Susceptibilities of Legionella spp. to Newer Antimicrobials In Vitro

The in vitro activities of 13 antimicrobial agents against 30 strains of Legionella spp. were determined. Rifapentine, rifampin, and clarithromycin were the most potent agents (MICs at which 90% of isolates are inhibited [MIC₉₀s], ≤0.008 μg/ml). The ketolide HMR 3647 and the fluoroquinolones levofloxacin and BAY 12-8039 (MIC₉₀s, 0.03 to 0.06 μg/ml) were more active than erythromycin A or roxithromycin. The MIC₉₀s of dalfopristin-quinupristin and linezolid were 0.5 and 8 μg/ml, respectively. Based on class characteristics and in vitro activities, several of these agents may have potential roles in the treatment of Legionella infections.The array of antimicrobial agents useful for the treatment of serious infections caused by Legionella spp. is limited. This is in part due to the relative resistance of Legionella spp. to a variety of antimicrobial agents and to the fact that these organisms are obligate intracellular pathogens and, thus, to be effective, the drugs must be able to penetrate into phagocytic cells (22).Erythromycin, rifampin, and fluoroquinolones have proven in vitro and in vivo efficacies and are used to treat clinical Legionella infections (23, 26). Mortality is still high in those with nosocomial pneumonia, especially immunocompromised and bacteremic patients (14), so there is a need for a wider range of suitable antibiotics to treat severe Legionella infections.This study examined the in vitro activities of several newer antimicrobial agents, including a ketolide, two fluoroquinolones, two oxazolidinones, rifapentine, and dalfopristin-quinupristin, against Legionella spp., an initial step in assessing their potential usefulness as therapeutic agents.(This work was presented in part at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997 [33]).Thirty clinical isolates of Legionella spp. were tested, including 21 L. pneumophila, 3 L. longbeacheae, 2 L. bozemanii, and 2 L. dumoffii isolates and one strain each of L. micdadei and L. gormanii. Strains were referred to our collection from various sources over several years and were kept frozen at −80°C.Antimicrobial test substances and their sources are as follows. HMR 3647, erythromycin A, clarithromycin, roxithromycin, rifampin, rifapentine, and levofloxacin were gifts from Hoechst-Marion-Roussel, Romainville, France; eperezolid (U-100592) and linezolid (U-100766) were gifts from Pharmacia & Upjohn Company Laboratories, Kalamazoo, Mich.; dalfopristin-quinupristin was provided by Rhône-Poulenc Rorer Pharmaceuticals, Collegeville, Pa.; and BAY 12-8039 was a gift of Bayer Inc., West Haven, Conn. Clindamycin hydrochloride and doxycycline hydrochloride were purchased from Sigma Chemical Company, St. Louis, Mo.Agar dilution susceptibility testing was performed on the buffered starch-yeast extract (BSYE) agar medium described by Saito et al. (32). Buffered charcoal-yeast extract medium (BCYE) has been shown to impair the activities of several antimicrobial substances (i.e., macrolides, rifampin, and fluoroquinolones) in earlier studies (3, 6, 12), so this medium was only used to subcultivate strains twice after thawing them from their −80°C storage temperature and as a growth control.To prepare inocula, several colonies were taken from BCYE plates (Remel, Lenexa, Kans.) after 48 h of incubation and were suspended in sterile water to a turbidity corresponding to a 0.5 McFarland standard, which yielded a cell density of approximately 10⁸ CFU/ml. Suspensions of bacteria were then further diluted 1:10 in sterile water for the smaller inoculum. Final inocula of 10⁵ and 10⁴ CFU/spot were applied to freshly made antibiotic-containing plates with a multiprong replicator device. Between each antibiotic, antibiotic-free plates were stamped to avoid carryover, and a blood agar plate was also inoculated at the end of each run to exclude contamination by other bacteria.Plates were incubated at 35°C in ambient air and were read after 48 and 96 h. Spots yielding the growth of single colonies and those with a faint haze were considered to be negative.Table ​Table11 shows the results for the 48-h incubation time for both inocula. For most agents, a twofold increase in the MIC at which 90% of the isolates were inhibited (MIC₉₀) was observed when the plates were examined after 96 h of incubation (Table ​(Table2).2). Such results may reflect either incomplete inhibition of growth at a particular antibiotic dilution or the loss of antimicrobial potency with prolonged incubation. Subsequent comments will be directed to results of the 48-h readings. With the larger inoculum, all strains grew on BSYE agar as well as on BCYE agar, whereas with the smaller inoculum, three to six strains yielded insufficient growth on control plates and therefore were excluded from the analysis. These findings are consistent with results from other studies, which showed that BSYE agar does not support the growth of some Legionella species as well as does BCYE agar (4, 15). Table ​Table33 compares the MICs of several antimicrobial agents tested against Legionella spp., obtained in different studies using different media and methods.

TABLE 1

Comparison of in vitro activities of 13 antimicrobial agents against Legionella spp., determined at 48 h of incubation

In Vitro Antibiotic Susceptibilities of Burkholderia mallei (Causative Agent of Glanders) Determined by Broth Microdilution and E-Test

In vitro susceptibilities to 28 antibiotics were determined for 11 strains of Burkholderia mallei by the broth microdilution method. The B. mallei strains demonstrated susceptibility to aminoglycosides, macrolides, quinolones, doxycycline, piperacillin, ceftazidime, and imipenem. For comparison and evaluation, 17 antibiotic susceptibilities were also determined by the E-test. E-test values were always lower than the broth dilution values. Establishing and comparing antibiotic susceptibilities of specific B. mallei strains will provide reference information for assessing new antibiotic agents.     

DNA microarray-based detection and identification of Burkholderia mallei,Burkholderia pseudomallei and Burkholderia spp.

We developed a rapid oligonucleotide microarray assay based on genetic markers for the accurate identification and differentiation of Burkholderia (B.) mallei and Burkholderia pseudomallei, the agents of glanders and melioidosis, respectively. These two agents were clearly identified using at least 4 independent genetic markers including 16S rRNA gene, fliC, motB and also by novel species-specific target genes, identified by in silico sequence analysis. Specific hybridization signal profiles allowed the detection and differentiation of up to 10 further Burkholderia spp., including the closely related species Burkholderia thailandensis and Burkholderia-like agents, such as Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia vietnamiensis, Burkholderia ambifaria, and Burkholderia gladioli, which are often associated with cystic fibrosis (CF) lung disease. The assay was developed using the easy-to-handle and economical ArrayTube? (AT) platform. A representative strain panel comprising 44 B. mallei, 32 B. pseudomallei isolates, and various Burkholderia type strains were examined to validate the test. Assay specificity was determined by examination of 40 non-Burkholderia strains.     

In Vitro Susceptibilities of Anaerobic Bacteria to Josamycin

A total of 132 strains of anaerobic bacteria were tested for susceptibility to josamycin, using a broth dilution technique. All strains of Peptococcus species, Peptostreptococcus species, and Bacteroides fragilis were inhibited by 2 μg or less per ml. Seventy percent of these susceptible strains were also killed by 2 μg or less of josamycin per ml. However, 2 of 12 Clostridium species and 6 of 10 Fusobacterium species had minimum inhibitory concentrations of 32 μg or more per ml.     

In Vitro Activity of the New Ketolide HMR3647 in Comparison with Those of Macrolides and Pristinamycins against Enterococcus spp.

Ninety-four erythromycin-susceptible and 107 erythromycin-resistant enterococcal strains (MIC of ≥512 μg/ml) were inhibited by the ketolide HMR3647 at MICs of ≤0.007 to 0.06 and 0.03 to 8 μg/ml, respectively. Eighteen vanA-positive isolates and 29 high-level-penicillin-resistant isolates, all of them erythromycin resistant, were inhibited by HMR3647 at an MIC range of 0.015 to 4 μg/ml. The new ketolide has excellent activity against Enterococcus species.     

In Vitro Susceptibilities of 27 Rickettsiae to 13 Antimicrobials

The MICs of 13 antibiotics (doxycycline, thiamphenicol, rifampin, amoxicillin, gentamicin, co-trimoxazole, ciprofloxacin, pefloxacin, ofloxacin, erythromycin, josamycin, clarithromycin, and pristinamycin) were determined for 27 available rickettsial species or strains. We used two in vitro cell culture methods described previously: the plaque assay and the microplaque colorimetric assay. Our results confirm the susceptibilities of rickettsiae to doxycycline, thiamphenicol, and fluoroquinolones. Beta-lactams, aminoglycosides, and co-trimoxazole were not active. Typhus group rickettsiae were susceptible to all macrolides tested, whereas the spotted fever group rickettsiae, R. bellii, and R. canada were more resistant, with josamycin, a safe alternative for the treatment of Mediterranean spotted fever, being the most effective compound. Strain Bar 29, R. massiliae, R. montana, R. aeschlimannii, and R. rhipicephali, which are members of the same phylogenetic subgroup, were more resistant to rifampin than the other rickettsiae tested. Heterogeneity in susceptibility to rifampin, which we report for the first time, may explain in vivo discrepancies in the effectiveness of this antibiotic for the treatment of rickettsial diseases. We hypothesize that rifampin resistance and erythromycin susceptibility may reflect a divergence during the evolution of rickettsiae.     

In Vitro Antifungal Susceptibilities of Scopulariopsis Isolates

MICs and minimum fungicidal concentrations of amphotericin B, miconazole, itraconazole, ketoconazole, fluconazole, and flucytosine against 17 isolates of Scopulariopsis spp. were determined by a broth microdilution method. All the isolates were resistant to itraconazole, fluconazole, and flucytosine, and amphotericin B, miconazole, and ketoconazole MICs were low for only a few.     

Susceptibilities of Gram-Negative Bacteria to Combinations of Antimicrobial Agents In Vitro

Combinations of either clindamycin or erythromycin with gentamicin, colimycin, or carbenicillin were studied for their antimicrobial effects on clinical isolates of Escherichia coli, Klebsiella sp., Proteus mirabilis, Serratia marcescens, Enterobacter aerogenes, and Pseudomonas aeruginosa. No antagonistic effects of the antibiotic combinations were observed. Synergistic effects of the antibiotic combinations were noted against most strains of E. coli tested, especially when either clindamycin or erythromycin was combined with gentamicin or colimycin. Of 16 other combinations of antibiotics examined with strains of E. coli as the test organisms, only the combination of penicillin G with either gentamicin or colimycin showed consistent synergy. The marked synergy of clindamycin combined with either gentamicin or colimycin was further demonstrated by following the kinetics of the bactericidal action of the antibiotic combinations and the effects on bacterial protein synthesis.     

In Vitro Activities of Cefminox against Anaerobic Bacteria Compared with Those of Nine Other Compounds

The agar dilution MIC method was used to test the activity of cefminox, a β-lactamase-stable cephamycin, compared with those of cefoxitin, cefotetan, moxalactam, ceftizoxime, cefotiam, cefamandole, cefoperazone, clindamycin, and metronidazole against 357 anaerobes. Overall, cefminox was the most active β-lactam, with an MIC at which 50% of isolates are inhibited (MIC₅₀) of 1.0 μg/ml and an MIC₉₀ of 16.0 μg/ml. Other β-lactams were less active, with respective MIC₅₀s and MIC₉₀s of 2.0 and 64.0 μg/ml for cefoxitin, 2.0 and 128.0 μg/ml for cefotetan, 2.0 and 64.0 μg/ml for moxalactam, 4.0 and >128.0 μg/ml for ceftizoxime, 16.0 and >128.0 μg/ml for cefotiam, 8.0 and >128.0 μg/ml for cefamandole, and 4.0 and 128.0 μg/ml for cefoperazone. The clindamycin MIC₅₀ and MIC₉₀ were 0.5 and 8.0 μg/ml, respectively, and the metronidazole MIC₅₀ and MIC₉₀ were 1.0 and 4.0 μg/ml, respectively. Cefminox was especially active against Bacteroides fragilis (MIC₉₀, 2.0 μg/ml), Bacteroides thetaiotaomicron (MIC₉₀, 4.0 μg/ml), fusobacteria (MIC₉₀, 1.0 μg/ml), peptostreptococci (MIC₉₀, 2.0 μg/ml), and clostridia, including Clostridium difficile (MIC₉₀, 2.0 μg/ml). Time-kill studies performed with six representative anaerobic species revealed that at the MIC all compounds except ceftizoxime were bactericidal (99.9% killing) against all strains after 48 h. At 24 h, only cefminox and cefoxitin at 4× the MIC and cefoperazone at 8× the MIC were bactericidal against all strains. After 12 h, at the MIC all compounds except moxalactam, ceftizoxime, cefotiam, cefamandole, clindamycin, and metronidazole gave 90% killing of all strains. After 3 h, cefminox at 2× the MIC produced the most rapid effect, with 90% killing of all strains.     

In Vitro Activity and Beta-Lactamase Stability of BL-S786 Compared with Those of Other Cephalosporins

In vitro activity of BL-S786, a new parenterally semisynthetic cephalosporin, was investigated against 570 bacterial isolates. BL-S786 inhibited most Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Salmonella. It inhibited some Enterobacter and indole-positive Proteus, but it was less active against these later species than was cefamandole, cefuroxime, or cefoxitin. It was not active against Serratia marcescens, Pseudomonas aeruginosa, or Bacteroides fragilis. BL-S786 was the least active new cephalosporin tested against staphylococci and was less active than cephalothin against streptococcal species. The activity of BL-S786 was not altered by the type of assay medium nor by 50% serum. The size of the test inoculum altered the minimal inhibitory and bactericidal concentrations for inhibition of some organisms, particularly those with Richmond type I β-lactamases. BL-S786 was not hydrolyzed by the R-factor-mediated, Richmond type III β-lactamase, but it was hydrolyzed by type I β-lactamases.     

In Vitro Susceptibilities of Neisseria gonorrhoeae to Fatty Acids and Monoglycerides

The susceptibility of Neisseria gonorrhoeae to several medium-chain fatty acids and their 1-monoglycerides was tested at a short inactivation time of 1 min. The results indicate that monocaprin, a monoglyceride of capric acid (10 carbon atoms, no double bonds), causes the fastest and most effective killing of all strains of N. gonorrhoeae tested.     

In Vitro Susceptibilities of Four Bartonella bacilliformis Strains to 30 Antibiotic Compounds

We have evaluated for the first time in vitro antibiotic susceptibilities of four human strains of Bartonella bacilliformis, the agent of Carrion's disease. Our results show that B. bacilliformis, like other Bartonella species, is highly susceptible to antibiotics, including most beta-lactams, aminoglycosides, chloramphenicol, rifampin, macrolides, tetracyclines, cotrimoxazole, and fluoroquinolones.     

In Vitro Susceptibilities of Aerobic and Facultative Non-Spore-Forming Gram-Positive Bacilli to HMR 3647 (RU 66647) and 14 Other Antimicrobials

The comparative in vitro activity of the ketolide HMR 3647 (RU 66647) and those of structurally related macrolide-lincosamide-streptogramin compounds (erythromycin, roxithromycin, azithromycin, clarithromycin, josamycin, lincomycin, pristinamycin, and quinupristin-dalfopristin) as well as those of benzylpenicillin, doxycycline, vancomycin, teicoplanin, levofloxacin, and rifapentine against 247 aerobic and facultative non-spore-forming gram-positive bacilli were determined by an agar dilution method. The ketolide was active against most organisms tested except Corynebacterium striatum, coryneform CDC group I2, and Oerskovia spp. The frequency of resistance to erythromycin and other macrolides as well as that to lincomycin was high. Pristinamycin and, to a lesser extent, quinupristin-dalfopristin were very active, but resistance to these agents was present in some strains of Rhodococcus equi, Listeria spp., C. striatum, Erysipelothrix rhusiopathiae, and Oerskovia spp. HMR 3647 was very active against all erythromycin-sensitive and many erythromycin-nonsusceptible strains, especially Corynebacterium minutissimum, Corynebacterium pseudodiphtheriticum, Corynebacterium amycolatum, and Corynebacterium jeikeium. In vitro resistance to benzylpenicillin was common, but doxycycline, vancomycin, and teicoplanin were very active against most organisms tested except E. rhusiopathiae, against which glycopeptide antibiotics were not active. The in vitro activity of levofloxacin was remarkable, but resistance to this agent was common for C. amycolatum, Corynebacterium urealyticum, C. jeikeium, and Oerskovia spp. strains. Rifapentine was also very active in vitro against many organisms, but resistance to this agent was always present in E. rhusiopathiae and was very common in C. striatum and C. urealyticum.     

In Vitro Activities of Terbinafine against Cutaneous Isolates of Candida albicans and Other Pathogenic Yeasts

Terbinafine is active in vitro against a wide range of pathogenic fungi, including dermatophytes, molds, dimorphic fungi, and some yeasts, but earlier studies indicated that the drug had little activity against Candida albicans. In contrast, clinical studies have shown topical and oral terbinafine to be active in cutaneous candidiasis and Candida nail infections. In order to define the anti-Candida activity of terbinafine, we tested the drug against 350 fresh clinical isolates and additional strains by using a broth dilution assay standardized according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) M27-A assay. Terbinafine was found to have an MIC of 1 μg/ml for reference C. albicans strains. For 259 clinical isolates, the MIC at which 50% of the isolates are inhibited (MIC₅₀) of terbinafine was 1 μg/ml (fluconazole, 0.5 μg/ml), and the MIC₉₀ was 4 μg/ml (fluconazole, 1 μg/ml). Terbinafine was highly active against Candida parapsilosis (MIC₉₀, 0.125 μg/ml) and showed potentially interesting activity against isolates of Candida dubliniensis, Candida guilliermondii, Candida humicola, and Candida lusitaniae. It was not active against the Candida glabrata, Candida krusei, and Candida tropicalis isolates in this assay. Cryptococcus laurentii and Cryptococcus neoformans were highly susceptible to terbinafine, with MICs of 0.06 to 0.25 μg/ml. The NCCLS macrodilution assay provides reproducible in vitro data for terbinafine against Candida and other yeasts. The MICs for C. albicans and C. parapsilosis are compatible with the known clinical efficacy of terbinafine in cutaneous infections, while the clinical relevance of its activities against the other species has yet to be determined.     

Antibiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents

OBJECTIVES: Fifty isolates of Burkholderia pseudomallei and 15 isolates of Burkholderia mallei were tested for their susceptibilities to 35 antimicrobial agents, including agents not previously tested against these bacteria. METHODS: MICs were determined by agar dilution in Mueller-Hinton medium. RESULTS: Among the antibiotics tested, lower MICs were obtained with imipenem, ceftazidime, piperacillin, piperacillin/tazobactam, doxycycline and minocycline. Fluoroquinolones and aminoglycosides had poor activities. A single clinical isolate of B. pseudomallei was resistant to ceftazidime, co-amoxiclav and doxycycline but remained susceptible to imipenem. CONCLUSIONS: Although B. mallei MICs are often lower, the overall results underline the importance of resistance in both species. The susceptibilities measured are consistent with the current recommendations for the treatment of B. pseudomallei and B. mallei infections.     

In Vitro Evaluation of Drug Susceptibilities of Babesia divergens Isolates

The susceptibilities of three bovine and two human Babesia divergens isolates to antimicrobial agents were evaluated in vitro by a tritiated hypoxanthine incorporation assay. The MICs at which 50% of isolates are inhibited (MIC₅₀s) for mefloquine (chlorhydrate), chloroquine (sulfate), quinine (chlorhydrate), clindamycin (phosphate), pentamidine (isethionate), phenamidine (isethionate) plus oxomemazine (chlorhydrate), lincomycin (chlorhydrate monohydrate), and imidocarb (dipropionate) were determined. Except for imidocarb, the MIC₅₀s observed for the different isolates were close. Imidocarb and the combination of phenamidine plus oxomemazine exhibited the highest in vitro activity, while antimalarial agents such as mefloquine, choroquine, and quinine were inactive. Other drugs had intermediate activities. The data support further in vitro evaluation of antimicrobial agents active against B. divergens for the improvement of therapeutic strategies.     

In Vitro and In Vivo Antifungal Susceptibilities of the Mucoralean Fungus Cunninghamella

We have determined the in vitro activities of amphotericin B (AMB), voriconazole, posaconazole (PSC), itraconazole (ITC), ravuconazole, terbinafine, and caspofungin against five strains of Cunninghamella bertholletiae and four of Cunninghamella echinulata. The best activity was shown by terbinafine against both species (MIC range = 0.3 to 0.6 μg/ml) and PSC against Cunninghamella bertholletiae (MIC = 0.5 μg/ml). We have also evaluated the efficacies of PSC, ITC, and AMB in neutropenic and diabetic murine models of disseminated infection by Cunninghamella bertholletiae. PSC at 40, 60, or 80 mg/kg of body weight/day was as effective as AMB at 0.8 mg/kg/day in prolonging survival and reducing the fungal tissue burden in neutropenic mice. PSC at 80 mg/kg/day was more effective than AMB at 0.8 mg/kg/day in reducing the fungal load in brain and lung of diabetic mice. Histological studies revealed an absence of fungal elements in organs of mice treated with either AMB at 0.8 mg/kg/day or PSC at 60 or 80 mg/kg/day in both models. ITC showed limited efficacy in both models. PSC could be a therapeutic option for the treatment of systemic infections caused by Cunninghamella bertholletiae.The genus Cunninghamella in the order Mucorales encompasses filamentous fungi that are inhabitants of soil and other environments and are common laboratory contaminants. Cunninghamella bertholletiae is the only member of the genus documented to cause human infections (8), although recently the species C. echinulata was also found in clinical samples (3). Infections caused by Cunninghamella are less frequent than those produced by other genera of Mucorales, e.g., Rhizopus and Mucor, but the mortality rate is higher (76%) (5, 19). In general, infections caused by the members of Mucorales are life-threatening and devastating, requiring prompt and aggressive treatment. There are several simultaneous approaches recommended for patient management, including surgical debridement, antifungal therapy, and correction of the underlying predisposing factors, when possible (5, 20).Amphotericin B (AMB) is the drug of choice, although very few data exist on the activity of this drug against some of the less frequent genera of Mucorales, such as Cunninghamella (1, 2). In a recent study in which a large set of clinical isolates of Mucorales was tested, C. bertholletiae demonstrated the highest resistance to AMB in vitro, with only 63% of the isolates tested showing MIC values under the working interpretative breakpoints described by the CLSI (2, 6). In addition, the failure of AMB therapy has been reported in clinical cases caused by Cunninghamella (4, 15, 18, 19). Posaconazole (PSC) appears to be a promising drug for the treatment of zygomycosis, having been successfully used as salvage therapy for patients who are refractory to or intolerant of AMB (10, 23). PSC is safer than AMB and shows good in vitro activity and in vivo efficacy against some zygomycetes (1, 2, 7, 22). There are only two clinical reports on the use of PSC for the management of Cunninghamella infection (9, 16), and there are no data on the use of this drug in animal models of C. bertholletiae infections. The aim of the present study, therefore, was to evaluate the in vitro activities of seven drugs against isolates of both C. bertholletiae and C. echinulata and to assess the in vivo efficacies of AMB, itraconazole (ITC), and PSC in neutropenic and diabetic murine models of disseminated infection by five strains of C. bertholletiae.     

In Vitro Activity of Doripenem against Burkholderia pseudomallei

The MIC₅₀ and MIC₉₀ values of doripenem, determined by Etest, for 110 isolates of Burkholderia pseudomallei were 0.5 and 0.75 μg/ml, respectively. There were significant correlations between MICs determined by Etest and MICs determined by agar dilution, MICs determined by Etest and inhibition zone size, and MICs determined by agar dilution and inhibition zone size.Burkholderia pseudomallei, a gram-negative bacterium, causes a disease called melioidosis in humans and animals (13). B. pseudomallei is usually resistant to many antibiotics. Antibiotics currently recommended as therapy for melioidosis are ceftazidime, imipenem, meropenem, amoxicillin-clavulanate, cefoperazone-sulbactam, trimethoprim-sulfamethoxazole, doxycycline, and chloramphenicol (13). The development of resistance by B. pseudomallei to the aforementioned antibiotics was recognized (3, 4, 10, 11, 14); hence, a search for new agents effective against B. pseudomallei is needed.Doripenem is a new parental 1-β-methyl carbapenem. Doripenem has demonstrated activity against a wide range of gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa (7, 8). Against P. aeruginosa, doripenem exhibits rapid bactericidal activity, with two- to fourfold-lower MICs than those of meropenem. However, the activity of doripenem against B. pseudomallei has not been reported. The present study was undertaken to explore the activity of doripenem against B. pseudomallei.One hundred ten clinical isolates of B. pseudomallei from different patients were selected from our collection. All isolates of B. pseudomallei were identified by API 20NE (bioMerieux, France). These isolates are susceptible to imipenem and meropenem, according to the inhibition zone diameter criteria for P. aeruginosa (≥16 mm). In vitro susceptibilities of all isolates were determined by Etest. Thirty isolates were randomly selected for determination of the MICs for doripenem by using the agar dilution and Kirby-Bauer disk diffusion methods. Paper disks containing doripenem (10 μg per disk; Becton Dickinson), Etest strips (AB Biodisk, Sweden), and doripenem powder (Johnson & Johnson Pharmaceutical Research & Development) were used. The methodology used for susceptibility testing was direct colony suspension, according to guidelines suggested by the CLSI (2). Quality control was performed by testing the susceptibility of P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922.The MICs of doripenem for the quality control organisms, namely P. aeruginosa ATCC 27853 and E. coli ATCC 25922, were 0.125 and 0.012 μg/ml, respectively. Both values were within the reference limits. A distribution of the MICs for doripenem determined by Etest for 110 strains of B. pseudomallei is shown in Table ​Table1.1. Etest results showed doripenem MICs ranging from 0.19 to 2 μg/ml, and the MIC₅₀ and the MIC₉₀ values were 0.5 and 0.75 μg/ml, respectively. The MIC₅₀ and MIC₉₀ values of doripenem, determined by agar dilution for 30 strains of B. pseudomallei, were 1 and 1.5 μg/ml, respectively. The inhibition zone diameters of doripenem determined for 30 strains of B. pseudomallei ranged from 24 to 36 mm. There was a significant correlation between the MICs determined by Etest and the MICs determined by agar dilution (r of 0.9; P of <0.001), the MICs determined by Etest and the inhibition zone diameters (r of −0.7; P of <0.001), and the MICs determined by agar dilution and the inhibition zone diameters (r of −0.7; P of <0.001), as shown in Fig. ​Fig.11.Open in a separate windowFIG. 1.Correlations between the MICs determined by Etest and agar dilution and the inhibition zone diameters. (A) Correlation between the MICs determined by Etest and the MICs determined by agar dilution. (B) Correlation between the MICs determined by Etest and the inhibition zone diameters. (C) Correlation between the MICs determined by agar dilution and the inhibition zone diameters.

TABLE 1.

Distribution of the MICs for doripenem determined by Etest for 110 isolates of B. pseudomallei

In Vitro Activities of Hexaazatrinaphthylenes against Leishmania spp.

The in vitro activity of a novel group of compounds, hexaazatrinaphthylene derivatives, against two species of Leishmania is described in this study. These compounds showed a significant dose-dependent inhibition effect on the proliferation of the parasites, with 50% inhibitory concentrations (IC₅₀s) ranging from 1.23 to 25.05 μM against the promastigote stage and 0.5 to 0.7 μM against intracellular amastigotes. Also, a cytotoxicity assay was carried out to in order to evaluate the possible toxic effects of these compounds. Moreover, different assays were performed to determine the type of cell death induced after incubation with these compounds. The obtained results highlight the potential use of hexaazatrinaphthylene derivatives against Leishmania species, and further studies should be undertaken to establish them as novel leishmanicidal therapeutic agents.