 2-4 times the MIC of anaerobic bacteria. Prolonged killing is observed when the area-under-the concentration-time-curve to MIC ratio is greater than 40. In clinical efficacy trials, levofloxacin has been effective in the treatment of patients with gynecologic, skin and skin-structure, and bone infections involving anaerobic pathogens. Both micro-biologic and pharmacodynamic studies support further evaluations of levofloxacin in the treatment of selective mixed aerobic/anaerobic infections.     

In vitro Activity of Gatifloxacin Against 238 Strains of Anaerobic Bacteria

The activity of gatifloxacin, a new 8-methoxy-fluoroquinolone, was tested against 208 pulmonary pathogens and against an additional 30 isolates of the Bacteroides fragilis group. Pulmonary isolates were from patients with documented anaerobic pleuropulmonary infections and were obtained by appropriate sampling methods. MICs were determined using the NCCLS-approved Wadsworth brucella laked blood agar method and compared to those of clindamycin, imipenem, metronidazole and trovafloxacin. Breakpoints used to define susceptible and [resistant] categories were (in μg/ml): Clindamycin-2, imipenem-4, metronidazole 8 and trovafloxacin. No breakpoint has been defined for gatifloxacin. Gatifloxacin inhibited 99% of all anaerobes tested at 4 μg/ml and 97% of all strains at 2 μg/ml. One strain of B. fragilis was resistant to gatifloxacin at 4 μg/ml; all strains of other B. fragilis group species were susceptible. One strain of Peptostreptococcus sp. was resistant to both gatifloxacin and trovafloxacin (MIC >4 μg/ml). All other strains were susceptible to all agents at ≤μg/ml. All of the non-sporeforming Gram-positive rods were susceptible to gatifloxacin at ≤μg/ml (three strains had an MIC of 4 μg/ml). Trovafloxacin had MICs of 4 μg/ml for two strains, and an MIC of 8 μg/ml for one strain. Five percent of B. fragilis, 21% of other B. fragilis group species and 20% of Clostridium species (other than C. difficile, C. perfringens or C. ramosum) were resistant to clindamycin. No imipenem resistant isolates were found in this study. Gatifloxacin appears to have excellentin vitro activity against pulmonary isolates of anaerobes and very good activity against strains of the B. fragilis group.     

Pathogenic synergy: mixed intra-abdominal infections

In this article we review our researches into the pathogenesis of mixed infections. These may conveniently be divided into in vitro and in vivo studies. In vitro we confirmed that interference with the killing of aerobes by polymorphonuclear leucocytes (PMN’s) is a property of theBacteroides strains tested and appears to depend on competition for opsonins i.e. complement factors. Further studies are in progress to define which complement factors and which bacterial structures are involved. The influence ofB. fragilis on chemotaxis has also been studied. Our preliminary data suggest thatB. fragilis is itself poorly chemotactic and reduces the chemoattractivity ofProteus mirabilis. This observation is surprising when we consider that abscess formation is the hall-mark ofB. fragilis infections and needs clarification. In vivo we have developed a skin infection model in mice which is economical and gives reproducible and quantitative results. In this model we have demonstrated pathogenic synergy betweenEscherichia coli andB. fragilis. Further studies are planned to assess the role of complement and bacterial factors in this in vivo synergy.     

Bacterial aggregation in sepsis

Previous studies have provided evidence that bacterial aggregation occurs in disease processes. In the present report, an assay system was used to demonstrate bacterial aggregation and coaggregation in vitro. The results showed that uropathogens aggregated in urine and saline and formed aggregates attached to uroepithelial cells. The presence of fimbriae and O, K, and H antigens on the cell surface did not appear to correlate with aggregation. Additional studies related to intraabdominal sepsis showed aggregation of aBacteroides fragilis strain and coaggregation betweenBacteroides fragilis andEscherichia coli, suggestive of in vivo interaction. Electron microscopy demonstrated the various aggregation reactions and was particularly effective in showing type 1 fimbriatedEscherichia coli coaggregating with theBacteroides fragilis. The ability of organisms to aggregate and coaggregate appears to have potential significance in health and disease.     

Sex- and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment

The efficacy of praziquantel against a Puerto Rican strain of Schistosoma mansoni was assessed using both in vivo and in vitro approach. The drug effective dose (50%) in the infected mouse model was about 30 times higher when determined against 28-day-old infections than against 7-week-old parasites. Single-sex female infections were also largely refractory to treatment and single-sex male infections moderately refractory, in comparison with bisexual infections. The in vitro approach consisted of overnight exposure of parasite cultures to various drug concentrations, followed by several days of culture in drug-free medium. In vitro results confirmed in vivo data and allowed for the observation of schistosome morphological phenomena after praziquantel exposure. Early worm contraction was observed in all cases, even after exposure to sub-lethal concentrations of praziquantel or upon exposure of the largely refractory 28-day-old schistosomes. In these instances, however, worms resumed movements and normal shape upon drug removal and were able to survive. The inference of these observations on the clinical use of praziquantel and on its mechanism of action is discussed.     

Comparison of Cytokine Induction by Lipopolysaccharide of Bacteroides fragilis with Salmonella typhimurium in Mice

Comparison of cytokine stimulation by lipopolysaccharide (LPS) of Bacteroides fragilis and Salmonella typhimurium was done to study the early events occurring in vivo. Mice injected intraperitoneally with either LPS demonstrated endogenous production of all the cytokines studied (tumor necrosis factor-alpha, interferon-gamma and interleukin-6) within 6 hr in the bloodstream. However induction of all the cytokines by B. fragilis LPS (50 μg/mouse) was much weaker compared with S. typhimurium LPS (50 μg/mouse). Even a dose of S. typhimurium LPS 40 times smaller (1.2 μg/mouse) induced cytokines more strongly compared with B. fragilis LPS. Thus, a weak biological response to B. fragilis LPS as evidenced by chick embryo lethality, limulus lysate gelation, LD₅₀ for mice and rabbit pyrogenicity could be due to weak induction of bioactive mediators by LPS.     

A prospective,randomized study of quadruple therapy and high-dose dual therapy for treatment of Helicobacter pylori resistant to both metronidazole and clarithromycin

Background and Aim. Failure of primary anti‐H. pylori therapy results in a high rate of antimicrobial resistance. Here, we investigated the efficacy of high‐dose dual therapy and quadruple therapy as salvage treatments for eradication of H. pylori resistant to both metronidazole and clarithromycin. Patients and Methods. Patients with at least one treatment failure and infected with H. pylori resistant to both metronidazole and clarithromycin, were randomized to receive either omeprazole 4 × 40 mg and amoxicillin 4 × 750 mg; or omeprazole 2 × 20 mg, bismuthcitrate 4 × 107 mg, metronidazole 4 × 500 mg and tetracycline 4 × 500 mg. Both regimens were given for 14 days. In cases of persistent infection, a cross‐over therapy was performed. Results. Eighty‐four patients were randomized. Cure of H. pylori infection was achieved in 31 patients after dual therapy and in 35 patients after quadruple therapy (per protocol: 83.8% (95% CI, 67.9–93.8) and 92.1% (95% CI, 78.6–98.3), respectively (p = 0.71); intention to treat: 75.6% (95% CI: 59.7–87.6) and 81.4% (95% CI: 66.6–91.6), respectively (p = 0.60)). Cross‐over therapy was performed in six of nine patients, four of whom were cured of the infection. Conclusion. Both high‐dose dual therapy and quadruple therapy are effective in curing H. pylori infection resistant to both metronidazole and clarithromycin in patients who experienced previous treatment failures.     

Rectal administration of metronidazole in severely ill patients.

Ten severely ill patients with life threatening sepsis received metronidazole as suppositories and blood concentrations of the drug were measured twice daily over five days. Therapeutic blood concentrations of metronidazole were maintained at all times in all patients. Rectal administration of metronidazole is accepted as effective prophylaxis against infection associated with surgery and as treatment of established infection. This study shows that in gravely ill patients metronidazole administered as suppositories gives perfectly adequate therapeutic serum concentrations of the drug, but that to achieve these concentrations rapidly the first suppository should be given with an intravenous loading dose.     

The in vitro and in vivo effects of metronidazole and chloroquine on Trypanosoma brucei brucei

Bloodstream forms of Trypanosoma brucei brucei were grown over baby hamster kidney cells in minimum essential medium with various concentrations of metronidazole (Flagyl) and chloroquine. Both drugs inhibited the multiplication of the parasite in vitro. The least effective concentrations for metronidazole and chloroquine were 0.003 mg/ml and 0.0024 mg/ml, respectively. Groups of 12-day-old female CDI mice were treated with 1 of the 2 drugs at 24, 48, and 72 hr after T. brucei infection. The drugs administered stat or daily reduced the number of parasites in the mice but did not effect a cure; they prolonged the survival period of the animals. However, metronidazole (0.1 mg/kg body weight) and chloroquine (0.08 mg/kg body weight) combined and given daily for 4 consecutive days cleared the infection. No trypanosomes were detected in the blood of these mice 3 mo after treatment. The dosages for both the in vitro (metronidazole 0.003 mg/ml; chloroquine 0.0024 mg/ml) and in vivo (metronidazole 0.1 mg/kg body weight; chloroquine 0.08 mg/kg body weight) were well below those prescribed for humans.     

S-thanatin in vitro prevents colistin resistance and improves its efficacy in an animal model of Pseudomonas aeruginosa sepsis

An experimental study was performed to evaluate the interaction between s-thanatin and colistin both in vitro and in vivo, using two Pseudomonas aeruginosa strains with different patterns of susceptibilities. We evaluated whether selecting for colistin-resistant P. aeruginosa could be prevented in vitro by combining colistin with s-thanatin. The strains were serially exposed in broth to twofold stepwise increasing concentrations of colistin alone or in combination with a fixed concentration [0.25× minimum inhibitory concentration (MIC)] of s-thanatin. We also performed an in vitro synergy study. For in vivo studies, a mouse model of Pseudomonas sepsis has been used. Main outcome measures were lethality and quantitative blood cultures. Exposure to colistin alone gradually selected for Pseudomonas strains with an increased MIC. In vitro studies, s-thanatin showed a positive interaction with colistin, and was able to prevent its resistance. In vivo studies, s-thanatin combined with colistin exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of this combination and provide a future therapeutic alternative in severe Pseudomonas infections.     

The antimicrobial peptide tachyplesin III coated alone and in combination with intraperitoneal piperacillin-tazobactam prevents ureteral stent Pseudomonas infection in a rat subcutaneous pouch model

We investigated the efficacy of Tachyplesin III alone or combined with piperacillin-tazobactam (TZP) to prevent biofilm formation in vitro and in a rat model of Pseudomonas aeruginosa ureteral stent infection. We have observed that in vitro TZP, in presence of Tachyplesin III, showed minimal inhibitory concentrations (MIC)s twofold and minimal bactericidal concentrations (MBC)s eightfold lower. The in vivo study showed that rats that received intraperitoneal TZP showed the lowest bacterial numbers. Tachyplesin III combined with TZP showed efficacies higher than that of each single compound. Coating ureteral stents with Tachyplesin III is able to inhibit bacterial growth up to 1000 times.     

Trypanocidal activity and selectivity in vitro of aromatic amidine compounds upon bloodstream and intracellular forms of Trypanosoma cruzi

Trypanosoma cruzi is the etiological agent of Chagas disease, an important neglected illness affecting about 12–14 million people in endemic areas of Latin America. The chemotherapy of Chagas disease is quite unsatisfactory mainly due to its poor efficacy especially during the later chronic phase and the considerable well-known side effects. These facts emphasize the need to search for find new drugs. Diamidines and related compounds are minor groove binders of DNA at AT-rich sites and present excellent anti-trypanosomal activity. In the present study, six novel aromatic amidine compounds (arylimidamides and diamidines) were tested in vitro to determine activity against the infective and intracellular stages of T. cruzi, which are responsible for sustaining the infection in the mammalian hosts. In addition, their selectivity and toxicity towards primary cultures of cardiomyocyte were evaluated since these cells represent important targets of infection and inflammation in vivo. The aromatic amidines were active against T. cruzi in vitro, the arylimidamide DB1470 was the most effective compound presenting a submicromolar LD₅₀ values, good selectivity index, and good activity at 4 °C in the presence of blood constituents. Our results further justify trypanocidal screening assays with these classes of compounds both in vitro and in vivo in experimental models of T. cruzi infection.     

In vitro Activity of Moxifloxacin Against 179 Strains of Anaerobic Bacteria Found in Pulmonary Infections

The activity of moxifloxacin (BAY 12-8039), a new 8-methoxyquinolone, was determined using the NCCLS-approved Wadsworth brucella laked blood agar method and compared to the activities of metronidazole, penicillin G, piperacillin/tazobactam and trovafloxacin. Breakpoints used to define susceptible and resistant categories were, respectively: ≤ 8 and ≥ 32 μg/mL for metronidazole, ≤ 2 and ≥ 8 μg/mL for moxifloxacin and trovafloxacin, ≤ 0.5 and ≥ 2 μg/mL for penicillin G and ≤ 32 and ≥128 μg/mL for piperacillin/tazobactam. A total of 179 anaerobic isolates from pulmonary infections were tested. Piperacillin/tazobactam was the most active antimicrobial, inhibiting 99% of strains at the susceptible breakpoint. Ninety-seven percent of these isolates were susceptible to moxifloxacin; 96% to trovafloxacin, 89% to metronidazole and 43% to penicillin G. Geometric mean moxifloxacin MIC values forBacteroides fragilis and the B. fragilis group were 0.5 and 0.8 μg/mL, respectively. Eighty-eight percent of B. fragilis and 100% of other B. fragilis group species were susceptible to both moxifloxacin and trovafloxacin. All of the strains of B. fragilis and most of the other B. fragilis group species were resistant to penicillin G. At least 99% of other Bacteroides species, Prevotella, and Fusobacterium strains were susceptible to moxifloxacin, metronidazole, piperacillin/tazobactam and trovafloxacin (88% were susceptible to trovafloxacin at 2 μg/mL and all were susceptible at 4 μg/mL). The strains of Clostridium difficile andClostridium ramosum found in these specimens were both resistant to penicillin G but susceptible to the other agents. All strains of Peptostreptococcus species were susceptible to all of the agents except penicillin G. Activities of the agents against non-spore-forming Gram-positive rods at the intermediate breakpoint were, respectively, moxifloxacin-100%, metronidazole-49%, penicillin G-86%, piperacillin/tazobactam-100%, and trovafloxacin-97%. The promising in vitro activity of moxifloxacin against anaerobic pulmonary isolates warrants further investigation, including clinical correlation studies.     

Anaerobic Bacterial Flora of Intra-abdominal Infections and their Antimicrobial Susceptibility Pattern in Kuwait

Clinical samples obtained from 200 patients with intra-abdominal infections were investigated for the presence of anaerobic bacteria. The majority of samples were from patients with appendicitis (108, 54%) followed by peritoneal abscess/peritonitis (37, 18.5%). A total of 153 anaerobes were isolated from 83 culture positive specimens with an isolation rate of 1.8 per sample. Ninety (59%) yielded Bacteroides fragilis group and B. fragilis stricto sensu accounted for half of them. Other isolates were 36 (23.5%) Prevotella species and 15 (9.8%)Peptostreptococcus micros . The susceptibility of the 153 isolates against eight antibiotics was determined by the E-test. All the isolates were susceptible to metronidazole, MIC₉₀s varying between 1–2 μg/mL. ThePrevotella spp., Peptostreptococcus spp., Fusobacterium spp. and Porphyromonas spp. were all susceptible to clindamycin (MIC₉₀s=0.25–2 μg/mL respectively), imipenem (MIC₉₀s=0.12–0.5μg/mL respectively) and meropenem (MIC₉₀=0.25 μg/mL each). About 25% of the B. fragilis group were resistant to clindamycin with MIC more than 256 μg/mL. Piperacillin-tazobactam also exhibited excellent in vitro activity against all the isolates (MIC₉₀=0.25 μg/mL).     

Molecular analysis of a gene fromBacteroides fragilis involved in metronidazole resistance inEscherichia coli

The region ofBacteroides fragilis DNA on the recombinant plasmid pMT100 responsible for conferring metronidazole resistance inEscherichia coli strains was characterized. An open reading frame (ORF1) of 195 bp encoded a protein of 64 amino acids with a predicted M_r, of 7.3 kDa. Deletion analysis indicated that ORF1 conferred the metronidazole resistance phenotype and encoded a protein with an apparent M_r of approximately 8–10 kDa.     

Experimental study on the efficacy of combination of alpha-helical antimicrobial peptides and vancomycin against Staphylococcus aureus with intermediate resistance to glycopeptides

An experimental study has been performed to compare the in vitro activity and the in vivo efficacy of magainin II and cecropin A, two alpha-helical antimicrobial peptides, and vancomycin against Staphylococcus aureus with intermediate resistance to glycopeptides. In vitro experiments included MIC determination, time-kill and synergy studies. For in vivo studies, a mouse model of staphylococcal sepsis has been used. Main outcome measures were: lethality, quantitative blood cultures and detection of TNF-alpha and interleukin-6 (IL-6) plasma levels. Combinations of alpha-helical antimicrobial peptides showed in vitro synergistic interaction. Significant increase in efficacy was also observed in vivo: combined-treated groups had significant lower bacteremia when compared to single-treated groups. Magainin II combined with vancomycin exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of these combinations and provide future therapeutic alternative in infections due to glycopeptides resistant staphylococci in the coming years.     

A case of cutaneous phaeohyphomycosis caused byExserohilum rostratum,its in vitro sensitivity and review of literature

A 40 year old woman presented with the infection of skin of 3 years duration on the upper anterior aspect of fore-arm. Histologic examination of the skin tissue revealed dematiaceous hyphae, aggregated structures or single celled elements. On detailed mycological examination the isolate was identified asExserohilum rostratum. Among the antimycotic tested in vitro amorolfine was found to be most effective with MIC value of 3 mcg/ml. This is the first report ofE. rostratum infection of man from India.     

Comparison between efficacy of allicin and fluconazole against Candida albicans in vitro and in a systemic candidiasis mouse model

The efficacy of allicin compared with fluconazole in alleviating systemic Candida albicans infections was evaluated both in vitro and in vivo through a systemic candidiasis mouse model. Determination of in vitro minimum inhibitory concentrations (MICs) for different C. albicans isolates revealed that both allicin and fluconazole showed different MICs that ranged from 0.05 to 12.5 μg mL(-1) and 0.25 to 16 μg mL(-1) , respectively. A time-kill study showed a significant effect of allicin (P<0.01) against C. albicans, comparable to that of fluconazole. Scanning electron microscopy observation revealed that, similar to fluconazole, allicin produced structural destruction of C. albicans cell surface at low MIC and lysis or puncture at high MIC concentrations. Treatment of BALB/c mice systemically infected with C. albicans showed that although the allicin treatment (at 5 mg kg(-1) day(-1) ) was slightly less efficacious than fluconazole treatment in terms of the fungal load reduction and host survival time, it was still effective against C. albicans in terms of mean survival time, which increased from 8.4 to 15.8 days. These results demonstrate the efficacy of anticandidal effects of allicin both in vitro and in an animal model of candidiasis and affirm the potential of allicin as an adjuvant therapy to fluconazole.     

Comparative efficacy of fluconazole and amphotericin B in the parenteral treatment of experimental paracoccidioidomycosis in the rat

Patients with severe and complicated paracoccidioidomycosis are treated with amphotericin B by the intravenous route. Fluconazole is active in vitro against Paracoccidioides brasiliensis and can also be administered intravenously, but few clinical or experimental data are available about its action against the infection caused by this fungus. In the present study, the efficacy of fluconazole andamphotericin B was assessed comparatively in rats inoculated parenterally with P. brasiliensis. The treatment was performed 3 times a week for 4 weeks starting one week after infection. Fluconazole administered intraperitoneally (14 mg/kg bodyweight/dose) was more effective (P > 0.001)than amphotericin B (2 mg/kg body weight/dose) in reducing the number of colony forming units in the lungs and spleen. When administered intravenously at the dose of 3 mg/kg body weight, fluconazole was as effective as amphotericin B (0.8 mg/kg body weight) in reducing the pulmonary fungal burden. Under these conditions, the rats treated with fluconazole had a smaller number of colony forming units than untreated animals (P > 0.001), but amphotericin B was more effective than fluconazole in reducing spleen infection (P > 0.005). Except for this result obtained with a low dose, fluconazole showed an antifungal action equal to or higher than that of amphotericin B. The activity of fluconazole at doses equivalent to those used for human treatment suggests that this antifungal agent may be an alternative to amphotericin B for the early intravenous treatment of patients with paracoccidioidomycosis. This revised version was published online in June 2006 with corrections to the Cover Date.