In Vitro Activities of Eight Antifungal Drugs against 55 Clinical Isolates of Fonsecaea spp.

The in vitro activities of eight antifungal drugs against clinical isolates of Fonsecaea pedrosoi (n = 21), Fonsecaea monophora (n = 25), and Fonsecaea nubica (n = 9) were tested. The resulting MIC₉₀s for all strains (n = 55) were as follows, in increasing order: posaconazole, 0.063 μg/ml; itraconazole, 0.125 μg/ml; isavuconazole, 0.25 μg/ml; voriconazole, 0.5 μg/ml; amphotericin B, 2 μg/ml; caspofungin, 2 μg/ml; anidulafungin, 2 μg/ml; and fluconazole, 32 μg/ml.Fonsecaea spp., anamorph members of the order Chaetothyriales (black yeasts and other melanized fungi), are principal agents of human chromoblastomycosis (16), a chronic cutaneous and subcutaneous infection characterized by slowly expanding skin lesions, a granulomatous immune response, and the presence of meristematic melanized muriform fungal cells in tissue scrapings (4). The last characteristic is a crucial diagnostic indicator that tends to be similar irrespective of the fungal pathogen. Chromoblastomycosis occurs worldwide in tropical and subtropical climates. Fonsecaea spp. are recoverable from environmental sources, so the disease is considered to be of traumatic origin (8, 9). The taxonomy of the genus Fonsecaea has been reviewed recently (12), and on the basis of sequence data, the following three species are recognized: Fonsecaea pedrosoi, Fonsecaea monophora, and Fonsecaea nubica. These species are morphologically identical, but their clinical spectra differ slightly: F. pedrosoi and F. nubica appear to be associated strictly with chromoblastomycosis, whereas F. monophora has also been isolated from brain abscesses, cervical lymph nodes, and bile (4, 13, 18).Therapy for chromoblastomycosis is challenging because there is no consensus regarding the treatment of choice. Several treatment options have been applied, but these tend to result in protracted disease, low cure rates, and frequent relapses (5, 9, 10, 16, 18). The therapeutic outcomes are variable and are allegedly dependent on the site of infection, lesion size, the etiological agent, and the patient''s health status (4). The specific identification of the causative pathogen is important for epidemiological reasons. The vast majority of cases of chromoblastomycosis in which the pathogen has been identified are caused by F. pedrosoi; for example, F. pedrosoi was isolated from 94% (66/69 cases) of patients with chromoblastomycosis in Sri Lanka (2) and from 98% (77/78 cases) of patients with culture-positive chromoblastomycosis in Brazil (17).The present study aimed at determining the in vitro susceptibilities of clinical isolates of Fonsecaea spp. to seven marketed antifungal drugs and the experimental 1,2,4-triazole antimycotic isavuconazole (11).Fifty-five Fonsecaea strains were obtained from the Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands) and comprised 21 F. pedrosoi strains, 25 F. monophora strains, and 9 F. nubica strains. Fifty isolates originated from patients with chromoblastomycosis, one isolate was recovered from a patient with a cerebral infection, two isolates were from diseased animals, and two isolates were clinical isolates from unknown sources. Seventeen strains came from southern China, 30 from South and Central America, and 8 from other countries (The Netherlands, Spain, Uruguay, Libya, France, United Kingdom). Strain identities were verified by sequencing the ribosomal internal transcribed spacer (ITS), tubulin (TUB1), and actin (ACT1) regions. In vitro susceptibility was determined as described in CLSI document M38-A2 (6). Briefly, the isolates were cultured on potato dextrose agar (35°C) for up to 7 days, and inocula were prepared by gently scraping the surface of the fungal colonies with a sterile cotton swab moistened with sterile physiological saline containing 0.05% Tween 40. Large particles in the cell suspensions were allowed to settle for 3 to 5 min at room temperature, and then the concentration of spores in the supernatant was adjusted spectrophotometrically (530 nm) to a percent transmission in the range 68 to 71, corresponding to 1.5 × 10⁴ to 4 × 10⁴ CFU/ml, as controlled by quantitative colony counts (6). Antifungal drugs were obtained as reagent-grade powders. The final concentrations of amphotericin B (AMB; Bristol-Myers Squibb, Woerden, The Netherlands), itraconazole (ITR; Janssen Research Foundation, Beerse, Belgium), voriconazole (VOR; Pfizer Central Research, Sandwich, United Kingdom), posaconazole (POS; Schering-Plough, Kenilworth, NJ), and caspofungin (CAS; Merck, Sharp & Dohme, Haarlem, The Netherlands) ranged from 0.016 to 16 μg/ml; the fluconazole (FLU; Pfizer) assay range was 0.063 to 64 μg/ml; and the isavuconazole (ISA; Basilea Pharmaceutica International AG, Basel, Switzerland) and anidulafungin (ANI; Pfizer) assay ranges were 0.008 to 8 μg/ml. After 72 h of incubation at 35°C, MICs and minimum effective concentrations (MECs) were determined visually by comparison of the growth in the wells containing the drug with the drug-free control. The MICs of AMB, ITR, VOR, POS, and ISA were defined as the lowest drug concentration that prevented any discernible growth (100% inhibition), whereas for FLU, the MIC was taken as the lowest concentration supporting ≥50% growth inhibition compared to the growth in the control wells. For CAS and ANI, MECs were determined microscopically as the lowest concentration of drug promoting the growth of small, round, compact hyphae relative to the appearance of the filamentous forms seen in the control wells. Quality control strains Paecilomyces variotii (ATCC 22319), Candida parapsilosis (ATCC 22019), and Candida krusei (ATCC 6258) were included in each assay run.The geometric mean MICs, MIC ranges, MIC₅₀s, and MIC₉₀s for the Fonsecaea isolates are presented in Table ​Table1.1. For each drug-species pair, the MIC₅₀ and geometric mean MIC values differed by <1 log₂ dilution step, indicating that in all cases the MIC₅₀ obtained by inspection reasonably reflected the central tendency of the antifungal susceptibility of the population. All isolates had low MICs (MIC₉₀s ≤ 0.5 μg/ml) for POS, ITR, ISA, and VOR; less active drugs (MIC₉₀s ≥ 2 μg/ml) were AMB, CAS, ANI, and FLU. There were no significant differences in the activities of the surveyed drugs against F. pedrosoi, F. monophora, and F. nubica. The MICs obtained in this study were similar to those obtained in other studies of Fonsecaea isolates (1, 3, 7, 14-16, 21).

TABLE 1.

Geometric mean MICs, MIC ranges, MIC₅₀s, and MIC₉₀s obtained by susceptibility testing of antimycotic agents against Fonsecaea isolates

Comparison of Anidulafungin MICs Determined by the Clinical and Laboratory Standards Institute Broth Microdilution Method (M27-A3 Document) and Etest for Candida Species Isolates

Anidulafungin Etest and CLSI MICs were compared for 143 Candida sp. isolates to assess essential (within 2 log₂ dilutions) and categorical agreements (according to three susceptibility breakpoints). Based on agreement percentages, our data indicated that Etest is not suitable to test anidulafungin against Candida parapsilosis and C. guilliermondii (54.4 to 82.4% essential and categorical agreements) but is more suitable for C. albicans, C. glabrata, C. krusei, and C. tropicalis (87.9 to 100% categorical agreement).The echinocandins are available for intravenous treatment of Candida infections, especially for patients with recent azole exposure (17, 26, 27). The Clinical and Laboratory Standards Institute (CLSI) has established guidelines and an interpretive susceptibility breakpoint (≤2 μg/ml) for testing echinocandins against Candida spp. (11, 12). We evaluated the suitability (essential and categorical agreements) of anidulafungin Etest MICs for 143 Candida sp. bloodstream isolates from the Hospital La Fe, Valencia, Spain. Categorical agreement was evaluated according to CLSI (11), Garcia-Effron et al. (21), and Desnos-Ollivier et al. (13, 14) susceptibility microdilution breakpoints (≤2, ≤0.5, and ≤0.25 μg/ml, respectively).The 143 isolates included caspofungin-resistant (six heterozygous and homozygous C. albicans mutants, one C. krusei isolate), caspofungin-susceptible (Table ​(Table1),1), and quality control (QC; C. parapsilosis ATCC 22019 and C. krusei ATCC 6258) isolates (8, 15, 23); anidulafungin MICs were within the established QC limits (12).

TABLE 1.

Echinocandin MICs for eight reference isolates of C. albicans and one of C. krusei determined by the CLSI M27-A3 broth microdilution and Etest methods^a

In Vitro Activity of Thimerosal against Ocular Pathogenic Fungi

The in vitro activity of thimerosal versus those of amphotericin B and natamycin was assessed against 244 ocular fungal isolates. The activity of thimerosal against Fusarium spp., Aspergillus spp., and Alternaria alternata was 256 times, 512 times, and 128 times, respectively, greater than that of natamycin and 64 times, 32 times, and 32 times, respectively, greater than that of amphotericin B. Thimerosal''s antifungal activity was significantly superior to those of amphotericin B and natamycin against ocular pathogenic fungi in vitro.The problem of keratomycosis in developing countries like China is more acute because of its higher incidence and the unavailability of effective antifungals (18, 28, 30). To date, only fluconazole and natamycin are commercially available for ocular use in China. Fluconazole has high bioavailability against Candida spp., but Fusarium spp. and Aspergillus spp. are resistant to it (3, 27, 29). Fusarium spp. and Aspergillus spp. are more commonly associated with keratomycosis, while Candida spp. are rarely implicated as etiological agents of keratomycosis in China (23, 26). Natamycin is the only topical ophthalmic antifungal compound approved in the United States (14). Natamycin is poorly soluble in water. After topical application, natamycin penetrates the cornea and conjunctiva poorly and effective drug levels are not achieved in either the cornea or the aqueous humor (15); it is therefore useful only in the treatment of superficial keratomycosis. Due to the relative unavailability of effective antifungals, keratomycosis fails to resolve in many of the patients who receive antifungal treatment; some patients experience vision loss and eventually corneal perforation, ultimately require penetrating keratoplasty, or even enucleation or evisceration (20, 28). Therefore, it is very important and urgent to explore broad-spectrum antifungals to effectively suppress a wide variety of ocular fungal pathogens and to develop new antifungal eye drops to combat this vision-threatening infection.Thimerosal is a preservative commonly used in ophthalmic solutions, otic drops, topical medicine, and vaccines because of its bactericidal property. However, the efficacy of thimerosal against ocular pathogenic fungi has not been evaluated so far. The present study was performed to determine the antifungal activity of thimerosal versus those of amphotericin B and natamycin against ocular pathogenic fungi in vitro. To our knowledge, this is the first study to determine the antifungal activity of thimerosal against main ocular pathogenic fungi. Results obtained in this study may contribute to the development of new antifungal eye drops.Two hundred forty-four strains of fungi isolated from patients with keratomycosis from the Henan Eye Institute in Zhengzhou, China, were investigated. These isolates were identified based on morphology by standard methods (22, 23, 25). They included 136 Fusarium isolates, 98 Aspergillus isolates, and 10 Alternaria alternata isolates. Candida parapsilosis ATCC 22019 was used as quality control for each test.Thimerosal (Yili Pharmaceutical Co. Ltd., Beijing, China), amphotericin B (Bristol-Myers Squibb, Princeton, NJ), and natamycin (Yinxiang Biotechnology Co. Ltd., Zhejing, China) were studied. They were all dissolved in 100% dimethyl sulfoxide. The stock solutions were prepared at concentrations of 400 μg/ml for thimerosal and 1,600 μg/ml for amphotericin B and natamycin. Drug dilutions were made in RPMI 1640 medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid. Final concentrations ranged from 0.0078 to 4 μg/ml for thimerosal and from 0.0313 to 16 μg/ml for amphotericin B and natamycin.A broth microdilution method was performed by following the Clinical and Laboratory Standards Institute M38-A2 document (13). The final inoculum was 0.4 × 10⁴ to 5 × 10⁴ CFU/ml. Following incubation at 35°C for 48 h, the MIC was determined as the lowest concentration of amphotericin B, natamycin, or thimerosal that prevented any discernible growth.The MIC range and mode, the MIC for 50% of the strains tested (MIC₅₀), and the MIC₉₀ were provided for the isolates with the SPSS statistical package (version 13.0). For calculation, any high off-scale MIC was converted to the next higher concentration.The in vitro activities of thimerosal, amphotericin B, and natamycin against the isolates are summarized in Tables ​Tables11 and ​and2.2. When comparing the MIC₉₀s of thimerosal with those of natamycin and amphotericin B, the activity of thimerosal against Fusarium spp. is 256 times greater than that of natamycin and 64 times greater than that of amphotericin B, the activity of thimerosal against Aspergillus spp. is 512 times greater than that of natamycin and 32 times greater than that of amphotericin B, and the activity of thimerosal against Alternaria alternata is 128 times greater than that of natamycin and 32 times greater than that of amphotericin B. Therefore, thimerosal''s effect was significantly superior to those of amphotericin B and natamycin against main ocular pathogenic fungi in vitro.

TABLE 1.

In vitro susceptibilities of ocular Fusarium isolates to thimerosal, amphotericin B, and natamycin

Prospective Open-Label Study of the Administration of Two-Percent Voriconazole Eye Drops

Thirteen human subjects scheduled for elective anterior segment eye surgery received hourly 2% voriconazole eye drops 4 hours presurgery. No side effects were reported. Significantly, the voriconazole concentration in the aqueous humor of the eye was similar to that reported for the 1% voriconazole solution, suggestive of concentration-independent absorption.Fungal keratitis accounts for 6 to 50% of all ocular infections and is one of the most difficult infections to treat (13). Seventy percent of fungal keratitis infections are attributed to Candida albicans, Aspergillus fumigatus, and some Fusarium species (2). At the Royal Victorian Eye and Ear Hospital (RVEEH), Melbourne, Victoria, Australia, Candida albicans is the causative fungus in 37% of the treated cases (2).Voriconazole (Vfend) is effective (Table ​(Table1)1) against common pathogens associated with fungal eye disease (i.e., Candida, Aspergillus, and Fusarium species [3, 6, 8]) and some less common keratitis-causative fungi such as Paecilomyces spp., Histoplasma spp., Scedosporium spp., Curvularia spp., and Acremonium (3, 8). The typical corneal fungal pathogens (8) are shown in Table ​Table1.1. Voriconazole is available commercially in oral and intravenous forms (Vfend package insert; Pfizer Inc., New York, NY). Case reports relating to the use of topical 1% voriconazole for ocular fungal infections are promising but limited (8). A recent study by Lau et al. reported that the concentration of voriconazole in the aqueous humor (after topical administration with 1% voriconazole solution) was sufficiently high to treat some common types of fungal infections but may be inadequate for common but less-sensitive keratitis-causative fungi (12).

TABLE 1.

In vitro MICs at which 90% of isolates are inhibited by voriconazole

Emergence of Quinolone-Resistant Bordetella pertussis in Japan

Six Bordetella pertussis strains isolated from children in Japan from 2004 to 2006 showed high-level resistance to nalidixic acid (NAL; MIC, >256 μg/ml) and decreased susceptibilities to fluoroquinolones. All of the NAL-resistant strains had the same D87G mutation in gyrA.Pertussis is an acute respiratory tract infection caused by Bordetella pertussis that is particularly serious for neonates and infants (2, 5, 19). Although the introduction of whole-cell and acellular vaccines caused a drastic decrease in the incidence of pertussis globally compared with that in the prevaccine era, developed countries have experienced a marked increase over the past 15 years (2). There has also been a recent shift in the age distribution of pertussis patients to adults and adolescents, an unrecognized but significant source of infection for neonates and infants (2, 5, 9, 19). Macrolides are widely used for antimicrobial treatment and postexposure prophylaxis of pertussis (2, 5). However, several erythromycin-resistant strains of B. pertussis have emerged in the United States (13, 14, 20) and alternative therapeutic agents are being sought to combat these strains. Several fluoroquinolones demonstrate excellent in vitro activity against B. pertussis (1, 3, 8, 11, 16, 22), and although contraindicated for children (1, 16), they might be candidate agents to treat adults with pertussis.However, as we found six nalidixic acid (NAL)-resistant B. pertussis strains isolated from 2004 to 2006 in Japan by disk diffusion test (no inhibitory zone detected around a 30-μg NAL disk on Bordet-Gengou agar), the MICs of erythromycin, NAL, norfloxacin, ciprofloxacin, sparfloxacin, levofloxacin, gatifloxacin, and chloramphenicol were determined by Etest (AB Biodisk, Solna, Sweden) on Bordet-Gengou agar (7, 10). The quality control strains used were B. pertussis CCUG 30837^T (= ATCC 9797^T) and Staphylococcus aureus ATCC 29213 (10). Strains for which the MICs of erythromycin and NAL were less than or equal to 0.12 and 32 μg/ml were considered susceptible (4, 10). Whole sequences of gyrA, gyrB, parC, and parE of the NAL-resistant strains were determined by PCR and direct sequencing. To prepare template DNA, one loopful colony of each strain was resuspended in 100 μl of 20 mM Tris-2 mM EDTA buffer (pH 7.5), incubated at 100°C for 15 min, and then centrifuged for 5 min at 23,200 × g. The 50-μl reaction mixture contained 2 μl of the supernatant fluid, 25 pmol of each primer, a 0.4 mM concentration of each deoxynucleoside triphosphate, 2.5 U of TaKaRa LA-Taq polymerase (TaKaRa, Shiga, Japan), and LA-Taq GC buffer I (TaKaRa). After an initial denaturation at 95°C for 5 min, amplification proceeded for 30 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 2 min 30 s, with a final 10-min extension at 72°C. Following cleanup with a QIAquick PCR purification column (Qiagen, Valencia, CA), the PCR product was sequenced by using a Big Dye terminator (version 3.1; Applied Biosystems, Foster City, CA). The primers used for PCR and direct sequencing are listed in Table ​Table11.

TABLE 1.

Primers used in this study

Comparative In Vitro Susceptibilities of Human Mycoplasmas and Ureaplasmas to a New Investigational Ketolide,CEM-101

MICs were determined for an investigational ketolide, CEM-101, and azithromycin, telithromycin, doxycycline, levofloxacin, clindamycin, and linezolid against 36 Mycoplasma pneumoniae, 5 Mycoplasma genitalium, 13 Mycoplasma hominis, 15 Mycoplasma fermentans, and 20 Ureaplasma isolates. All isolates, including two macrolide-resistant M. pneumoniae isolates, were inhibited by CEM-101 at ≤0.5 μg/ml, making CEM-101 the most potent compound tested.Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Mycoplasma fermentans, and Ureaplasma spp. isolates are responsible for infections in the respiratory and urogenital tracts (17, 18). Macrolides have historically been the treatments of choice for M. pneumoniae respiratory infections of adults and children because they have the advantages of being safe and well tolerated in oral formulations and of possessing antiinflammatory properties independent of their antibacterial activity and activity against other microorganisms that may cause clinically similar illness. These properties have also made macrolides attractive for empirical treatment, since mycoplasmal infections are rarely confirmed by microbiological testing. Macrolides are also active against some other Mycoplasma spp., as well as Ureaplasma spp. M. fermentans and M. hominis, however, are resistant to some members of this class, such as erythromycin, but are susceptible to clindamycin (19).During the past several years, concerns have arisen over the impact of the widespread use of macrolides on antimicrobial resistance in respiratory pathogens, such as Streptococcus pneumoniae isolates, among which 30% or more of clinical isolates are no longer susceptible to macrolides and may not respond to treatment with these drugs (7). Recent publications from Japan have confirmed the emergence in 10 to 33% of M. pneumoniae isolates of macrolide resistance that may have implications for patient outcomes (8, 10, 11, 14). These isolates typically have mutations in domain V of the 23S rRNA gene and erythromycin MICs of 32 to >64 μg/ml. A recent report from Shanghai, China, documented that 39 of 50 (78%) M. pneumoniae strains isolated there were macrolide resistant (6). Macrolide-resistant M. pneumoniae has also been reported from France (12) and the United States (20, 21). The Centers for Disease Control and Prevention recently described 3 of 11 cases (27%) of M. pneumoniae infections from an outbreak in Rhode Island that were macrolide resistant (20). We have encountered two children in Birmingham, AL, with macrolide-resistant M. pneumoniae infections of the lower respiratory tract who did not respond initially to treatment with azithromycin and required several days of hospitalization (21). Fluoroquinolone resistance has been described in genital mycoplasmas (1, 3), and tetracycline resistance may now exceed 40% in some populations (17). Azithromycin resistance associated with clinical treatment failure has also been documented in M. genitalium (4). These findings clearly indicate the need for new drug classes or improvements in drugs of existing classes for treatment of mycoplasmal and ureaplasmal infections.CEM-101 is a new ketolide with activity against many bacteria that cause respiratory and/or urogenital infections, such macrolide-resistant S. pneumoniae, chlamydiae, Haemophilus influenzae, Moraxella catarrhalis, and Neisseria gonorrhoeae (2, 5, 9, 13). To investigate further the antimicrobial spectrum of CEM-101, we studied its vitro activities against human mycoplasmas and ureaplasmas in comparison to the activities of other antimicrobial agents (Table ​(Table11).

TABLE 1.

In vitro activities of CEM-101 and other antimicrobials against Mycoplasma and Ureaplasma species isolated from humans

Pulmonary Epithelial Lining Fluid Concentrations after Use of Systemic Amphotericin B Lipid Formulations

Amphotericin B (AMB) concentrations were determined in pulmonary epithelial lining fluid (ELF) of 44 critically ill patients, who were receiving treatment with liposomal AMB (LAMB) (n = 11), AMB colloidal dispersion (ABCD) (n = 28), or AMB lipid complex (ABLC) (n = 5). Mean AMB levels (± standard errors of the means) in ELF amounted to 1.60 ± 0.58, 0.38 ± 0.07, and 1.29 ± 0.71 μg/ml in LAMB-, ABCD-, and ABLC-treated patients, respectively (differences are not significant).Invasive pulmonary mycoses exhibit a high mortality, particularly in critically ill patients (19). Amphotericin B (AMB) lipid formulations—liposomal AMB (AmBisome; Gilead) (LAMB), AMB colloidal dispersion (Amphotec [Three Rivers] and Amphocil [Torrex-Chiesi]) (ABCD), and AMB lipid complex (Abelcet; Zeneus) (ABLC)—display differences in plasma pharmacokinetics and tissue distribution (15, 26, 28). During treatment with AMB lipid formulations, AMB concentrations were investigated in epithelial lining fluid (ELF), which is a well-established model for pulmonary drug penetration (1-4, 6-10, 12, 17).This study was approved by the local ethics committee. Patients on lipid-formulated AMB requiring bronchoalveolar lavage (BAL) were enrolled (Table ​(Table1).1). AMB concentrations were assessed in 8-ml aliquots of BAL samples obtained by a standard procedure (16). BAL fluid was concentrated by evaporation, and AMB was quantified by high-performance liquid chromatography as described previously, with modifications for BAL samples (13). The concentrations were assessed by using a linear standard curve (R between 0.995 and 0.999), obtained from standards comprising 0.9% saline solution spiked with AMB. The lower detection limit of AMB in BAL fluid was 0.005 μg/ml. The assay has been found to be linear over the concentration range of 0.005 to 2.5 μg/ml for AMB in BAL fluid. The intraday and interday precisions were 3.2% and 4.7%, respectively. AMB concentrations in ELF were calculated by the urea dilution method (23), AMB_ELF = AMB_BAL × (urea_PLA/urea_BAL), where AMB_ELF is the AMB concentration in ELF, AMB_BAL is the AMB concentration in BAL fluid, urea_PLA is the urea concentration in plasma, and urea_BAL is the urea concentration in BAL fluid (23). Two ml of the BAL fluid was separated for urea quantification, which was performed using an enzymatic assay (urea/blood urea nitrogen; Roche) as with plasma.

TABLE 1.

Demographic and clinical characteristics of patients^a

Carbapenem-Resistant KPC-2-Producing Escherichia coli in a Tel Aviv Medical Center, 2005 to 2008

All of the carbapenem-resistant Escherichia coli (CREC) isolates identified in our hospital from 2005 to 2008 (n = 10) were studied. CREC isolates were multidrug resistant, all carried bla_KPC-2, and six of them were also extended-spectrum beta-lactamase producers. Pulsed-field gel electrophoresis indicated six genetic clones; within the same clone, similar transferable bla_KPC-2-containing plasmids were found whereas plasmids differed between clones. Tn4401 elements were identified in all of these plasmids.Carbapenem resistance in Escherichia coli is usually attributed to the acquisition of β-lactamases such as AmpC (14, 23, 24, 27, 31), metallo β-lactamases (4, 17, 25, 33), or KPC-type carbapenemases (2, 3, 26, 32). In 2005, KPC-2-mediated carbapenem-resistant E. coli (CREC) clinical strains were first identified in our hospital (21).The increasing prevalence of carbapenem-resistant Enterobacteriaceae in Israel (30), along with concerns regarding the emergence of highly epidemic clones, led to the study of carbapenem resistance in E. coli in our hospital. We determined CREC prevalence, elucidated the molecular mechanisms contributing to carbapenem resistance, and explored the molecular epidemiology and plasmids associated with this resistance.All of the CREC isolates identified in our hospital from February 2005 to October 2008 were included in this study. Strains were identified as resistant to at least one carbapenem using the Vitek-2 and agar dilution (MIC of imipenem or meropenem, >4 μg/ml; MIC of ertapenem, >2 μg/ml). Antibiotic susceptibilities were determined by Vitek-2 (bioMérieux Inc., Marcy l''Etoile, France), and MICs of carbapenems were determined by agar dilution according to the Clinical and Laboratory Standards Institute (CLSI) protocols (8). MICs of tigecycline and colistin and MICs of imipenem, meropenem, and ertapenem lower than 0.5 μg/ml were determined by Etest (AB Biodisk, Solna, Sweden). The criteria used for the interpretation of carbapenem MICs were based on the CLSI 2010 guidelines (9). The interpretive criterion used for tigecycline was based on FDA breakpoint values for Enterobacteriaceae that define a MIC of ≤2 as susceptible.β-Lactamases were analyzed by analytical isoelectric focusing (IEF) (16) on crude enzyme preparations from sonicated cell cultures as described elsewhere (21). The following β-lactamases were used as controls: TEM-1, pI = 5.4; TEM-26, pI = 5.6; K1, pI = 6.5; SHV-1, pI = 7.6; P99, pI = 7.8; ACT-1, pI = 9.The genetic relatedness between isolates was determined using pulsed-field gel electrophoresis (PFGE) as previously described (7). DNA macrorestriction patterns were compared according to the Dice similarity index (1.5% tolerance interval) (9a) using GelCompar II version 2.5 (Applied Maths, Kortrijk, Belgium). A PFGE clone was defined as a group of strains showing >85% banding pattern similarity (19).Multilocus sequence typing (MLST) was performed on two representative E. coli clones according to the protocol at the E. coli MLST website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi.html).Epidemiological links and potential contact between patients were analyzed using data on room location, consulting physicians, and other procedures performed during their hospitalization.PCR molecular screening of β-lactamase genes and Tn4401 elements (18) was performed using the primers listed in Table ​Table1.1. PCR products were sized on an agarose gel and sequenced using an ABI PRISM 3100 genetic analyzer (PE Biosystems). Nucleotide and deduced protein sequences were identified using the BLAST algorithm (www.ncbi.nlm.nih.gov/).

TABLE 1.

Primers used in this study

Prevalence of Fluoroquinolone Resistance among Tuberculosis Patients in Shanghai,China

We determined the prevalence of fluoroquinolone resistance among the isolates of Mycobacterium tuberculosis from 605 pulmonary tuberculosis patients in Shanghai, China. Mutations in gyrA were found in 81.5% of phenotypically fluoroquinolone-resistant isolates and were used as a molecular marker of fluoroquinolone resistance. gyrA mutations were detected in 1.9% of strains pan-susceptible to first-line drugs and 25.1% of multidrug-resistant strains. Fluoroquinolone resistance was independently associated with resistance to at least one first-line drug and prior tuberculosis treatment.Fluoroquinolones are among the most promising antibiotic drugs for tuberculosis (TB) treatment and have the potential to become part of a new first-line treatment regimen against TB (12, 17). Fluoroquinolones were introduced into clinical practice in China nearly 20 years ago and have been widely used to treat common bacterial infections, TB patients infected with Mycobacterium tuberculosis strains resistant to first-line drugs, and TB patients with severe adverse reaction to first-line agents (2, 13). Although high levels of fluoroquinolone resistance have been detected among many common bacterial pathogens (16, 19), little is known about the fluoroquinolone resistance of M. tuberculosis. A previous study reported that the risk that a TB patient would acquire fluoroquinolone resistance was correlated with the patient''s previous exposure to fluoroquinolones (14). If TB patients are infected with M. tuberculosis strains that are resistant to fluoroquinolones, it will not be possible to use fluoroquinolones in anti-TB treatment regimens.To estimate the prevalence and to identify the risk factors associated with fluoroquinolone resistance among pulmonary TB patients in Shanghai, we performed a retrospective case control study using specimens and data collected and stored in the Tuberculosis Reference Laboratory, Shanghai Municipal Center for Disease Control and Prevention. The incidence rate of pulmonary TB in Shanghai in 2005 was 39.4 per 100,000 persons. From March 2004 through November 2007, clinical isolates from 4,663 patients with pulmonary TB were collected. Drug susceptibility testing for the major first-line drugs, specifically, isoniazid (0.2 μg/ml), rifampin (rifampicin) (40 μg/ml), ethambutol (2 μg/ml), and streptomycin (4 μg/ml), were routinely performed on each isolate by using the proportion method (4). A total of 85.1% of the TB patients were infected with M. tuberculosis strains susceptible to isoniazid, rifampin, ethambutol, and streptomycin; these patients are hereafter referred to as pan-susceptible. A total of 14.9% of the strains were resistant to at least one first-line drug, and 5.6% were multidrug resistant (MDR) (18). We selected a random sample of TB patients infected with pan-susceptible strains (n = 257), a random sample of TB patients infected with a strain monoresistant to isoniazid (n = 60), and a random sample of TB patients infected with a strain that was polyresistant but not MDR (n = 77), all TB patients infected with a strain monoresistant to rifampin (n = 36), and all 175 (70.9%) strains available from 247 reported MDR-TB patients. In total, the initial clinical isolates of M. tuberculosis and sociodemographic data from 605 pulmonary TB patients were included in the study. The study was approved by the ethics committee of Fudan University.Mutations in the fluoroquinolone resistance-determining region of gyrA gene are the most important mechanism of fluoroquinolone resistance in M. tuberculosis (1, 7, 8, 11). To confirm that mutations in the fluoroquinolone resistance-determining region of gyrA can be used as a reliable molecular marker for detection of fluoroquinolone-resistant M. tuberculosis in Shanghai, we compared ofloxacin (2 μg/ml) susceptibility testing and gyrA sequencing results for 175 MDR strains. The sensitivity of gyrA mutations among 54 isolates with the ofloxacin-resistant phenotype was 81.5% (95% confidence interval [CI], 68.6% to 90.8%). The specificity of gyrA sequencing was 100% (97.5% CI, 97.0% to 100.0%). Next, we sequenced the gyrA gene of the initial isolate from 605 pulmonary TB patients (Table ​(Table1).1). The prevalence of gyrA mutations was lowest among pan-susceptible strains of M. tuberculosis (1.9%) and higher among strains that were resistant to one or more first-line drugs (17.0%), particularly MDR strains (25.1%). By univariate analysis, gyrA mutations were more likely to occur among strains resistant to first-line anti-TB drugs, especially MDR strains, than among pan-susceptible strains (Table ​(Table2).2). By multivariate logistic regression modeling, with adjustment for age, MDR was the strongest independent predictor of a gyrA mutation (Table ​(Table3).3). We tested the multivariate model for goodness of fit (P = 0.607), and interaction terms did not significantly improve the model.

TABLE 1.

Estimates of prevalence levels and 95% CIs for gyrA mutations by drug susceptibility test result

In Vitro Activities of Panduratin A against Clinical Staphylococcus Strains

In vitro antistaphylococcal activities of panduratin A, a natural chalcone compound isolated from Kaempferia pandurata Roxb, were compared to those of commonly used antimicrobials against clinical staphylococcal isolates. Panduratin A had a MIC at which 90% of bacteria were inhibited of 1 μg/ml for clinical staphylococcal isolates and generally was more potent than commonly used antimicrobials.Staphylococci are frequently refractory to many new and commonly used antimicrobial agents and have become a problem in recent years (8, 12, 17). Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) infections have emerged as a worldwide problem, and clinical strains of MRSA exhibit reduced susceptibility to antimicrobial agents (18). Moreover, coagulase-negative staphylococci are well established due to nosocomial bacteremia and indwelling medical device-associated infection, showing increased multidrug resistance (1, 14). Thus, the identification of novel agents effective in inhibiting these strains has gained renewed urgency (7). In addition, there is renewed interest in plants with antimicrobial properties as a consequence of current problems associated with the use of antibiotics (4, 9). Panduratin A, a natural chalcone compound isolated from the rhizome of fingerroot (Kaempferia pandurata Roxb.), has been reported to possess antibacterial activity against Prevotella intermedia, Prevotella loescheii, Porphyromonas gingivalis, Propionibacterium acnes, and Streptococcus mutans, as well as antibiofilm activity against multispecies oral biofilms in vitro (6, 13, 15, 16). However, antimicrobial activities of panduratin A against other pathogenic bacteria, such as staphylococci, have not yet been investigated.In this study, we compared the in vitro activities of panduratin A against MRSA, methicillin-susceptible S. aureus (MSSA), methicillin-resistant coagulase-negative staphylococci (MRCNS), and methicillin-susceptible coagulase-negative staphylococci (MSCNS) with those of treatments with available antimicrobial agents, such as ampicillin, erythromycin, gentamicin, levofloxacin, linezolid, oxacillin, tetracycline, thymol, and vancomycin.Clinical MRSA (n = 27), MSSA (n = 27), MRCNS (n = 28), and MSCNS (n = 26) were obtained from the Research Institute of Bacterial Resistance, College of Medicine, Yonsei University, South Korea. The clinical Staphylococcus strains were collected in 2008 from patients at a Korean tertiary-care hospital. The strains were isolated from body fluids, blood, genital secretions, pus, or sputum, of the patient. The species were identified by conventional methods (2) or by using the Vitek system (bioMerieux SA, Marcy l''Etoile, France) according to the manufacturer''s instructions. Reference strains S. aureus ATCC 29213 and Staphylococcus epidermidis ATCC 12228 from the American Type Culture Collection (Rockville, MD) were included as controls.Panduratin A (FIG. ​(FIG.1)1) was isolated in pure form from an ethanol extract of Kaempferia pandurata Roxb. according to the method of Park et al. (13). Panduratin A was dissolved in 10% dimethyl sulfoxide (DMSO) to obtain a 1,024-μg/ml stock solution. Ampicillin, erythromycin, gentamicin, tetracycline, thymol, and vancomycin were purchased from Sigma-Aldrich. Co. (St. Louis, MO). Levofloxacin and oxacillin were purchased from Sigma-Fluka Co. (Steinheim, Germany), and linezolid was provided by Dong-A Pharmaceutical Co. (Seoul, South Korea). Stock solutions of commercial antimicrobial agents were prepared according to the manufacturer''s instructions.Open in a separate windowFIG. 1.Structure of panduratin A.In vitro susceptibility tests were performed in a 96-well microtiter plate to determine MICs of panduratin A and other antimicrobial agents against 108 isolates of clinical staphylococci using standard broth microdilution methods with an inoculum of 5 × 10⁵ CFU/ml, according to the guidelines of CLSI standard M7-A6 (3). A twofold dilution of panduratin A stock solution or other antimicrobial agent preparation was mixed with the test organisms (5 × 10⁵ CFU/ml) in Mueller-Hinton broth (MHB) medium (Difco Becton Dickinson, Sparks, MD). Column 12 of the microtiter plate contained the highest concentrations of panduratin A or other antimicrobial agents, and column three contained the lowest concentrations of panduratin A or other antimicrobials agents. Column 2 served as the positive control for all samples (only medium and inoculum or antimicrobial agent-free wells), and column 1 was the negative control (only medium, no inoculum, and no antimicrobial agent). Microtiter plates were incubated aerobically at 37°C for 24 h. The MIC was defined as the lowest concentration of antimicrobial agent that resulted in the complete inhibition of visible growth.Panduratin A was diluted in 10% DMSO, followed by twofold dilutions in the test wells; thus, the final concentration of DMSO would be serially decreased. We examined the effect of DMSO on the growth and viability of all staphylococci tested. DMSO at ≤10% was found not to affect growth or viability of the staphylococci tested. These results suggest that DMSO had no effect on activity and that all the antimicrobial activity was due to panduratin A.Minimal bactericidal concentrations (MBCs) were determined for each antimicrobial agent per Staphylococcus strain as outlined for MICs (5). Briefly, medium (approximately 100 μl) from each well showing no visible growth was spread onto MHA (MHB supplemented with 1.5% bacterial agar) plates. Wells in column 2, the positive controls (antimicrobial agent-free wells), and wells in column 1, growth-negative controls, were included for the MBC test. Plates were incubated at 37°C for 24 h or until growth was seen in the growth-positive control plates. MBC was defined as the lowest concentration of antimicrobial agent at which all bacteria in the culture are killed or the lowest concentration at which no growth occurs on MHA plates (5, 10).Table ​Table11 shows the MICs and MBCs of panduratin A in comparison to those of ampicillin, erythromycin, gentamicin, levofloxacin, linezolid, oxacillin, tetracycline, thymol, and vancomycin for clinical staphylococci isolates. In this study, all isolates were susceptible to panduratin A, with MICs of ≤2 μg/ml. In our previous report (13), the MIC of panduratin A against P. gingivalis, P. loescheii, and S. mutans was 4 μg/ml while that of panduratin A against P. intermedia and P. acnes was 2 μg/ml (6, 13, 15). These results show that panduratin A has activities against clinical staphylococci stronger than those against P. gingivalis, P. loescheii, and S. mutans and comparable or equal to those against P. intermedia and P. acnes. Moreover, panduratin A has the capability of preventing the biofilm formation of primary multispecies oral bacteria (Actinomyces viscosus, S. mutans, and Streptococcus sanguis) in vitro (16). This report suggests that panduratin A might also have the ability to inhibit staphylococcal biofilm formation. Hence, future research is necessary to determine the inhibition activity of panduratin A against staphylococcal biofilm formation.

TABLE 1.

Comparative in vitro activities of panduratin A and other antimicrobial agents against clinical staphylococcal isolates

Comparative Efficacies of Two Antimony Regimens To Treat Leishmania braziliensis-Induced Cutaneous Leishmaniasis in Rhesus Macaques (Macaca mulatta)

This study compared the efficacies of two N-methylglucomine antimoniate (MA) dose regimens for treating macaques with Leishmania braziliensis-induced chronic skin disease. Whereas all animals treated with the full dose (20 mg MA/kg/day) were cured, 50% of the monkeys receiving a low-dose regimen (5 mg MA/kg/day) relapsed. The antimony concentrations in macaque plasma and tissue samples were greater in the full-dose group than in that receiving a subtherapeutic MA regimen. Our data also suggest the presence of drug-induced hepatic pathology.Leishmaniasis is a cause of significant morbidity and mortality throughout the world, with two million new cases of human infection worldwide each year. However, an approved vaccine to prevent leishmaniasis does not exist (7). Treatment for leishmaniasis relies largely on pentavalent antimony (Sb^v) compounds (meglumine antimoniate and sodium stibogluconate). A number of other therapeutic agents may be employed, but high costs have limited the large-scale use of the most potent drugs (4). Factors limiting the usefulness of Sb^V therapy include their adverse toxic effects (arthralgias, myalgias, cardiac arrhythmia, pancreatitis, and hepatic or renal function impairment) and the increasing occurrence of parasite resistance (2, 4). Nevertheless, responses to Sb^V vary considerably, depending on both the parasite''s intrinsic drug sensitivity and the host''s immune status (1, 4, 15). Poor clinical responses can also be attributed to inadequately dosed antimony regimens (1, 2) and problems concerning drug pharmacokinetics (PK) or biodisposal (5, 13).The Leishmania-macaque model has proven to be a valuable in vivo system for anti-infectious disease drug and vaccine development studies (7, 14). The aim of the present study was to compare the pharmacological parameters (PK, toxicity, and efficacy) of a low dose of MA with those of a standard dose of MA in macaques with L. braziliensis infection. All animal studies were performed under the guidance and with the approval of the Institutional Animal Care and Use Committee. Groups of six outbred adult rhesus macaques (Macaca mulatta) were used in this study. A high dose (10⁷ promastigotes) of virulent L. braziliensis (MHOM/BR/2000/CP13396 strain) was injected intradermally above the left upper eyelid of each monkey. The infection was allowed to proceed until the macaques reached skin disease progression. At 9 weeks postinfection, macaques received a 21-day course of a low dose (5 mg/kg/day) or a full dose (20 mg/kg/day) of MA administered through an intramuscular route. A vehicle-only-treated control group was included to assess the development of a local skin lesion caused by the infection. Animals were euthanized on days 55 (140, 142, O6, L30, M2, and O34) and 95 (S62, T32, U48, U12, U46, and X53) after the completion of treatment, and selected necropsy tissue specimens (liver, spleen, and kidneys) were removed from drug-cured and control macaques to determine residual tissue antimony concentrations and for histological examination to assess antimony-induced histopathological changes. Antimony concentrations in macaque plasma and tissues were determined by inductively coupled plasma mass spectrometry (ICP-MS) under the optimized conditions previously described (11).Although L. braziliensis-infected macaques showed a high degree of variability in lesion size (area range, 20 to 540 mm²) before antimonial therapy, the ulcerative cutaneous lesion persisted in untreated animals until the end of the observation period (Fig. ​(Fig.1).1). Treatment with both therapeutic schemes rapidly reduced the lesion size (in comparison with that of untreated lesions) after treatment. However, while complete healing was achieved in all animals receiving a regular MA schedule, three out of six monkeys treated with a low-dose regimen relapsed with the presentation of macroscopic wound inflammation after a clinical cure and wound reopening 3 to 4 months after the cessation of therapy. The concentration-time profiles of Sb in macaque plasma (Fig. ​(Fig.2)2) confirmed that drug exposure was much lower in that group (range, 11.3 to 149.3 ng Sb/g) than in macaques treated with a full-dose regimen (range, 36.4 to 150.5 ng Sb/g). Except for days 16, 23, 61, and 68, the differences in plasma Sb concentrations between the two groups were statistically significant (P < 0.001 to 0.05).Open in a separate windowFIG. 1.Response of L. braziliensis cutaneous infections in rhesus macaques to either low-dose (5 mg/kg/day; group A) or standard-dose (20 mg/kg/day; group B) MA treatment. To assess therapy, lesion size development was scored weekly following infection and treatment. All values represent the mean ± standard deviation. Before treatment, there was no statistically significant difference (P > 0.05) in mean lesion size over time between the macaque groups.Open in a separate windowFIG. 2.Time course of plasma Sb concentrations after intramuscular injection of either a low dose (group A) or a full dose (group B) of MA into L. braziliensis-infected macaques. For detection of total Sb, heparin-anticoagulated blood samples were analyzed by ICP-MS as described previously (11). *, Significant differences (P < 0.001 to 0.05) in plasma Sb concentrations between the two groups.Sustained nadir blood levels of Sb gave rise to residual drug accumulation in different soft tissues. Approximately 2 and 3 months after the last dose of MA, Sb was detected in the livers, spleens, and kidneys of treated macaques, with concentrations in renal tissue significantly lower than those found in hepatic tissue (Table ​(Table1).1). No overt clinical signs of toxicity were observed in any macaque, regardless of the administration scheme. Nevertheless, histological evidence of MA-induced hepatic injury (Fig. ​(Fig.3)3) was observed in all treated macaques. There was a clear correlation (r = 0.94; P = 0.001) between tissue Sb levels and the extent to which hepatocytes were affected. In contrast, no histopathological alterations were found in any other tissue examined.Open in a separate windowFIG. 3.Histopathology of liver samples from representative drug-cured L. braziliensis-infected macaques (A, L30; B, 140; C, U46; D, O34). Micrographs show focal hepatocellular acidophilic necrosis (arrows) with surrounding inflammatory infiltrates (circled). These obliterate the sinusoids and protrude into the parenchyma and are associated with fatty changes in stellate cells (arrowheads). Hypotrophy of the hepatic parenchyma at the center of the lobules (boxed) and numerous hemosiderin deposits within liver cells and Kupffer cells (inset in panel D) are also illustrated. Hematoxylin-and-eosin staining was used.

TABLE 1.

Residual concentrations of antimony found in selected necropsy tissue specimens from drug-cured L. braziliensis-infected macaques^a

Capsular Type and Antibiotic Resistance in Streptococcus agalactiae Isolates from Patients,Ranging from Newborns to the Elderly,with Invasive Infections

Streptococcus agalactiae isolates (n = 189) from patients with invasive infections were analyzed for capsular type by PCR, for antimicrobial susceptibility, and for the presence of resistance genes. In contrast to the predominance of capsular type III in children, types Ib and V were most common among adults. All 45 levofloxacin-resistant strains had two amino acid substitutions, Ser₈₁Leu in the gyrA gene and Ser₇₉Phe in the parC gene, and showed similar pulsed-field gel electrophoresis patterns.Streptococcus agalactiae (a group B streptococcus [GBS]) is the main microorganism causing meningitis and sepsis in infants and also sepsis in nonpregnant adults (12, 14).GBS infection in infants is classified as early onset, occurring in newborns within the first week of life, or late onset, developing in infants more than 1 week old, with most infections arising in the first 3 months and only extremely rarely in older infants (18). In the 1970s, morbidity and mortality from these GBS infections were high (3, 4, 9). In 1996, however, recommendations for the prevention of perinatal GBS infection were issued by the American College of Obstetricians and Gynecologists (2), the Centers for Disease Control and Prevention (7), and later also the American Academy of Pediatrics (1). As a result, preventive efforts increased and the incidence of early-onset disease decreased substantially (6, 23). A more detailed revised guideline, based on prenatal bacterial cultures and epidemiologic studies, was recommended in 2002 (17).Recently, Phares et al. (15) reported on a 7-year epidemiologic survey of invasive GBS disease in the United States that demonstrated a significant decline in the incidence of early-onset disease in infants, contrasting with an increase in GBS disease among adults ≥65 years old.In the present paper, we describe details concerning patient age, disease, and underlying diseases associated with invasive GBS infection, as well as the capsular types, antimicrobial susceptibilities, and resistance genes of isolates in Japan.Between August 2006 and July 2007, our laboratory received 189 GBS strains from the bacteriologic laboratories of 97 medical institutions participating in the Invasive Streptococcal Disease Working Group at the 19th Annual Meeting of the Japanese Society for Clinical Microbiology. All isolates were from sterile sites: blood (n = 124), cerebrospinal fluid (n = 54), pustule fluid (n = 7), joint fluid (n = 3), and tissue (n = 1).To identify the capsular type of GBS by PCR, we used nine sets of primers from types Ia to VIII as reported by Poyart et al. (16). We also applied our newly designed dltS primers for the identification of GBS (Table ​(Table11).

TABLE 1.

Primers for PCR and sequencing for FQ resistance in S. agalactiae

In Vitro Activity of Tebipenem,a New Oral Carbapenem Antibiotic,against β-Lactamase-Nonproducing,Ampicillin-Resistant Haemophilus influenzae

In vitro activity of tebipenem, a new oral carbapenem antibiotic, against clinical Haemophilus influenzae isolates was compared with those of 8 reference agents. Isolates were classified into 6 resistance classes after PCR identification of β-lactamase genes and ftsI gene mutations. For all isolates, the minimal concentration at which 90% of isolates were inhibited was lower for tebipenem than for the reference oral antibiotics, except for cefditoren. Tebipenem also showed excellent bactericidal activity against β-lactamase-nonproducing, ampicillin-resistant isolates.Tebipenem-pivoxil (TBM-PI) is a new oral carbapenem agent whose active metabolite, tebipenem (TBM), shows broad-spectrum activity against Gram-positive and -negative bacteria, except for Pseudomonas aeruginosa (4, 7). Unlike other clinically available oral β-lactam antibiotics, TBM displays excellent activity against Streptococcus pneumoniae strains, including penicillin-resistant strains (5). In consideration of these characteristics, the clinical efficacy of TBM-PI for treatment of community-acquired pediatric infections such as pneumonia, acute otitis media (AOM), and sinusitis was investigated. In a phase III clinical study of AOM, the efficacy of TBM-PI (3.5 to 5 mg/kg twice a day [b.i.d.]) was equal to that of high-dose cefditoren-pivoxil (CDN-PI; 4.2 to 6 mg/kg three times a day [t.i.d.]) (11). Among the important pathogens causing pneumonia and AOM, the antimicrobial characteristics of TBM against Haemophilus influenzae are not yet verified. Furthermore, β-lactamase-nonproducing, ampicillin-resistant (BLNAR) strains are increasing in Japan and various other countries (9). In the present study, we evaluated in vitro antibacterial and bactericidal activities of TBM and reference agents against H. influenzae strains, including BLNAR strains.A total of 232 H. influenzae strains were collected between October 2005 and December 2008 from pediatric patients at random. The details of the strains were as follows: 112 were obtained from tympanic effusions in patients with AOM, 30 were obtained from patients with pneumonia, and 90 were obtained from cerebrospinal fluid samples collected from patients with meningitis. To clarify the genetic background as related to β-lactam resistance, we used PCR identification to place H. influenzae strains in 1 of 6 genetic resistance classes (classes indicated with a “g” prefix) (Table ​(Table1)1) (2). Susceptibility testing was performed using an agar dilution method (3). Muller-Hinton (MH) agar (Becton Dickinson, Sparks, MD) with 0.5% yeast extract, 2% defibrinated, heat-treated horse blood, and 15 μg of β-NAD⁺ per ml (subsequently referred to as “the 3 supplements”) was used. Plates were examined after incubation at 37°C in a 5% CO₂ atmosphere for 20 h. Table ​Table22 shows the MIC ranges, MIC₅₀s, and MIC₉₀s of TBM and 8 reference antibiotics against H. influenzae strains in each of the 6 genetic resistance classes (total n = 232). Antimicrobial activities of TBM for genetically β-lactamase-nonproducing, ampicillin-susceptible (gBLNAS), genetically low β-lactamase-nonproducing, ampicillin-resistant (gLow-BLNAR), and genetically β-lactamase-nonproducing, ampicillin-resistant (gBLNAR) strains were essentially equal to those of meropenem. The increased ratios of MIC₉₀s between gBLNAS and gBLNAR strains for TBM, cefditoren, and meropenem were lower (4 to 8 times) than those for cefotaxime, amoxicillin, and cefdinir (32 to 64 times). The susceptibility distribution of the 232 H. influenzae strains to TBM according to resistance class is shown in Fig. ​Fig.1.1. The MIC peaks of TBM against gBLNAS and gBLNAR strains were 0.063 and 0.5 μg/ml, respectively. Antimicrobial activities of cephalosporins, except for cefditoren, were greatly decreased by amino acid substitutions in penicillin-binding protein 3 (PBP3) of BLNAR strains (9). The reason why TBM was affected relatively little by these amino acid substitutions appears to involve the binding affinities of TBM for PBPs; TBM was found to bind not only to PBP3 but also to PBP1B, PBP2, and PBP4 (10).Open in a separate windowFIG. 1.Distribution of MICs of TBM for clinical isolates of H. influenzae (n = 232) classified into groups consisting of gBLNAS, genetically β-lactamase-producing ampicillin-resistant (gBLPAR), gLow-BLNAR, gBLNAR, genetically β-lactamase-producing, amoxicillin-clavulanic acid-resistant I (gBLPACR-I), and gBLPACR-II strains (“I” and “II” indicate different substitutions in PBP3) (Table ​(Table11).

TABLE 1.

Identification of 6 resistance classes based on PCR

Potency and Bactericidal Activity of Iclaprim against Recent Clinical Gram-Positive Isolates

The in vitro activity of iclaprim, a novel diaminopyrimidine derivative, was evaluated against 5,937 recent gram-positive clinical isolates collected in the United States and Europe. Iclaprim demonstrated potent activity against Staphylococcus aureus (including methicillin-resistant S. aureus [MRSA]), beta-hemolytic Streptococcus spp., and Enterococcus faecalis strains tested. In addition, iclaprim exhibited bactericidal activity against all S. aureus strains tested, including MRSA.Staphylococcus aureus strains, including methicillin-resistant S. aureus (MRSA) strains, beta-hemolytic streptococci (most commonly Streptococcus pyogenes and Streptococcus agalactiae), and Enterococcus spp. are the principal gram-positive pathogens responsible for complicated skin and skin-structure infections (cSSSI). The increasing prevalence of MRSA in hospital and community settings (5, 20), as well as the potential for resistance or emergence of resistance during therapy to drugs such as vancomycin (1, 19), linezolid (9), and daptomycin (12), underscores the urgent need for additional well-differentiated therapeutic agents (17). Iclaprim is a new-generation diaminopyrimidine that potently and selectively inhibits bacterial dihydrofolate reductase (DHFR) (11, 18, 21). It was designed by a rational drug design approach based on structural information available on trimethoprim (TMP), the best known compound of the diaminopyrimidine class which, either alone or in its synergistic 1:19 combination with sulfamethoxazole, has been widely used in medical practice for over four decades. Iclaprim has been shown to have very potent activity against gram-positive bacteria that are susceptible to TMP. Iclaprim inhibits bacterial DHFR in a similar manner to TMP but possesses higher affinity due to increased hydrophilic interactions between iclaprim and DHFR (15). Thus, iclaprim may retain activity against some TMP-resistant isolates, but it would be lower than that against TMP-susceptible strains.Unlike TMP, the spectrum of activity of iclaprim is more focused against gram-positive pathogens, including MRSA strains. Iclaprim has been shown to be rapidly bactericidal against these strains and to possess a low potential for resistance development when used on its own, without the synergistic combination of a sulfonamide agent (8). For these reasons, iclaprim is under development as a monotherapy, and an intravenous formulation of iclaprim has completed two phase 3 trials for the treatment of cSSSI caused by gram-positive pathogens (11, 16). In addition, an oral formulation of iclaprim as a step-down therapy for patients with cSSSI is ongoing (16).We evaluated the potency and bactericidal activity of iclaprim against a large collection of contemporary gram-positive isolates from hospitalized patients in the United States (26 centers), the European Union (22 centers), Israel (1 center), and Turkey (1 center) from 2004 to 2006. In total, 5,937 clinical isolates representative of predominant gram-positive pathogens in cSSSI were tested. All organisms were collected from skin and soft tissue, bloodstream, and respiratory clinical specimens. The numbers of individual strains for each species tested are shown in Tables ​Tables11 and ​and2.2. Susceptibility testing by broth microdilution, including the appropriate quality controls, was performed according to the documents CLSI M7-A7 (2) and CLSI M100-S18 (3). Antimicrobial agents tested included iclaprim (Arpida Ltd., Reinach, Switzerland) and comparators. Minimum bactericidal concentration (MBC) values were determined for 101 randomly selected strains. MBC experiments were performed by plating the broth from wells from at least five log₂ dilutions from the MIC (13, 14) onto growth medium. The lowest concentration of the antimicrobial that killed ≥99.9% of the starting test inoculum was defined as the MBC endpoint. Cidality was defined as an MBC/MIC ratio of ≤4. In all cases, the thymidine content of the test medium was assessed to ensure that no artifactual inhibition of iclaprim activity occurred (2, 6).

TABLE 1.

Activity of iclaprim and comparator agents tested against Staphylococcus aureus isolates from the United States and the European Union

Pharmacokinetics of Ertapenem following Intravenous and Subcutaneous Infusions in Patients

Steady-state pharmacokinetics of ertapenem were compared in patients after 1-g intravenous and subcutaneous (s.c.) infusions. Bioavailability was 99% ± 18% after s.c. administration, but peaks were reduced by about (43 ± 29 versus 115 ± 28 μg/ml) and times to peak were delayed. Simulations based on unbound concentrations show that time over the MIC should always be longer than 30% to 40% of the dosing interval, suggesting that s.c. infusion could be an alternative in patients with reduced vascular access.Ertapenem is a recent long-acting, parenteral carbapenem antibiotic mainly indicated in the treatment of community-acquired infections or hospital-acquired infections without suspicion of Pseudomonas or Acinetobacter (5), as an alternative to penicillin-β-lactamase inhibitor combination (10, 19, 23). The pharmacokinetics of ertapenem has been extensively described (2, 3, 6-8, 15, 16, 18, 21). Ertapenem may be administered intravenously (i.v.) or intramuscularly (i.m.) for several days (13), but for many hospitalized patients the i.m. route might be contraindicated due to anticoagulant therapy. Subcutaneous (s.c.) administration is daily safely used with drugs and fluids mostly for dehydrated elderly patients or patients in palliative care when oral or i.v. administration is impossible (20) and could then appear as an interesting alternative. Advantages for the s.c. route over the i.v. route include a similar number of or even fewer complications, cost savings, greater patient comfort, and less nursing time to start and maintain the infusion (1, 22). The aim of this study was to compare the pharmacokinetics of ertapenem at steady state following 30-min i.v. and s.c. infusions in order to determine if s.c. administration of ertapenem, which is not yet approved, could be a viable alternative for i.v. infusion in patients with limited vascular sites.The study was conducted at the University Hospital of Poitiers (France) after its approval by the local ethics committee (Region Poitou-Charentes CCPPRB, protocol no. 05.12.26). Written informed consent was obtained from each subject or their closest relative if the patient was unconscious. The study enrolled 6 adult male patients suspected of having an infection due to ertapenem-susceptible bacteria (Table ​(Table1).1). Ertapenem was the only antibiotic used, sedation was obtained with propofol and sufentanil, and no other drugs that could have been suspected of interacting with ertapenem pharmacokinetics such as vasopressors or midazolam were used. At study enrollment, patients were mechanically ventilated and exhibited a systemic inflammatory response syndrome. Infection sites justifying ertapenem administration were early-onset ventilator-associated pneumonia (n = 5) and surgical wound infection (patient no. 2). The microorganisms isolated at those infection sites were methicillin-susceptible Staphylococcus aureus (n = 2), Haemophilus influenzae (n = 1), Escherichia coli (n = 1), and Klebsiella pneumoniae (n = 2). Local tolerance was assessed during the 24 h following subcutaneous infusion by checking for erythema, pruritus, hematoma, or necrosis at the insertion site. Ertapenem (Invanz) was purchased from the pharmaceutical company Merck Sharp & Dohme-Chibret (Paris, France) as a dry powder and reconstituted in 50 ml normal saline just before being infused with a pump (Orchestra DPS; Fresenius Vial, Brezins, France). The administration sites were a central vein (i.v.) or the anterior side of a thigh (s.c.). Initially 1 g of ertapenem was administered i.v. over 30 min once daily, and blood samples for the i.v. pharmacokinetic study were collected between the 4th day and the 7th day. The next day, treatment was shifted to the s.c. route and a second series of blood samples was collected during the following 24 h for the s.c. pharmacokinetic study. Ertapenem administration was shifted back to the i.v. route until the end of therapy. Blood samples were drawn via an arterial catheter in heparinized tubes and immediately centrifuged for 10 min at 2,500 × g and 4°C to separate plasma, which was then transferred to storage vials and diluted (1:1) with a stabilizing solution consisting of 1:1 ethylene glycol and 2-(4-morpholino) ethylsulfonic acid at 0.1 mol/liter (pH 6.5). Plasma ultrafiltrates were obtained from plasma samples collected at times 0.5 h and 24 h postdosing by centrifugation with a Centrifree system (CF50A model; Amicon, Molsheim, France). Samples were stored at −80°C until analysis. Ertapenem concentrations were measured using a liquid chromatography method with tandem mass spectrometry detection (12). Within- and between-day variability of the method at various concentrations led to coefficients of variation no greater than 16.4% (n = 11) and accuracies ranging between 98.5% and 110.9%. A compartmental pharmacokinetic analysis for total plasma concentrations was conducted with WinNonLin version 4.0.1. (Pharsight Corporation, Mountain View, CA), using a two-open-compartment model with multiple zero order infusions after i.v. administrations followed by a one-open-compartment model with one zero order infusion after s.c. administration. Duration of infusion was set at a fixed value equal to 0.5 h after i.v. administrations, but estimated by the modeling after s.c. administration. A 1/y weight was used for all the analysis. Unbound concentrations were derived from measured total concentrations using a saturable two-class binding site model with one specific binding site and one nonspecific binding site, previously validated for ertapenem (7). The rate constants for specific and nonspecific binding sites were estimated from the 12 pairs of total and unbound concentrations measured at 0.5 and 24 h and using the mean concentration values of albumin (14.1 g/liter) and the remaining proteins (38.7 g/liter) characteristic of these patients. The same compartmental pharmacokinetic analysis as for total concentrations was conducted with unbound concentrations, and simulations were derived for each subject and each route of administration in order to estimate the percentage of dosing interval during which unbound concentrations would be higher than various breakpoint values, chosen as 1, 2, 4, and 8 mg/liter, in agreement with the Clinical Laboratory Standards Institute. Results are presented as means ± standard deviation (SD), and nonparametric Wilcoxon''s rank test was used for statistical comparisons, with P < 0.05 considered as significant.

TABLE 1.

Patient characteristics and individual and mean ± SD pharmacokinetic parameters based on total ertapenem concentrations measured after i.v. and s.c. 30-min infusions of ertapenem (1 g/24 h) to 6 adult patients