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

In Vitro and In Vivo Activities of LCB01-0371, a New Oxazolidinone

LCB01-0371 is a new oxazolidinone with cyclic amidrazone. In vitro activity of LCB01-0371 against 624 clinical isolates was evaluated and compared with those of linezolid, vancomycin, and other antibiotics. LCB01-0371 showed good activity against Gram-positive pathogens. In vivo activity of LCB01-0371 against systemic infections in mice was also evaluated. LCB01-0371 was more active than linezolid against these systemic infections. LCB01-0371 showed bacteriostatic activity against Staphylococcus aureus.The emergence of multidrug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative staphylococci (MRCNS), penicillin-resistant Streptococcus pneumoniae (PRSP), and vancomycin-resistant enterococci (VRE), has generated worldwide concern in the medical community (11). The requirement for effective new antimicrobial agents to treat infections caused by Gram-positive organisms is becoming urgent as resistance to existing agents arises and spreads around the world.The oxazolidinones, a totally synthetic class of novel antibiotics, have strong activity against nearly all Gram-positive organisms, including those resistant to other agents (1, 10). They inhibit protein synthesis by binding to domain V of the 23S rRNA and thereby blocking formation of the initiation complex (6). Linezolid is the first member of the oxazolidinone class approved by the FDA in the United States. The success of linezolid and the occurrence of strains resistant to linezolid in clinical isolates of Enterococcus faecium (4, 5) and S. aureus (12) have inspired further efforts toward developing new oxazolidinones with improved safety and antibacterial activity.LCB01-0371 (Fig. ​(Fig.1),1), a novel oxazolidinone with cyclic amidrazone, was synthesized by LegoChem BioSciences Inc. (Daejeon, Republic of Korea). In this study, in vitro activity of LCB01-0371 was compared with those of eight different antibacterial agents against 624 clinical isolates that were collected from several general hospitals in the Republic of Korea. In vivo activity of LCB01-0371 against systemic infections in mice and time-kill studies of LCB01-0371 against S. aureus giorgio (methicillin-susceptible S. aureus [MSSA]) and S. aureus p125 (MRSA) were also investigated.Open in a separate windowFIG. 1.Chemical structure of LCB01-0371.(This study was presented in part at the 49th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 2009 [7].)In vitro MICs were determined by the 2-fold agar dilution method as described by the Clinical and Laboratory Standards Institute (CLSI) (3). Mueller-Hinton agar (MHA) medium was used for testing aerobic and facultative organisms. Streptococcus pneumoniae, Streptococcus pyogenes, and Moraxella catarrhalis were grown on Mueller-Hinton agar supplemented with 5% defibrinated sheep blood (Hanil Komed Ltd., Sungnam City, Republic of Korea). Mueller-Hinton agar supplemented with 3% Fildes enrichment (Oxoid Ltd., Basingstoke, Hampshire, England) was used for Haemophilus influenzae. Bacteria (10⁴ to 10⁵ CFU) were spotted onto plates containing the appropriate concentration of drug. Plates were incubated at 35°C for 18 h and examined for growth. The MIC was considered to be the lowest concentration that completely inhibited growth on agar plates, disregarding a single colony or a faint haze caused by the inoculum.Time-kill studies were performed by the M26-A method of the NCCLS (8). Test organisms incubated on tryptic soy agar (TSA) for 18 h at 37°C were diluted with fresh Mueller-Hinton broth to ∼10⁵ CFU/ml, and the diluted cultures were preincubated for 2 h. Each drug was added to the cultures at concentrations of 0.25×, 0.5×, 1×, 2×, 4×, and 8× MIC. Aliquots (0.1 ml) of the cultures were removed at 0, 2, 4, 6, and 24 h of incubation, and serial 10-fold dilutions were prepared in saline as needed. Drug carryover effects were reduced by 100-fold dilution of the sample with agar. The numbers of viable cells on drug-free MHA plates after 24 h of incubation were determined. The compound was considered bactericidal at the concentration that reduced the original inoculum by 3 log₁₀ CFU/ml (99.9%) at each of the time periods or considered bacteriostatic if the inoculum was reduced by ∼0 to 3 log₁₀ CFU/ml.In vivo activity of LCB01-0371 against systemic infections caused by S. aureus giorgio (MSSA), S. aureus p125 (MRSA), Enterococcus faecalis u810, S. pneumoniae ATCC 6305, and Haemophilus influenzae hd2 in mice was determined. Four-week-old male ICR mice weighing 18 to 22 g (Daehan Bio Link Co., Ltd., Eum-sung Gun, Republic of Korea) were used for the systemic infection model. They were maintained in animal rooms kept at 23 ± 2°C with 55% ± 20% relative humidity. Test organisms for infection were cultured in Mueller-Hinton agar medium (Difco) at 37°C for 18 h. For S. pneumoniae, Muller-Hinton agar medium was supplemented with 5% defibrinated sheep blood. For use as inocula, bacterial strains were suspended in 0.9% saline solution containing 5% gastric mucin (Sigma), except for S. pneumoniae, which was suspended in 0.9% saline solution. Mice were used in groups of six for each dose and were challenged intraperitoneally with a single 0.5-ml portion of the bacterial suspension, corresponding to an inoculum range of 10 to 100 times the minimal lethal dose of bacteria. Four dose levels were used for each antibiotic, depending on the in vitro antimicrobial activity of the compound. Antibiotics at various dose regimens were administered orally twice, at 1 and 4 h postinfection. Mortality was recorded for 7 days, and the median effective dose needed to protect 50% of the mice (ED₅₀) was calculated by the Probit method (2). The challenge inoculum was sufficient to kill 100% of the untreated control mice, which died within 48 h postinfection.All animal experiments were approved by the Ethics Review Committee of Handong Global University, Republic of Korea.The comparative in vitro antibacterial activities of LCB01-0371 are shown in Table ​Table1.1. The MIC₉₀ of LCB01-0371 for MSSA and MRSA was 2 μg/ml. LCB01-0371 was as active as linezolid. Against methicillin-susceptible coagulase-negative staphylococci (MSCNS) (MIC₉₀, 0.5 μg/ml) and MRCNS (MIC₉₀, 0.5 μg/ml), LCB01-0371 was at least 2-fold more active than linezolid. LCB01-0371 was equally active irrespective of whether the strains were methicillin susceptible or resistant. Against S. pneumoniae (MIC₉₀, 1 μg/ml) and S. pyogenes (MIC₉₀, 2 μg/ml), LCB01-0371 showed antibacterial activity comparable to that of linezolid. LCB01-0371 was as active as linezolid against E. faecalis (MIC₉₀, 2 μg/ml) and E. faecium (MIC₉₀, 2 μg/ml). Against VRE (MIC₉₀, 1 μg/ml), LCB01-0371 was 2-fold more active than linezolid. LCB01-0371 showed weak activity against the fastidious Gram-negative aerobes H. influenzae and M. catarrhalis. Against H. influenzae, LCB01-0371 yielded a MIC₉₀ of 16 μg/ml, while slightly better activity against M. catarrhalis (MIC₉₀, 8 μg/ml) was seen. The MIC₉₀ of LCB01-0371 against H. influenzae was 2-fold lower than that of linezolid, but the MICs of LCB01-0371 against H. influenzae and M. catarrhalis were too high for clinical efficacy.

TABLE 1.

In vitro antibacterial activities of LCB01-0371 against clinical isolates

In Vitro Chemosensitization of Plasmodium falciparum to Antimalarials by Verapamil and Probenecid

We tested the effect of probenecid and verapamil in chemosensitizing Plasmodium falciparum to 14 antimalarials using the multidrug-resistant strain V1S and the drug-sensitive 3D7. Verapamil chemosensitizes V1S to quinine and chloroquine. Interestingly, probenecid profoundly chemosensitizes V1S to piperaquine. Thus, probenecid could be used to increase piperaquine efficacy in vivo.The modulation of chloroquine (CQ) resistance with the calcium channel blocker verapamil (VPM), antipsychotic drugs, histamine receptor antagonists, and antidepressant agents among others was the first case of resistance modulation to be reported (reviewed in references 6, 8, and 28).Clinical evaluation of some of these agents has been carried out in areas where CQ resistance is moderate (22, 24-26). However, these agents have the disadvantage of being pharmacologically active, with systemic effects that may result in a variety of side effects. In addition, the minimum concentrations of these agents needed to chemosensitize parasites to CQ (usually more than 1 μM of free drug) (1, 4, 11-13) are not achievable in vivo when normal doses are used; therefore, high doses have to be used, with all of the attendant risks of toxicity. All of these limitations may explain why the reversal of CQ resistance has never attained widespread application.We have demonstrated that the uricosuric drug probenecid (PBN) chemosensitizes parasites to antifolate and CQ (16, 18). In this study, we tested the effect of PBN against the aminoquinolines CQ, piperaquine (PPRQ), primaquine (PMQ), desethylamodiaquin (DEAQ), and amodiaquin (AQ); the amino alcohols lumefantrine (LM), mefloquine (MFQ), halofantrine (HLF), and quinine (QN); the antifolates pyrimethamine (PM), chlorcycloguanil (CCG), and methotrexate (MTX); the benzonaphthyridine pyronaridine (PRN); and the sesquiterpene dihydroartemisinin (DHA). We used the chemosensitizer VPM as a comparator.CQ, PMQ, AQ, MFQ, QN, MTX, PBN, and VPM were purchased from Sigma (Poole, Dorset, United Kingdom). PPRQ was a gift from Universal Corporation Limited, Kikuyu, Kenya. DEAQ, DHA, LM, PRN, and HLF were gifts from Steve Ward, Liverpool School of Tropical Medicine, Liverpool, United Kingdom. Drugs were dissolved as suggested by manufacturers. For PBN, after dilution in dimethyl sulfoxide (500 mg/ml), a subsequent serial dilution was carried out; this consisted of fivefold dilution in absolute ethanol followed by twofold dilution in 2% sodium bicarbonate and then dilution in RPMI 1640 medium (PBN crystallizes when diluted directly from dimethyl sulfoxide to RPMI 1640 medium).We employed two reference P. falciparum laboratory strains: V1S, a multidrug-resistant strain (resistant to CQ, PM, and QN) and 3D7, a strain fully sensitive to all tested antimalarials, except LM and PMQ. Cultures were carried out in RPMI 1640 (GIBCO BRL, United Kingdom), and antimalarial activities were expressed as the drug concentration required for 50% inhibition of [³H]hypoxanthine incorporation (IC₅₀) during the 66-h assay (19).Chemosensitization consisted of testing the activity of the antimalarials in the presence of PBN and VPM at the following ratios: 0.8:0.2, 0.6:0.4, 0.4:0.6, and 0.2:0.8. Chemosensitization was measured geometrically by construction of isobolograms and algebraically, by calculating the sum of the minimum fractional inhibitory concentrations (FICs) (2). FIC values <0.5 and falling between 0.5 and 1 denote pronounced and moderate chemosensitization, respectively. FIC values >1.0 indicate the absence of chemosensitization, and antagonism is denoted by FIC values >4.Our data show that FIC of VPM/CQ, VPM/PMQ, and VPM/LM for V1S ranged between 0.5 and 0.71, a clear indication of chemosensitization or a VPM effect, though moderate (Table ​(Table1).1). The most pronounced effect was observed with DEAQ and QN, with a FIC of <0.5 (Table ​(Table1).1). No chemosensitization was observed when 3D7 was used.

TABLE 1.

In vitro analysis, using the multidrug-resistant strain V1S, of the combination of VPM and PBN with 14 antimalarial drugs^a

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

Susceptibilities of 23 Desulfovibrio Isolates from Humans

Antimicrobial susceptibilities of 23 strains of Desulfovibrio spp. were tested by Etest. Generally, Desulfovibrio spp. were highly susceptible to sulbactam-ampicillin, meropenem, clindamycin, metronidazole, and chloramphenicol: MIC₉₀s of 6, 4, 0.19, 0.25, and 8 μg/ml, respectively. In addition, these strains generally showed high MICs to piperacillin and piperacillin-tazobactam. Desulfovibrio fairfieldensis (eight strains) was the species least susceptible to most agents, especially β-lactams, and was the only species resistant to fluoroquinolones. Desulfovibrio desulfuricans strain Essex 6 isolates were less susceptible to β-lactams than D. desulfuricans strain MB isolates.Desulfovibrio spp. are gram-negative anaerobes and a type of dissimilatory sulfate-reducing bacteria. Most established species of Desulfovibrio are distributed in the environment, but some Desulfovibrio spp. reside in oral cavities and intestinal tracts of animals, including humans (1, 17). In 1996, Tee et al. first reported the isolation of Desulfovibrio species from a blood culture of a patient with cholecystitis and suggested that Desulfovibrio species might act as an opportunistic pathogen (16). Since then, several case reports that suggested Desulfovibrio may be the causative organism have been published (5, 7, 8, 10, 11, 14, 15). In the published case reports, most Desulfovibrio strains were isolated from the blood of patients who suffered from a brain abscess, appendicitis, intra-abdominal abscess, or abdominal wall abscess, while some were isolated from peritoneal fluid or the pus from abscesses of various origins. Gibson et al. have also reported that Desulfovibrio spp. might be associated with ulcerative colitis (4). Furthermore, Langendijk et al. found that some Desulfovibrio spp. may be associated with the early onset of periodontitis, rapidly progressive periodontitis, adult periodontitis, and refractory periodontitis (6).Presently, four Desulfovibrio spp. (D. fairfieldensis, D. desulfuricans, D. piger, and D. vulgaris) are recognized to be associated with humans (5, 8). They are slow growers and relatively difficult to isolate from clinical specimens by a conventional approach. Identification to the species level without molecular techniques is considerably difficult. Lozniewski et al. tested the susceptibility of 16 clinical isolates of Desulfovibrio spp. and showed the broad MIC range of some antimicrobial agents (9). However, they did not identify their isolates to the species level. Furthermore, Warren et al. tested 18 clinical Desulfovibrio isolates, which were identified to the species level; however, only scattered strains of D. desulfuricans were included in their study (18). Therefore, the information available regarding the antibiogram of human Desulfovibrio isolates is extremely limited and incomplete.Considering the present situation, it is important and necessary to obtain additional information concerning the antibiogram of Desulfovibrio spp. for the empirical treatment of anaerobic infections in which Desulfovibrio spp. might be involved. Our laboratory has collected Desulfovibrio strains from various human specimens, both clinical and nonclinical, over the past several years. After molecular identification by 16S rRNA sequencing, 13 isolates of D. desulfuricans strains Essex 6 and MB were included in our collection. Therefore, in this study, we performed the Etest to obtain additional knowledge regarding the antimicrobial susceptibilities of Desulfovibrio species.Twenty-three strains of Desulfovibrio spp. were tested. These strains were isolated from human specimens and identified by classical phenotypic and molecular methods such as 16S rRNA gene sequencing. Specimens from which isolates were obtained and the number of isolates are as follows: mucosal swab of a patient with pouchitis, 6; stool specimens,7; tongue coating, 6; appendicitis, 3; and blood culture, 1. The following four Desulfovibrio strains were used as reference strains: D. fairfieldensis ATCC 700045, D. desulfuricans Essex 6 (ATCC 29577^T), D. desulfuricans MB (ATCC 27774), and D. piger ATCC 29098^T. Bacteroides fragilis ATCC 25285^T was used as the quality control strain. A special agar medium for Desulfovibrio, which was formulated and named “Desulfovibrio agar” (DA) by the author, was also used as a susceptibility test medium. The ingredients of DA were as follows: polypeptone, 15 g; soya-peptone, 7.5 g; yeast extract, 7.5 g; beef extract, 7.5 g; l-cysteine HCl, 0.75 g; ferric ammonium citrate, 0.75 g; dextran sodium sulfate, 10 g; and agar powder, 15 g per liter of distilled water (pH 7.0). In our preliminary experiment, it was confirmed that Desulfovibrio spp. grew more rapidly on the surface of DA than on the surface of Brucella blood agar (BBA) and formed a blackish or black halo around the colonies on this medium due to H₂S production followed by FeS formation. DA was also used for a pour plate method as described below.Nine antianaerobic antimicrobial agents were used: sulbactam-ampicillin, piperacillin, piperacillin-tazobactam, cefoxitin, cefotaxime, meropenem, clindamycin, chloramphenicol, and metronidazole. Eleven non-antianaerobic antimicrobial agents were also included in this study: ampicillin, cefoperazone, sulbactam-cefoperazone, cephalothin, ceftazidime, cefpirome, erythromycin, minocycline, ciprofloxacin, levofloxacin, and sulfamethoxazole-trimethoprim.The Etest was first performed according to a commonly used protocol. Namely, the bacterial suspension was inoculated on the surface of BBA by swabbing. The bacterial suspension was adjusted to McFarland no. 1 standard solution. However, this protocol is not always appropriate for testing Desulfovibrio species because achieving confluent growth of the inoculated organisms is difficult on the surface of BBA. Then we used the DA medium. Although Desulfovibrio spp. grew better on the surface of this medium, it was still difficult to achieve confluent growth by swab inoculation. Finally, we used the pour plate method described by Wilkins et al. (19). Briefly, 100 μl of bacterial suspension (McFarland no. 1 standard) was mixed with 20 ml DA medium, which was maintained at 50°C and then poured into petri dishes. When this pour plate method was used, a clear transparent elliptical zone of growth inhibition was observed within 48 h of incubation, with another zone of blackened medium surrounding this clear zone due to H₂S formation. After 48 h of anaerobic incubation (atmosphere of 80 to 85% N₂, 5 to 10% CO₂, and 10% H₂), the MIC was read as the concentration at which the border of the elliptical zone of growth inhibition intersected the scale in the Etest strips. Results for reference strains using the DA agar method were comparable to those of the standard BBA method (data not shown).β-Lactamase production was tested using the nitrocefin hydrolysis test (Cefinase; Becton-Dickinson Co., Ltd.) according to the manufacturer''s instructions.Regardless of the species, all of the Desulfovibrio spp. tested in this study were susceptible to five antianaerobic agents with low MIC₉₀s, i.e., sulbactam-ampicillin (6 μg/ml), clindamycin (0.19 μg/ml), meropenem (4 μg/ml), metronidazole (0.75 μg/ml), and chloramphenicol (8 μg/ml). On the other hand, these strains showed high MIC₉₀s toward the other antianaerobic agents: piperacillin (>256 μg/ml), piperacillin-tazobactam (>256 μg/ml), cefoxitin (>256 μg/ml), and cefotaxime (>256 μg/ml) (Table ​(Table1).1). The high susceptibilities of Desulfovibrio spp. to clindamycin, metronidazole, chloramphenicol, and imipenem have been noted in previously published reports (9, 18). In this study, one D. fairfieldensis strain that was intermediately resistant to meropenem was recognized. With regard to the susceptibility of Desulfovibrio spp. to carbapenems, previous reports have demonstrated their uniform susceptibility to imipenem (9, 18). However, another report demonstrated that D. fairfieldensis strains were less susceptible to ertapenem, with fairly high MIC₉₀ (>32 μg/ml) and MIC₅₀ (>32 μg/ml) values (18). Further studies are required to determine the susceptibilities of D. fairfieldensis strains to an array of carbapenems, which are now very often chosen as empirical treatment for serious infections.

TABLE 1.

Susceptibilities of Desulfovibrio isolates from humans to 20 antimicrobial agents

Analysis of blaSHV Codon 238 and 240 Allele Mixtures Using Sybr Green High-Resolution Melting Analysis

Klebsiella pneumoniae isolates frequently contain complex mixtures of bla_SHV alleles. A high-resolution melting-based method for interrogating the extended-spectrum activity conferring codon 238 and 240 polymorphisms was developed. This detects minority extended-spectrum β-lactamase-encoding alleles, allows estimation of allele ratios, and discriminates between single and double mutants.High-resolution melting (HRM) analysis is showing considerable promise as a method for rapid and cost-effective interrogation of single nucleotide polymorphisms (SNPs) (5). There have been numerous reports of the successful use of HRM to discriminate homozygotes and heterozygotes in humans (5). However, the use of HRM to analyze allele mixtures that are not 1:1 and to reveal the presence of minority alleles has been little explored.The increasing prevalence of bacteria producing extended-spectrum β-lactamases (ESBLs) has become a significant problem facing health care across the world. Many ESBLs are derived from plasmid-encoded SHV or TEM β-lactamases (12-14). The conversion of a non-ESBL SHV enzyme into an ESBL is nearly always associated with a G238S (GGC→AGC) substitution (numbering according to that of Ambler et al. [1]), while a further extension of the spectrum of activity is mediated by an E240K (GAG→AAG) substitution (12). Although there are many other substitutions reported, mutations of codons 238 and 240 are by far the most significant for ESBL activity of the bla_SHV family (http://www.lahey.org/Studies/).An unusual aspect of the biology of the SHV-encoding gene, bla_SHV, is that it is present on the chromosome of most Klebsiella pneumoniae strains (2, 3). However, it is also disseminated on plasmids, and most SHV ESBLs are plasmid encoded. It appears that there have been two recent mobilizations of non-ESBL bla_SHV from the K. pneumoniae chromosome onto plasmids, the mobilized genes being bla_SHV-1 and bla_SHV-11, which differ only at codon 35 (7). The G238S mutant-encoding derivatives of these are bla_SHV-2 and bla_SHV-2a, respectively, while the G238S and E240K double-mutant-encoding derivatives are bla_SHV-5 and bla_SHV-12, respectively. These six bla_SHV variants are all abundant.The existence of both chromosome and plasmid-borne bla_SHV means that many K. pneumoniae strains harbor mixtures of bla_SHV alleles, and the ratios between alleles can vary widely (6, 8, 9). It has also been determined that the presence of a minority ESBL-encoding allele confers an ESBL-positive phenotype (9). This provides a diagnostic challenge.An allele-specific real-time PCR (sometimes known as kinetic PCR) method for interrogating the bla_SHV codon 238 and 240 SNPs has previously been reported (8, 9). This has proven effective, but it requires four separate PCRs. Also, it is difficult to calibrate for interlab comparison because of the effects of small batch-to-batch variations in primer concentration and quality (unpublished data). In contrast, the allele discrimination in an HRM-based assay takes place at the end of the PCR, so the format is inherently more robust.Here, we demonstrate a single-tube HRM-based assay for codon 238 and 240 mutations in the bla_SHV gene and its comparison with allele-specific real-time PCR. To support this assay, data analysis methods were developed for the inference of confidence limits regarding whether or not an analyte contains a mutant allele. This procedure is similar in some respects to the probe-based melting analyses of Chia et al. and Randegger and Hachler (4, 15), but it is inherently simpler and provides more information.Two isolate collections of K. pneumoniae were used in this study (Table ​(Table1).1). Isolates A1 to L1 are clinical isolates from the Princess Alexandra Hospital (PAH) in Brisbane, Australia, have previously been characterized, and contain various mixtures of bla_SHV-11, bla_SHV-2a, and bla_SHV-12 (8, 9, 11, 16). Isolates 14 to 121 are derived from the SENTRY collection (10). All strains were cultured in Luria-Bertani (LB) broth and stored in cryovials with 12% glycerol at −80°C. The isolates that are asterisked in Table ​Table11 are derivatives of the clinical isolates that have been subjected to selection for resistance to 128 μg/ml cefotaxime (8).

TABLE 1.

bla_SHV allele distributions and results from allele-specific real-time PCR and HRM assays

In Vitro Activity of Ceftaroline against 623 Diverse Strains of Anaerobic Bacteria

The in vitro activities of ceftaroline, a novel, parenteral, broad-spectrum cephalosporin, and four comparator antimicrobials were determined against anaerobic bacteria. Against Gram-positive strains, the activity of ceftaroline was similar to that of amoxicillin-clavulanate and four to eight times greater than that of ceftriaxone. Against Gram-negative organisms, ceftaroline showed good activity against β-lactamase-negative strains but not against the members of the Bacteroides fragilis group. Ceftaroline showed potent activity against a broad spectrum of anaerobes encountered in respiratory, skin, and soft tissue infections.With the continuing emergence of novel patterns of resistance to commonly used antimicrobial agents, alternative therapies are needed to treat serious infections. Ceftaroline is a novel, parenteral, broad-spectrum cephalosporin that exhibits bactericidal activity against Gram-positive organisms, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus, and multidrug-resistant Streptococcus pneumoniae (MDRSP) strains, as well as common Gram-negative pathogens (8, 12, 14, 16, 18-22). Ceftaroline is currently in development for the treatment of complicated skin and skin structure infections and community-acquired pneumonia.Anaerobic bacteria are common pathogens in a variety of pleuropulmonary infections, including aspiration pneumonia, lung abscesses, and empyema (1, 3, 6, 15). However, many laboratories do not culture for anaerobes (9), diminishing awareness of the role of anaerobes in these infections. The main anaerobic pathogens isolated from these infections include Prevotella melaninogenica (∼25%), Prevotella intermedia (∼30%), Fusobacterium species (∼39%), Gram-positive cocci (∼30%), and Veillonella species (∼35%) (7). Cephalosporins such as cefoxitin have been used for the therapy of aspiration pneumonias. Although cefoxitin is active against most respiratory anaerobes, it has poor activity against the newer resistant strains of members of the family Enterobacteriaceae and MRSA. The activity of ceftaroline against Gram-positive anaerobes is similar to that of amoxicillin-clavulanate, and non-β-lactamase-producing Gram-negative strains generally have low ceftaroline MICs (present study), suggesting that ceftaroline might have an adequate spectrum of activity for therapy for some cases of aspiration pneumonia.To investigate the broader potential of ceftaroline, we compared its in vitro activity against 623 unique clinical isolates of anaerobic bacteria representing 5 Gram-negative bacterial genera and 17 Gram-positive bacterial genera to the activities of ceftriaxone, metronidazole, clindamycin, and amoxicillin-clavulanate.The reference agar dilution procedure described in CLSI document M11-A7 was used (5). The organisms were recovered from a variety of clinical specimens and were stored at −70°C in 20% skim milk. Identification was accomplished by standard phenotypic methods or by partial 16S rRNA gene sequencing for strains that could not be identified phenotypically (13, 17). Quality control strains Bacteroides fragilis ATCC 25285, Clostridium difficile ATCC 700057, and Staphylococcus aureus ATCC 29213 were included on each day of testing.The antimicrobial agents were obtained as follows: ceftaroline was from Forest Laboratories, Inc. (New York, NY); ceftriaxone, vancomycin, and metronidazole were from Sigma-Aldrich, Inc. (St. Louis, MO); and amoxicillin and clavulanate were from GlaxoSmithKline (Research Triangle Park, NC). The agar dilution plates were prepared on the day of testing.The strains were taken from the freezer and transferred twice to ensure purity and good growth. Cell paste from 48-h cultures was suspended in brucella broth to achieve the turbidity of a 0.5 McFarland standard, and the mixture was applied to plates with a Steers replicator to deliver approximately 10⁵ CFU/spot. The plates were incubated for 44 h at 37°C in an anaerobic chamber. The MIC was the lowest concentration that completely inhibited growth or that resulted in a marked reduction in growth compared with that for the drug-free growth control (5).A summary showing the MIC range, MIC₅₀, MIC₉₀, and percent susceptibility is presented in Table ​Table1.1. The cumulative ceftaroline MIC distributions for all groups of strains are displayed in Table ​Table22.

TABLE 1.

Summary of ceftaroline and comparator agent MICs, by species or group

Comparative In Vitro Activities of Nemonoxacin (TG-873870), a Novel Nonfluorinated Quinolone,and Other Quinolones against Clinical Isolates

The in vitro antibacterial activities of nemonoxacin (TG-873870), a novel nonfluorinated quinolone, against 770 clinical isolates were investigated. Nemonoxacin (tested as its malate salt, TG-875649) showed better in vitro activity than ciprofloxacin and levofloxacin against different species of staphylococci, streptococci, and enterococci, Neisseria gonorrhoeae, and Haemophilus influenzae. The in vitro activity of TG-875649 was also comparable to or better than that of moxifloxacin against these pathogens, which included ciprofloxacin-resistant, methicillin-resistant Staphylococcus aureus and levofloxacin-resistant Streptococcus pneumoniae.Antimicrobial resistance is a global public health threat (6, 7). In Taiwan, multidrug and fluoroquinolone resistances are common in both Gram-negative and Gram-positive pathogens from inpatients as well as outpatients (3, 4, 8-10). For example, as many as 80% of nosocomial Staphylococcus aureus strains in Taiwan are methicillin-resistant S. aureus (MRSA) strains, of which 80% are fluoroquinolone resistant (9). Based on a national surveillance program of >1,200 pneumococcal isolates from recent years, the levofloxacin resistance level was 10% among non-penicillin-susceptible strains (isolates with penicillin MIC > 2 μg/ml) (3).Development of new antibiotics is one of the means of combating multidrug-resistant bacteria (6). Nemonoxacin (TG-873870) is a novel nonfluorinated quinolone, and TG-875649 is a malate salt of nemonoxacin. The chemical structure of TG-875649 is shown in Fig. ​Fig.1.1. The present study examined the in vitro antibacterial activity of TG-875649 against a spectrum of Gram-positive and Gram-negative clinical isolates in Taiwan.Open in a separate windowFIG. 1.Chemical structure of TG-875649, a malate salt of nemonoxacin (TG-873870). Me, methyl.(This study was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 17 to 20 September 2007 [5].)Nonduplicate bacterial strains were selected from a pool of clinical isolates previously studied under a national surveillance program in Taiwan (4, 9). Isolates were selected to include those with known resistance to different classes of antibiotics in addition to fluoroquinolones (levofloxacin and/or ciprofloxacin); thus, multidrug-resistant bacteria comprised a larger proportion than the wild type. A total of 770 isolates were tested, among which 688 (89.4%) were isolated in the year 2004, with the remainder isolated from 1998 to 2002. MICs were determined by the broth microdilution (BMD) method, using custom-made 96-well microtiter panels containing different antimicrobial agents at various concentrations (Trek Diagnostics, West Essex, England). The test procedure followed the instructions of the MIC panel manufacturer and guidelines of the Clinical and Laboratory Standards Institute (CLSI) (1, 2). For most species, 50 μl of the 0.5 McFarland standard organism suspension was transferred to 10 ml of cation-adjusted Mueller-Hinton broth (CAMHB) to obtain a final inoculum of 5 × 10⁵ CFU/ml at 100 μl/well. For Proteus mirabilis, a 1 × 10⁵-CFU/ml inoculum was used to avoid an inoculum effect. For fastidious organisms, 100 μl of the 0.5 McFarland standard suspension was transferred to 10 ml of MHB containing 3% lysed horse blood for Neisseria gonorrhoeae and streptococci and to 10 ml of Haemophilus Test Medium (HTM) broth for Haemophilus influenzae. All MIC plates were incubated in 35°C ambient air overnight except those for N. gonorrhoeae, which were incubated in 5% CO₂. Appropriate quality control strains were included during each test run. The CAMHB and HTM were purchased from Trek Diagnostics. All other media were purchased from BBL (Becton Dickinson Microbiology System, Cockeysville, MD).Table ​Table11 lists the antibacterial activities (MIC range, MIC₅₀, and MIC₉₀) of TG-875649 against various species of Gram-positive and Gram-negative pathogens in comparison with those of 3 fluoroquinolones (FQs) (ciprofloxacin, levofloxacin, and moxifloxacin) and nonquinolone agents. TG-875649 was more active than ciprofloxacin and levofloxacin against all staphylococcal isolates, including 150 S. aureus, with 4- to >32-fold lower MIC₉₀s. In addition, against ciprofloxacin-resistant (CIP^r) MRSA, the MIC₉₀ of TG-875649 (1 μg/ml) was 4-fold lower than that of moxifloxacin. Of the 47 CIP^r MRSA isolates, only 2 (4.3%) and 3 (6.4%) isolates had levofloxacin and moxifloxacin MICs of ≤1 μg/ml, respectively (Table ​(Table2),2), while the majority (45 isolates; 95.7%) had TG-875649 MICs of ≤1 μg/ml (Table ​(Table2).2). Among the 50 enterococci tested, 17 (7 Enterococcus faecalis strains and 10 E. faecium strains) were vancomycin resistant. The MIC₉₀s of TG-875649 were at least 2-fold lower than those of the 3 FQs for both E. faecalis and E. faecium.

TABLE 1.

Antibacterial activities of TG-875649, a malate salt of nemonoxacin (TG-873870), and reference compounds against Gram-positive and Gram-negative bacteria

Comparative In Vitro Activities of Torezolid (DA-7157) against Clinical Isolates of Aerobic and Anaerobic Bacteria in South Korea

Resistance of Gram-positive pathogens to first-line antimicrobial agents has been increasing in many parts of the world. We compared the in vitro activities of torezolid with those of other antimicrobial agents, including linezolid, against clinical isolates of major aerobic and anaerobic bacteria. Torezolid had an MIC₉₀ of ≤0.5 μg/ml for the Gram-positive bacterial isolates tested and was more potent than either linezolid or vancomycin.Antimicrobial resistance in Gram-positive cocci has become a major problem in recent years. Oxazolidinones, a new therapeutic class of synthetic drugs, are active against Gram-positive pathogens. Linezolid, the only marketed oxazolidinone, inhibits the initiation of bacterial protein translation by binding to the 23S rRNA peptidyl transferase region (15). The widely used drug linezolid is effective against most Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus spp., and penicillin-resistant Streptococcus pneumoniae (1, 2). However, several recent studies have reported the emergence of linezolid-resistant staphylococci and enterococci in Brazil, China, France, Germany, Italy, and Sweden. The dominant resistance mechanisms are mutations of the 23S rRNA gene and the recently described mobile chloramphenicol-florfenicol resistance (cfr) methyltransferase gene (9).The antibacterial activity of oxazolidinones depends on their affinity for the site of action on the ribosome. Therefore, by modifying their chemical structure, novel oxazolidinones with improved antimicrobial activity can be obtained. Accordingly, it is important to find more useful and less toxic oxazolidinones. Torezolid [TR-700, DA-7157; R-3-(4-(2-(2-methyltetrazol-5-yl)pyridine-5-yl)-3-fluorophenyl)-5-hydroxymethyl oxazolidin-2-on] is the active moiety of the prodrug torezolid phosphate (TR-701, DA-7218) (Fig. ​(Fig.1).1). In a recent study, torezolid was 4- to 8-fold more active than linezolid against Gram-positive bacteria collected from the United States (3). In another study, torezolid demonstrated an 8- to 16-fold increase in potency against all of the linezolid-resistant isolates tested, including MRSA, MRSA carrying the mobile cfr methyltransferase gene, and vancomycin-resistant enterococci (14). However, as far as we know, the activities of torezolid against anaerobic bacteria have not been reported.Open in a separate windowFIG. 1.Chemical structure of torezolid.Human plasma protein binding of torezolid was about 80% (data not shown), and the MIC was unaffected by the presence of 20% human plasma (4). Torezolid has a better pharmacokinetic profile than linezolid. After oral administration of torezolid at 200 mg once a day, the maximum concentration of the drug in serum, half-life, and area under the curve were 2.0 μg/ml, 11.2 h, and 25.4 μg·h/ml, respectively (13). In another study, torezolid phosphate was safe and effective with once-daily 200-mg dosing over 5 to 7 days of treatment for severe complicated skin and skin structure infections caused by Gram-positive bacteria (16). In this study, we compared the in vitro activities of torezolid with those of other antimicrobial agents, including linezolid, against clinical isolates of major aerobic and anaerobic Gram-positive and Gram-negative bacteria.(Part of this study was presented at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2004 [12]).Five hundred ten nonduplicate aerobic and anaerobic bacterial isolates were collected between 2002 and 2004 from patients at a South Korean tertiary-care hospital. The species were identified by conventional methods or by using either the ID 32 GN or the ATB 32A system (bioMérieux, Marcy-l''Etoile, France). Antimicrobial susceptibility was tested by the CLSI agar dilution method (5, 6, 7). The media used were Mueller-Hinton agar (Becton Dickinson, Sparks, MD) for testing of Staphylococcus spp., Enterococcus spp., and Moraxella catarrhalis; Mueller-Hinton agar supplemented with 5% sheep blood for Streptococcus spp.; Haemophilus test medium for Haemophilus influenzae; and brucella agar (Becton Dickinson) supplemented with 5 μg hemin, 1 μg vitamin K₁ per ml, and 5% laked sheep blood for anaerobic bacteria.The antimicrobial agents used were torezolid and linezolid (Dong-A, Seoul, South Korea); erythromycin, tetracycline, oxacillin, penicillin G, and cefuroxime (Sigma Chemical, St. Louis, MO); piperacillin and tazobactam (Yuhan, Seoul, South Korea); azithromycin and sulbactam (Pfizer Korea, Seoul, South Korea); clindamycin (Korea Upjohn, Seoul, South Korea); levofloxacin (Daiichi, Tokyo, Japan); ampicillin, gentamicin, and chloramphenicol (Chong Kun Dang, Seoul, South Korea); cefotaxime (Han-Dok, Seoul, South Korea); cefoxitin and imipenem (Merck Sharp & Dohme, Rahway, NJ); cefotetan (Je Il, Seoul, South Korea); metronidazole (Choong Wae, Seoul, South Korea); trimethoprim and sulfamethoxazole (Dong Wha, Seoul, South Korea); cefaclor and vancomycin (Daewoong, Seoul, South Korea); and teicoplanin (Sanofi Aventis, Bridgewater, NJ).American Type Culture Collection strains of S. aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), S. pneumoniae (ATCC 49619), H. influenzae (ATCC 49247), Bacteroides fragilis (ATCC 25285), and Bacteroides thetaiotaomicron (ATCC 29741) were used as reference strains. The meningeal breakpoints of penicillin G and cefotaxime were used for S. pneumoniae.MRSA continues to be prevalent in South Korea, accounting for 64% of the S. aureus strains in one study (10). In this study, all of the isolates of staphylococci tested were inhibited by torezolid at ≤1 μg/ml and the MIC for 90% of the strains tested (MIC₉₀) was 4- to 8-fold lower than that of linezolid (Table ​(Table1).1). The majority of the MRSA isolates was resistant to erythromycin, clindamycin, gentamicin, levofloxacin, and tetracycline.

TABLE 1.

Comparative antimicrobial activities of torezolid and other antimicrobial agents against aerobic and anaerobic bacteria

Antimicrobial Susceptibility Patterns for Recent Clinical Isolates of Anaerobic Bacteria in South Korea

We determined the antimicrobial susceptibilities of 255 clinical isolates of anaerobic bacteria collected in 2007 and 2008 at a tertiary-care hospital in South Korea. Piperacillin-tazobactam, cefoxitin, imipenem, and meropenem were highly active β-lactam agents against most of the isolates tested. The rates of resistance of Bacteroides fragilis group organisms and anaerobic Gram-positive cocci to moxifloxacin were 11 to 18% and 0 to 27%, respectively.Anaerobic bacterial resistance trends may vary greatly, depending on regions or institutions (1). The Clinical and Laboratory Standards Institute (CLSI) does not recommend routine susceptibility testing of all clinical isolates of anaerobic bacteria, except for the management of patients with serious infections (4). A recent survey indicated that only a few laboratories in the United States performed antimicrobial susceptibility testing of anaerobic bacteria due to the complex techniques and predictable susceptibilities involved (5). However, regional susceptibility patterns are pivotal in the empirical treatment of infected patients because these patterns are related to clinical outcomes (13). Therefore, periodic monitoring of the regional or institutional resistance trends of clinically important anaerobe isolates is recommended (4). Our investigation of resistance trends of Bacteroides fragilis group organisms from South Korea has been taking place since 1989 (9, 15). However, few studies have focused on the susceptibilities of other anaerobes. Therefore, the aim of this study was to determine the recent antimicrobial resistance patterns of frequently isolated anaerobes at a tertiary-care hospital in South Korea.Anaerobes were isolated from blood, normally sterile body fluid, and abscess specimens, but Clostridium difficile was isolated from stool specimens of suspected C. difficile-associated disease patients at Severance Hospital in 2007 and 2008. The isolates were identified by either conventional methods (19) or the ATB 32A system (bioMérieux, Marcy l''Etoile, France). A total of 255 nonduplicated isolates were used in this study, including 63 of B. fragilis, 57 of other B. fragilis group species, 28 of Prevotella spp., 9 of other Gram-negative bacilli, 15 of Anaerococcus prevotii, 15 of Peptoniphilus asaccharolyticus, 15 of Finegoldia magna, 13 of Peptostreptococcus spp., 15 of C. perfringens, 12 of C. difficile, and 13 of other Gram-positive bacilli.Antimicrobial susceptibility testing was performed using the CLSI agar dilution method (4). The medium used was Brucella agar (Becton Dickinson, Cockeysville, MD) supplemented with 5 μg hemin and 1 μg vitamin K₁ per ml and 5% laked sheep blood. The antimicrobial powders used were piperacillin and tazobactam (Yuhan, Seoul, South Korea), cefoxitin (Merck Sharp & Dohme, West Point, PA), cefotetan (Daiichi Pharmaceutical, Tokyo, Japan), clindamycin (Korea Upjohn, Seoul, South Korea), imipenem, metronidazole (Choong Wae, Seoul, South Korea), chloramphenicol (Chong Kun Dang, Seoul, South Korea), meropenem (Sumitomo, Tokyo, Japan), moxifloxacin (Bayer Korea, Seoul, South Korea), and vancomycin (Eli Lilly & Co., Indianapolis, IN). For the combination of piperacillin and tazobactam, a constant tazobactam concentration of 4 μg/ml was added.An inoculum of 10⁵ CFU was applied with a Steers replicator (Craft Machine Inc., Woodline, PA), and the plates were incubated in an anaerobic chamber (Forma Scientific, Marietta, OH) for 48 h at 37°C. The MIC of each antimicrobial agent was defined as the concentration at which there was a marked reduction in growth, such as from confluent colonies to a haze, <10 tiny colonies, or 1 to 3 normal-sized colonies. B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741 were used as controls.β-Lactamase production by anaerobic Gram-negative bacilli, with the exception of B. fragilis group organisms, was determined by applying test organisms to the Cefinase disks and recording the results after 30 min (Becton Dickinson, Cockeysville, MD).Table ​Table11 shows the MICs of antimicrobial agents and the resistance rates of the anaerobes tested. Among the 255 isolates, B. fragilis group organisms were the most prevalent (47%). These organisms are more virulent and more resistant to antimicrobial agents than most other anaerobes (3). In this study, piperacillin-tazobactam, cefoxitin, imipenem, and meropenem were highly active against B. fragilis group organisms, with resistance rates of less than 7%. The rates of resistance to imipenem and piperacillin-tazobactam were 4% and 7%, respectively, for other B. fragilis group organisms. However, much higher resistance rates were observed for piperacillin (27 to 51%), cefotetan (14 to 68%), and clindamycin (33 to 86%). These values were similar to those observed in 1997 to 2004 in the same hospital: piperacillin, 33 to 49%; cefotetan, 14 to 60%; clindamycin, 51 to 76% (15). A higher prevalence of resistance, in particular to clindamycin, was observed than in the United States, i.e.,19 to 35% (17). CLSI added a recommendation to test susceptibility to moxifloxacin in 2004 and 2007. In this study, the moxifloxacin resistance rates were 11% for B. fragilis and 18% for other B. fragilis group organisms. These rates were slightly higher than the 7 to 9% reported in Taiwan (11) but lower than those in Greece (14) and the United States (16 to 75% and 26 to 55%, respectively) (17).

TABLE 1.

Antimicrobial activities against 255 anaerobic bacteria isolated in 2007 to 2008

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 Ceftaroline against Community-Associated Methicillin-Resistant,Vancomycin-Intermediate,Vancomycin-Resistant,and Daptomycin-Nonsusceptible Staphylococcus aureus Isolates

This study assessed the in vitro activities of ceftaroline and five comparator agents against a collection of Staphylococcus aureus isolates. Ceftaroline demonstrated potent activity against community-associated methicillin-resistant S. aureus (CA-MRSA) isolates and showed bactericidal activity against vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), heteroresistant VISA (hVISA), and daptomycin-nonsusceptible S. aureus (DNSSA) isolates. Ceftaroline may represent a bactericidal treatment option for infections caused by these pathogens.The increasing prevalence of resistant Staphylococcus aureus strains, including methicillin-resistant S. aureus (MRSA), community-associated MRSA (CA-MRSA), and S. aureus strains with reduced susceptibility to vancomycin, emphasizes the need for innovative antimicrobials with activity against these pathogens (19, 24). Although the prevalence of S. aureus strains with reduced vancomycin susceptibility remains low, such strains have been associated with vancomycin treatment failure, limiting the treatment options for patients with such infections (3, 8). Ceftaroline is a novel, parenteral, broad-spectrum cephalosporin exhibiting bactericidal activity against Gram-positive organisms, including MRSA and multidrug-resistant Streptococcus pneumoniae (MDRSP), as well as common Gram-negative pathogens (6, 9, 21). Ceftaroline is currently in phase 3 development for the treatment of complicated skin and skin structure infections and community-acquired bacterial pneumonia. The study described here evaluated the in vitro activities of ceftaroline and five comparator agents against CA-MRSA, vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), heteroresistant VISA (hVISA), and daptomycin-nonsusceptible S. aureus (DNSSA) isolates.(A preliminary report of these results was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy-Infectious Diseases Society of America Annual Meeting, Washington, DC, 25 to 28 October 2008.)A total of 132 MRSA strains were selected for evaluation. CA-MRSA strains (n = 92) were isolated from patients admitted to St. John Hospital and Medical Center in Detroit, MI. These patients had positive MRSA cultures within 48 h of admission, in accordance with the definition of CA-MRSA described by the Centers for Disease Control and Prevention (2). DNSSA strains (n = 7) and hVISA strains (n = 3) were obtained from blood collected from patients at the same hospital. The hVISA isolates were identified by a modified population analysis profile method (12). VISA isolates (n = 20) and VRSA isolates (n = 10) were obtained via the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) program, supported under NIAID/NIH contract HHSN272200700055C.All CA-MRSA isolates were typed by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI, followed by visual interpretation and categorization. PCR methods were used to determine the staphylococcal cassette chromosome mec (SCCmec) type, the presence of Panton-Valentine leukocidin (PVL) genes lukS-PV and lukF-PV, and the presence of the arginine catabolic mobile element (ACME) via detection of the arcA locus.In vitro susceptibility tests were performed with the antimicrobials ceftaroline (lot CI 170-07; Forest Laboratories, Inc., New York, NY), vancomycin-HCl (Sigma), daptomycin (Cubist), clindamycin-HCl (Sigma), linezolid (Pfizer), and trimethoprim-sulfamethoxazole (Sigma), which were obtained individually and reconstituted to a 1:19 ratio. All antibiotics were received in powder form and were reconstituted according to guidelines of the Clinical and Laboratory Standards Institute (CLSI). MICs and minimum bactericidal concentrations (MBCs) were determined according to the guidelines of the CLSI (4, 5). Microdilution tests with cation-adjusted Mueller-Hinton broth were used to identify the MICs of all antimicrobial agents tested. The percentages of susceptible isolates were determined by using the CLSI breakpoints. For the testing of daptomycin, additional calcium was added to the broth for a final concentration of 50 mg/liter. The MICs were read visually and corresponded to the concentration in the well with the lowest drug concentration with no visible bacterial growth. The MBC was defined as the antibiotic concentration that reduced the number of viable cells by ≥99.9%. This was based on colony counts from the growth control well and rejection values determined by tables provided by Pearson (18).S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were used as the control strains for the MIC determinations, and S. aureus ATCC 25923 was used as the control strain for the MBC determinations.Approximately 58% of the CA-MRSA isolates were SCCmec type IV/IVa, whereas the majority of the other isolates were SCCmec type II (Table ​(Table1).1). All SCCmec type IVa isolates were positive for the PVL and ACME genes, whereas isolates of all other SCCmec types were negative for these genes. Of all the other S. aureus strains, only two DNSSA isolates were SCCmec type IVa and positive for the PVL and ACME genes.

TABLE 1.

SCC mec elements, PVL and ACME genes, and ceftaroline MIC₉₀s and MBC₉₀s of CA-MRSA isolates^a

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

Activity Profile In Vitro of Micafungin against Spanish Clinical Isolates of Common and Emerging Species of Yeasts and Molds

A collection of 2,278 isolates belonging to 86 different fungal species was tested with micafungin and eight other drugs using the EUCAST procedures. Micafungin was active against species of Candida and Aspergillus (even azole-resistant species) as well as Penicillium spp., Scedosporium apiospermum, and Acremonium spp. It was inactive for species of Basidiomycota and Mucorales and for multiresistant species such as those of Fusarium.Micafungin is a new drug that belongs to the echinocandin class of antifungal agents. Its mechanism of action is by means of the inhibition of 1,3-β-d-glucan synthesis in the fungal cell wall (10).Micafungin has been recently approved in Europe and the United States for the treatment of candidemia, acute disseminated candidiasis, Candida peritonitis and abscesses, esophageal candidiasis, and recently for the prophylaxis of Candida infections in patients undergoing hematopoietic stem cell transplantation.The in vitro activity of micafungin against most common species of Candida is well known (4, 11-13). However, information is limited for uncommon species of yeasts as well as for molds.The aim of this study is to analyze the in vitro activity of micafungin and eight other antifungal agents against a collection of clinical isolates of yeasts and molds from human beings using the methods approved by AFST-EUCAST.The strains were recovered from 115 Spanish hospitals through a period of 3 years, from 2005 to 2007. A total of 2,278 clinical isolates were included in the analysis. Isolates were identified by morphological and biochemical methods and sequencing of DNA targets if necessary. They belonged to 86 different species of common and emerging fungal pathogens. The isolates were obtained from blood (559; 24.5%), biopsies and other deep sites (217; 9.5%), respiratory tract specimens (751; 33%), skin samples (180; 7.9%), and other locations (707; 25.1%).The following drugs were used: amphotericin B (range, 16.0 to 0.03 μg/ml; Sigma-Aldrich Quimica S.A., Madrid, Spain), flucytosine (64.0 to 0.12 μg/ml; Sigma-Aldrich), fluconazole (64.0 to 0.12 μg/ml; Pfizer S.A. Madrid, Spain), itraconazole (8.0 to 0.015 μg/ml; Janssen S.A., Madrid, Spain), voriconazole (8.0 to 0.015 μg/ml; Pfizer S.A.), posaconazole (8.0 to 0.015 μg/ml; Schering-Plough, Kenilworth, NJ), caspofungin (16.0 to 0.03 μg/ml; Merck & Co., Inc., Rahway, NJ), micafungin (16.0 to 0.03 μg/ml; Astellas Pharma Inc., Tokyo, Japan), and anidulafungin (16.0 to 0.03 μg/ml; Pfizer S.A.).Susceptibility testing was performed by using broth microdilution. For Candida species, MICs were determined using the reference procedure for testing fermentative yeasts described by AFST-EUCAST (7, 17). For Cryptococcus neoformans and other species of nonfermentative yeasts, such as Trichosporon and Rhodotorula spp., susceptibility testing strictly followed the recommendations by the EUCAST with a minor modification in order to improve the growth of microorganisms (3). For filamentous fungi, broth microdilution testing was performed following the AFST-EUCAST reference method (18). For testing echinocandins against molds, the MIC was defined as the lowest drug concentration resulting in aberrant hyphal growth by examination with an inverted microscope, that is, the minimum effective concentration (MEC) (2).Tables ​Tables11 and ​and22 display the susceptibility results obtained when the collection of clinical isolates was tested.

TABLE 1.

Summary of susceptibility results of antifungal agents tested in vitro against yeast species^a

Species Identification and Antifungal Susceptibility Patterns of Species Belonging to Aspergillus Section Nigri

A phylogenetic analysis was performed for 34 Aspergillus strains belonging to section Nigri. Molecular methods allowed for the correct classification into three different clades (A. niger, A. tubingensis, and A. foetidus). Correlation with in vitro itraconazole susceptibility distinguished the following three profiles: susceptible, resistant, and showing a paradoxical effect. A number of different species whose morphological features resemble those of A. niger showed unusual MICs to itraconazole that have never been described for the Aspergillus genus.Black aspergilli are widely distributed in nature (16); they are common food spoilers but are also well used for industrial purposes (15). Among Aspergillus species of the Nigri group, A. niger constitutes the most frequent etiological agent of otomycosis (13) and is considered the third cause of pulmonary aspergillosis (10). Nevertheless, the clinical implications of other species are rarely reported, and they are generally identified as A. niger (14, 22).Clinically, identification of unknown Aspergillus clinical isolates to the species level may be important given that different species have dissimilar susceptibilities to antifungal drugs. Thus, the knowledge of the species identity may influence the choice of appropriate antifungal therapy (2, 4). Furthermore, since the antifungal susceptibility patterns for most of the species within section Nigri have been poorly investigated, their identification and antifungal susceptibility profiles appear to be of clinical interest for further research.Black aspergilli belong to one of the most difficult groups concerning classification and identification (18), and so a number of different techniques have been developed in order to solve this issue. Among them, molecular tools are the gold standard (1, 18), as the sequencing of the β-tubulin or calmodulin gene is suitable, and enough, to discriminate between species within section Nigri (3, 18, 21).Thirty-four Aspergillus section Nigri strains belonging to the Mold Collection of the Centro Nacional de Microbiologia and collected since 2004 were analyzed. Thirty-three strains were independent clinical isolates, and 1 had an environmental origin. All strains were identified as A. niger using conventional methods of morphology at the macroscopic as well as microscopic levels (9). Species identification analysis was addressed using sequences of the β-tubulin gene from all the strains included in this study together with the sequences of different Aspergillus section Nigri type strains and others that were available at GenBank as follows: A. tubingensis {"type":"entrez-nucleotide","attrs":{"text":"AY820007","term_id":"65307061","term_text":"AY820007"}}AY820007^T, {"type":"entrez-nucleotide","attrs":{"text":"AY820009","term_id":"65307063","term_text":"AY820009"}}AY820009, and {"type":"entrez-nucleotide","attrs":{"text":"AY585527","term_id":"53830386","term_text":"AY585527"}}AY585527; A. foetidus {"type":"entrez-nucleotide","attrs":{"text":"AY585533","term_id":"53830397","term_text":"AY585533"}}AY585533^T, {"type":"entrez-nucleotide","attrs":{"text":"AY585534","term_id":"53830399","term_text":"AY585534"}}AY585534, and {"type":"entrez-nucleotide","attrs":{"text":"DQ768454","term_id":"111609935","term_text":"DQ768454"}}DQ768454; and A. niger {"type":"entrez-nucleotide","attrs":{"text":"FJ629288","term_id":"225904264","term_text":"FJ629288"}}FJ629288^T, {"type":"entrez-nucleotide","attrs":{"text":"EF422213","term_id":"126105445","term_text":"EF422213"}}EF422213, and {"type":"entrez-nucleotide","attrs":{"text":"AY585537","term_id":"53830403","term_text":"AY585537"}}AY585537. Partial sequences of the β-tubulin gene were amplified using the primer set βtubAniger1 and βtubANiger2 (11) and were carried out according to standard PCR guidelines (Applied Biosystems). Sequences were assembled and edited using the SeqMan II and EditSeq software packages (Lasergene 8.0; DNAStar, Inc., Madison, WI).All phylogenetic analyses were conducted with InfoQuest FP software, version 4.50 (Bio-Rad). The methodology used was maximum parsimony clustering. Phylogram stability was assessed by using parsimony bootstrapping with 2,000 simulations and by using the Aspergillus clavatus {"type":"entrez-nucleotide","attrs":{"text":"AY214441","term_id":"32441964","term_text":"AY214441"}}AY214441^T sequence as the out-group.The phylogenetic tree grouped the 34 clinical isolates into three different clades consisting of 13 A. niger isolates, 18 A. tubingensis isolates, and 3 A. foetidus isolates (Fig. ​(Fig.1).1). Table ​Table11 shows β-tubulin gene identification, as well as the origin and susceptibility profiles of the isolates.Open in a separate windowFIG. 1.Phylogenetic tree using maximum parsimony phylogenetic analysis and 2,000 bootstrap simulations based on β-tubulin gene sequences from all the Aspergillus section Nigri strains included in the study. Percentages indicate the bootstrap support for each group of sequences. (T), type strain.

TABLE 1.

Source, molecular identification, MICs, and MECs for species of Aspergillus section Nigri^a

Antimicrobial Susceptibilities of a Worldwide Collection of Stenotrophomonas maltophilia Isolates Tested against Tigecycline and Agents Commonly Used for S. maltophilia Infections

Antimicrobial susceptibilities were determined for 1,586 isolates of Stenotrophomonas maltophilia from globally diverse medical centers using the Clinical Laboratory Standards Institute broth microdilution method. The combination trimethoprim-sulfamethoxazole (96.0% of isolates susceptible at ≤2 μg/ml trimethoprim and 38 μg/ml sulfamethoxazole) and tigecycline (95.5% of isolates sussceptible at ≤2 μg/ml) were the only antimicrobials tested with >94% susceptibility in all regions. Susceptibility rates for other commonly used were lower than expected and varied geographically. This in vitro data supports tigecycline as a potential candidate for clinical investigations into S. maltophilia infections.Stenotrophomonas maltophilia is a Gram-negative bacillus, inherently multidrug resistant (MDR) and frequently recovered from environmental sources. It has been associated with severe nosocomially acquired bacteremia and pneumonia, usually among immunocompromised patients, as well as meningitis, endocarditis, and urinary tract, skin/soft tissue, and ocular infections. S. maltophilia infections are associated with high morbidity and mortality, with estimated crude mortality rates ranging from 20 to 70% and with the risk of mortality highest among patients receiving inappropriate initial antimicrobial therapy (5). Treatment of S. maltophilia infections represents a significant challenge because of the organism''s high levels of intrinsic resistance to many antimicrobial agents, difficulties in susceptibility testing, the development of resistance during therapy, and the paucity of clinical trials to determine optimal therapy (8, 12).The combination trimethoprim-sulfamethoxazole (TMP/SMX) is the recognized antimicrobial of choice for the treatment of infections caused by S. maltophilia with ceftazidime, ticarcillin-clavulanate, minocycline, tigecycline, fluoroquinolones, and the polymyxins being described as alternative therapies. It is important to note that all recommended therapy options have been based on in vitro studies and anecdotal experience rather than appropriately structured clinical trials (11). Resistance to TMP/SMX has been described and varies geographically, being shown by as many as 10% of isolates in Europe (7). In addition, allergic reactions to the combination TMP/SMX are common and can be severe, which further compromises its application (1). Clearly, therapeutic alternatives are needed to treat infections caused by S. maltophilia.Tigecycline is a 9-t-butylglycylamido derivative of minocycline and is the first glycylcycline licensed for clinical use. Tigecycline binds to the 30S ribosomal subunit, resulting in inhibition of protein synthesis (13). It exhibits a wide range of activity against Gram-positive and -negative organisms, including MDR strains. Tigecycline is approved by the United States Food and Drug Administration (USFDA) for the treatment of complicated skin and skin structure infections (cSSSI), intra-abdominal infections, and, more recently, community-acquired bacterial pneumonia. Tigecycline has demonstrated good in vitro activity against S. maltophilia in several studies (6, 9, 14). The aim of this study was to assess antimicrobial resistance in S. maltophilia against commonly used agents by using the largest and most geographically diverse collection of contemporary isolates available, with the rationale being the paucity of such information in the face of a clear need for clinical and research options.From January 2003 to December 2008, a total of 1,586 unique clinical S. maltophilia strains were recovered and identified from 119 medical centers located across Asia and the Pacific (Asian-Pacific), Europe, Latin America, and North America. Bacterial identification was confirmed by the central monitoring site (JMI Laboratories, North Liberty, IA) using standard algorithms (microscopy, culture characteristics, and oxidase reaction) followed by an automated system (Vitek 2; bioMerieux, Hazelwood, MO). MIC values were determined for all isolates based on the Clinical Laboratory Standards Institute (CLSI) broth microdilution method using commercially prepared and validated panels (TREK Diagnostic Systems, Cleveland, OH) in fresh cation-adjusted Mueller-Hinton broth (2). Tigecycline breakpoints established by the USFDA for Enterobacteriaceae (≤2 μg/ml for susceptibility and ≥8 μg/ml for resistance) as well as the polymyxin B breakpoints established by the CLSI for P. aeruginosa (≤2 μg/ml for susceptibility and ≥8 μg/ml for resistance), were applied for comparison only (Tygacil; Wyeth Pharmaceuticals, Philadelphia, PA). CLSI quality control ranges and interpretive criteria were used for comparator compounds (3).Clinical sites of infection for S. maltophilia were primarily bloodstream (51%) and respiratory tract (37%). Tigecycline activities were similar across the four geographic regions (94.5 to 96.5% of isolates inhibited at ≤2 μg/ml) and were most similar to those of TMP/SMX (90.8 to 98.9% of isolates susceptible) (Tables ​(Tables11 and ​and2).2). When tested against S. maltophilia isolates from North America and Europe, TMP/SMX was the most active compound (MIC₅₀, ≤0.5 μg/ml and MIC₉₀, 1 μg/ml; 97.6 to 98.9% of isolates susceptible), followed by tigecycline (MIC₅₀, 1 μg/ml, and MIC₉₀, 2 μg/ml; 94.5 to 95.3% o isolates susceptible) and levofloxacin (MIC₅₀, 1 μg/ml, and MIC₉₀, 4 μg/ml; 82.5 to 83.7% of isolates susceptible) (Table ​(Table2).2). Tigecycline was the most active compound tested against S. maltophilia isolates from the Asian-Pacific and Latin American regions (MIC₅₀, 0.5 μg/ml, and MIC₉₀, 2 μg/ml; 96.1 to 96.5% of isolates susceptible), followed by TMP/SMX (MIC₅₀, ≤0.5 μg/ml, and MIC₉₀, 1 μg/ml; 90.8 to 95.5% of isolates susceptible) (Table ​(Table2).2). Levofloxacin exhibited good in vitro activity against S. maltophilia isolates from Latin America (91.3% susceptible), but its activity was more restricted when tested against isolates from other geographic regions (78.0 to 83.7% of isolates susceptible) (Table ​(Table2).2). In general, ceftazidime (32.6 to 51.0% of isolates susceptible), ticarcillin-clavulanate (27.0 to 46.1% of isolates susceptible), and polymyxin B (33.4 to 76.4% of isolates susceptible) showed the most limited in vitro activities against S. maltophilia.

TABLE 1.

Regional MIC distributions for tigecycline tested against 1,586 S. maltophilia strains, stratified by geographic region

In Vitro Activity of Micafungin against Planktonic and Sessile Candida albicans Isolates

Planktonic and sessile susceptibilities to micafungin were determined for 30 clinical isolates of Candida albicans obtained from blood or other sterile sites. Planktonic and sessile MIC₉₀s for micafungin were 0.125 and 1.0 μg/ml, respectively.Candida albicans device-related infections are associated with growth of organisms in a biofilm state (3, 6). Device removal is often considered necessary for cure (10), since antimicrobial agents have been considered to have poor activity against microbial biofilms. If, however, antimicrobial agents were active against microbial biofilms, device removal might be avoidable.Cell walls are integral to C. albicans biofilms; therefore, antifungal agents that target cell wall synthesis may be active against fungal biofilms (1). We previously showed that caspofungin and anidulafungin had MIC₉₀s of 2 and ≤0.03 μg/ml, respectively, against 30 C. albicans isolates in biofilms (7, 12). We also demonstrated that caspofungin was active in vivo in an experimental intravascular catheter infection model (13).Herein, we evaluated the activity of micafungin against planktonic and sessile forms of the 30 clinical isolates of C. albicans against which we had previously studied caspofungin, anidulafungin, amphotericin B deoxycholate, and voriconazole (7, 12). One isolate per patient was included; isolates were included only if ≤3 types of organisms were cultured from the specimen from which C. albicans was isolated. Isolates were from blood cultures (n = 10), peritoneal fluid (n = 6), abscess fluid (n = 5), soft tissue (n = 5), bone (n = 2), pleural fluid (n = 1), and urine (n = 1). C. albicans GDH 2346 was used as a positive control.Planktonic MICs were determined using broth microdilution (5). Isolates were grown on Sabouraud dextrose agar for 24 h at 37°C. C. albicans was titrated to 76.6% transmittance at 530 nm in sterile saline and then diluted 1/1,000 in RPMI. Serial twofold micafungin dilutions ranging from 16 to 0.03 μg/ml were assayed. Drug dilution and titrated organism (100 μl each) were placed into corresponding wells of a 96-well, round-bottomed microtiter plate and incubated at 37°C. Forty-eight hours later, MICs were read using a reading mirror and scored according to CLSI guidelines. The lowest concentration associated with a ≥50% reduction in turbidity compared with that for the positive-control well was reported as the MIC. Planktonic MICs for micafungin ranged from ≤0.03 to 0.25 μg/ml (Table ​(Table1).1). The MIC₅₀ and MIC₉₀ were 0.125 μg/ml. The GDH 2346 MIC was 0.06 μg/ml.

TABLE 1.

Comparison of planktonic and sessile susceptibilities of 30 C. albicans isolates^a

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

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