Extended-Spectrum Cephalosporinase in Acinetobacter baumannii

An AmpC-type β-lactamase conferring high-level resistance to expanded-spectrum cephalosporins and monobactams was characterized from an Acinetobacter baumannii clinical isolate. This class C β-lactamase (named ADC-33) possessed a Pro210Arg substitution together with a duplication of an Ala residue at position 215 (inside the Ω-loop) compared to a reference AmpC cephalosporinase from A. baumannii. ADC-33 hydrolyzed ceftazidime, cefepime, and aztreonam at high levels, which allows the classification of this enzyme as an extended-spectrum AmpC (ESAC). Site-directed mutagenesis confirmed the role of both substitutions in its ESAC property.Acinetobacter baumannii is commonly associated with serious nosocomial infections (4, 11, 23). The emergence of multidrug-resistant A. baumannii strains, specifically those resistant to carbapenems, has created severe therapeutic challenges. A growing number of β-lactamases conferring resistance to expanded-spectrum cephalosporins have been identified in Acinetobacter spp. Resistance to oxyiminocephalosporins (ceftazidime and cefotaxime) is usually related to the overproduction of the resident AmpC-type β-lactamase (1, 2, 10) encoded by the gene bla_AmpC. The overexpression of that gene has been associated with the insertion sequence ISAba1 providing a strong promoter (2, 9). Recently, the AmpC variants have been named according to a nomenclature specific to A. baumannii (ADC, for Acinetobacter-derived cephalosporinases [10]).Most AmpC-type β-lactamases naturally produced by Gram-negative bacteria hydrolyze amino- and ureidopenicillins, cephamycins (cefoxitin and cefotetan), and, at a lower level, oxyiminocephalosporins, such as ceftazidime, cefotaxime, and ceftriaxone, and monobactams, such as aztreonam (3). Zwitterionic cephalosporins (cefepime and cefpirome) together with carbapenems are usually excluded from the spectrum of activity of AmpC β-lactamases (8). However, natural cephalosporinases possessing broadened substrate activity in the taxa Enterobacteriaceae and Pseudomonas aeruginosa have been reported (12, 14, 22). These extended-spectrum AmpCs (ESACs) confer reduced susceptibility to all cephalosporins (12, 14, 22). They differ from “regular” cephalosporinases by amino acid substitutions or insertions/deletions in four specific regions that are all located in the vicinity of the active site: the Ω-loop, the H-10 helix, the H-2 helix, and the C-terminal extremity of the protein (12, 14, 22).Here, we describe the first ESAC-type β-lactamase from A. baumannii with a broadened substrate activity toward expanded-spectrum cephalosporins and monobactams.The A. baumannii KI clinical isolate was obtained from a rectal screening of a patient who had been transferred from the Guadeloupe Islands (French Caribbean islands) and admitted at the Raymond Poincaré Hospital (Garches, Paris, France) in January 2009. This patient did not receive any antibiotic treatment. A. baumannii KI was selected for further study on the basis of its uncommon pattern of resistance to β-lactam antibiotics, including high-level resistance to expanded-spectrum cephalosporins and reduced susceptibility to carbapenems. In addition, it was resistant to cotrimoxazole and fluoroquinolones and susceptible to aminoglycosides.Susceptibility testing was performed by use of the Etest (17) and interpreted according to the CLSI guidelines (5). It showed that A. baumannii KI was resistant to amino-, carboxy-, and ureidopenicillins, to expanded-spectrum cephalosporins, including ceftazidime, cefotaxime, ceftriaxone, cefepime, and cefpirome, and to aztreonam and had reduced susceptibilities to imipenem and meropenem (see Table ​Table2).2). By use of cloxacillin-containing plates as described previously (22), the susceptibilities to ceftazidime and cefepime were restored, suggesting (i) overproduction of the AmpC β-lactamase and (ii) the likelihood for this AmpC to possess ESAC properties. Mating-out assays and electrotransformation were performed as described previously (20) but failed to transfer any cephalosporin resistance marker from A. baumannii KI to A. baumannii BM4547 or to Escherichia coli TOP10 recipient strains, suggesting that this resistance to expanded-spectrum cephalosporins was chromosomally conferred. Whole-cell DNA of A. baumannii KI was extracted as described previously (22). Primers PreAmpC-PISAba1 and PreAmpC-Ab1 were used in combination with primer PreAmpC-Ab2 to amplify 1,521-bp and 1,254-bp fragments, respectively, encompassing the entire bla_AmpC gene with and without the P_ISAba1 promoter, respectively (Table ​(Table11 ). All the inserts containing the P_ISAba1 promoter are named with “P+” accordingly. The amplification products were cloned into E. coli TOP10 by using the ZeroBluntTOPOPCR cloning kit (Invitrogen, Cergy-Pontoise, France) followed by selection on plates containing 50 μg/ml of amoxicillin and 30 μg/ml of kanamycin. A. baumannii strain AYE and the CIP7010 reference strain were used to clone two regular bla_AmpC β-lactamase genes (7, 18). Strain AYE corresponds to a multidrug-resistant isolate from France (19) whose complete genome sequence was determined (6, 24). DNA sequence analysis showed that the β-lactamase from strain KI, named ADC-33, had 8 and 7 amino acid changes compared to regular β-lactamases ADC-50 and ADC-11, corresponding to the ADC β-lactamases of strains CIP7010 and AYE, respectively (Fig. ​(Fig.1).1). No amino acid change was identified among the conserved SVSK, YSN, and KTG motifs, neither in helix H-2 nor in helix H-10, previously associated with the extended-spectrum activity of ESACs (12, 14, 22). However, we identified a single amino acid substitution, Pro210Arg, associated with a duplication of the Ala residue at position 215, both of them located inside the Ω-loop. Thus, the molecular basis of the extended-spectrum hydrolysis profile of ADC-33 might be related to those specific substitutions. The presence of the insertion sequence ISAba1 providing strong promoter sequences (−35 [TTAGAA] and −10 [TTATTT]) immediately upstream of the bla_AmpC gene was noticed, as previously observed when the bla_AmpC gene was overexpressed (9).Open in a separate windowFIG. 1.Amino acid sequence alignment including ADC-33 (-33) as an ESAC, ADC-50 (-50) and ADC-11 (-11) as regular AmpCs, and ADC-7 (-7) taken as the reference sequence as published (10). Conserved amino acid identities are indicated by dashes. The typical AmpC β-lactamase domains (SVSK, YSN, and KTG) are underlined. Helices H-2 and H-10 are boxed in gray. The Ω-loop is boxed in gray and double underlined. Differences observed inside the Ω-loop are in boldface. The vertical arrow indicates the position of the +1 amino acid (cleavage site for signal peptide). Numbering is according to the sequence of the mature protein.

TABLE 1.

Primers used in this work

Mode of Action of Van-M-02, a Novel Glycopeptide Inhibitor of Peptidoglycan Synthesis,in Vancomycin-Resistant Bacteria

Van-M-02, a novel glycopeptide, was revealed to exert potent activities against Gram-positive bacteria, including vancomycin-resistant enterococci (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA). A crude assay system was then used to study the mode of action of Van-M-02 as a peptidoglycan synthesis model of both vancomycin-susceptible and -resistant strains. The results suggested that Van-M-02 inhibits the synthesis of lipid intermediates irrespective of their termini. This inhibitory activity may contribute to the anti-VRE and anti-VRSA activities observed.The increasing incidence of vancomycin resistance in clinical settings has prompted research into new antibiotics against vancomycin-resistant strains (7, 12). We previously reported the synthesis of a novel glycopeptide, Van-M-02 (Fig. ​(Fig.1),1), during the course of our study of a vancomycin dimer (8). In this report, we describe the potent activities of Van-M-02 against the Gram-positive bacteria, including vancomycin-resistant enterococci (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA), and the investigation of its mode of action using a crude assay system.Open in a separate windowFIG. 1.Structures of Van-M-02, ΔN-Van-M-02, vancomycin, and ΔN-vancomycin.MICs were determined by the broth dilution method in accordance with CLSI document M7-A7 (2). Van-M-02 showed potent activities against VRSA, VanA-type VRE, and constitutive VanB-type VRE, with MICs of 4 μg/ml or less despite its structural similarity to vancomycin (Table ​(Table1).1). In order to assess the contribution of the d-Ala-d-Ala binding pocket to the activity of Van-M-02, N-terminally degraded Van-M-02 (ΔN-Van-M-02, Fig. ​Fig.1)1) was prepared (see Materials and Methods in the supplemental material). In a previous report, the corresponding compound ΔN-vancomycin (6) lost its antibacterial activities even against vancomycin-sensitive strains due to the lack of tight binding to d-Ala-d-Ala. In the present study, however, ΔN-Van-M-02 was effective against both vancomycin-sensitive (MIC = 2 μg/ml) and -resistant strains (MICs of 4 to 16 μg/ml).

TABLE 1.

Antibacterial activities of Van-M-02 and ΔN-Van-M-02

Modifying Culture Conditions in Chemical Library Screening Identifies Alternative Inhibitors of Mycobacteria

In this study, application of a dual absorbance/fluorescence assay to a chemical library screen identified several previously unknown inhibitors of mycobacteria. In addition, growth conditions had a significant effect on the activity profile of the library. Some inhibitors such as Se-methylselenocysteine were detected only when screening was performed under nutrient-limited culture conditions as opposed to nutrient-rich culture conditions. We propose that multiple culture condition library screening is required for complete inhibitory profiling and for maximal antimycobacterial compound detection.Recent data from the World Health Organization show that there are more than 9 million new cases of tuberculosis (TB) each year, half a million of which are caused by drug-resistant Mycobacterium tuberculosis (26, 27, 28). The spread of antibiotic resistance has necessitated the identification of new anti-infective molecules for the treatment of TB (25). TB drug discovery research is often dependent on the robustness of the upstream biological assay used. In terms of whole-cell antimycobacterial assays, screens are commonly performed under optimal growth conditions such as in the presence of excess nutrients. Evidence suggests, however, that M. tuberculosis persists in a nutrient-deprived state in the host lung (4, 12-14, 21). Screening under nutrient-rich conditions may fail to detect compounds that are preferentially active against nutrient-limited M. tuberculosis during infection.Traditionally, antimycobacterial assays use optical density (OD) as an indicator of growth, which can be distorted by the intrinsic absorbance of some compounds and the propensity of mycobacterial cells to form aggregates. Alternatives to OD measurement include the use of reporter molecules, such as the green fluorescent protein (GFP). Collins et al. (10) demonstrated that when expressed in M. tuberculosis, the levels of GFP paralleled the numbers of CFU during growth. The MICs of a range of antitubercular drugs determined using GFP were consistent with those obtained using Alamar Blue (7) and the BACTEC 460 system (9). In this work, we used both OD and GFP fluorescence to screen the library of pharmacologically active compounds (LOPAC) (LO1280; Sigma-Aldrich, St. Louis, MO) for antimycobacterial compounds.For expression of GFP, a vector was constructed by PCR amplification of the gfpmut2 gene (11) from pOT11 (19) using primers GFP_RBS_F1 (5′-GGGGGTACCTTTAAGAAGATATACATATGAGTAAAGGAGAA-3′) and GFP_R1 (5′-GGGGGCATGCTTATTATTTGTATAGTTCATCCATGCC-3′). The product was cloned into the KpnI and SphI restriction sites of pTKmx (16), generating plasmid pTKmxGFP. The pAL5000 origin of replication of pTKmxGFP was excised by NheI restriction digestion and replaced with the replicon of the high-copy-number plasmid pHIGH100 (5), amplified using PCR primers OriM_F (5′-GGGGGCTAGCAACGAGGACAGTCGCACGAC-3′) and OriM_R (5′-GGGGGCTAGCATCGAGCCGAGAACGTTATC-3′), generating plasmid pSHIGH. The hsp60 gene promoter from Mycobacterium bovis BCG, amplified using PCR primers Hsp60_F (5′-GGGGGGTACCGGTACCGGTGACCACAACGACGCGCCCGCT-3′) and Hsp60_R (5′-GGGGGGTACCCGCAATTGTCTTGGCCATTGCGAA-3′), was cloned into the KpnI site of pSHIGH, generating plasmid pSHIGH+hsp60. (Underlining indicates position of restriction site for each primer.)Mycobacterium smegmatis mc²155 harboring plasmid pSHIGH+hsp60 was inoculated into Luria-Bertani broth (LB) containing 50 μg/ml kanamycin and supplemented with 0.1% (vol/vol) Tween 80 and 100 μg/ml d-arabinose to reduce cell aggregation as previously described (2, 20). A selection of first- and second-line antitubercular drugs and tetracycline were used to test the validity of the antimycobacterial assay. The cultures were grown to the mid-logarithmic phase and diluted to an OD at 600 nm of 0.2 (10-mm path length). Two hundred microliters of sterile deionized water was added to each well on the perimeter of the plate to minimize evaporation of the growth medium during the assay. Fifty microliters of LB containing 50 μg/ml kanamycin, 0.1% Tween 80, and 100 μg/ml d-arabinose were added to the remaining wells. Starting at 50 μM, twofold serial dilutions were performed for the experimental compounds and control antibiotics. Fifty microliters of the cell culture, corresponding to approximately 5 × 10⁶ CFU per well, was added to each one of the inner wells, except the medium control wells. The plates were sealed, wrapped in parafilm, and incubated at 37°C for 96 h with 200 rpm shaking. OD and GFP fluorescence measurements were performed at 0- and 96-h incubation using a Wallac Envision multilabel plate reader (Perkin-Elmer). Data were analyzed with SigmaPlot 11 (SYSTAT) using four-parameter logistic standard curve analysis. The MIC and 50% inhibitory concentration (IC₅₀) values were determined with respect to the controls at 96 h. For each of the drugs tested, use of OD and GFP measurements produced identical MICs and good correlation for the IC₅₀s (Table ​(Table11 and Fig. ​Fig.11).Open in a separate windowFIG. 1.(A) Correlation of the data from the OD and GFP fluorescence-based library screens. The LOPAC inhibitory data from the OD and GFP fluorescence assays were compared to determine the level of correlation in the presence of different growth media. Pearson coefficients of r = 0.64 (nutrient-rich culture conditions), r = 0.5 (carbon-limited culture conditions), and r = 0.33 (nitrogen-limited culture conditions) were obtained indicating medium to large positive correlation between the two assays. (B) Profile of LOPAC inhibitory activity under various growth conditions. Examination of the LOPAC library index determined that a number of compounds were not detected in all of the culture conditions. This resulted in modifications in the inhibitory profile of the LOPAC library as a function of various growth conditions. Mycobacterium smegmatis was grown under nutrient-rich (LB), carbon-limited (C−), and nitrogen-limited (N−) culture conditions with each LOPAC compound present at a single concentration of 20 μM. Inhibitory activity is expressed as percentage inhibition of M. smegmatis growth. Symbols: ○, LB medium; ▵, carbon-limited culture conditions; □, nitrogen-limited culture conditions. The opacity of symbols increases with increasing inhibition. Significant results in both axes are displayed with solid symbols, i.e., LB medium (•), carbon-limited culture conditions (▴), nitrogen-limited culture conditions (▪). Scatter plots were drawn using Scatterplot3d (17).

TABLE 1.

Comparison of the optical density- and fluorescence-based inhibitor assays using known antibiotics in Mycobacterium smegmatis^a

Interaction of Ceftobiprole with the Low-Affinity PBP 5 of Enterococcus faecium

Ceftobiprole is a new cephalosporin that exhibits a high level of affinity for methicillin-resistant Staphylococcus aureus PBP 2a. It was reported that ceftobiprole did not interact with a mutated form of the low-affinity protein Enterococcus faecium PBP 5 (PBP 5fm) that, when overexpressed, confers a β-lactam resistance phenotype to the bacterium. Our results show that ceftobiprole binds to unmutated PBP 5fm to form a stable acyl-enzyme and that ceftobiprole is able to efficiently kill a penicillin-resistant Enterococcus faecium strain that produces this protein.β-Lactam antibiotics (penicillins, cephalosporins, carbapenems, and monobactams) are the most frequently prescribed antibacterial agents used to fight serious bacterial infections. They inactivate the membrane-bound d,d-transpeptidases essential for peptidoglycan synthesis by forming with them stable acyl-enzymes (9). This explains why these enzymes are generally designated penicillin-binding proteins (PBPs) (17, 19). In Gram-positive cocci, the resistance to β-lactam antibiotics is primarily conferred by the presence or the overproduction of a low-affinity PBP. In enterococci, among which is the opportunistic human pathogen Enterococcus faecium (3), the resistance to penicillin may be associated with overproduction of the intrinsic low-affinity protein PBP 5 (PBP 5fm) or to other alterations affecting PBP 5fm (15).Ceftobiprole (BPR) (BAL9141) is a novel broad-spectrum cephalosporin that is active against Gram-positive and Gram-negative bacterial groups, including methicillin-resistant Staphylococcus aureus (MRSA) (2, 6, 10, 12) and Enterococcus faecalis (1). It has been demonstrated to be a good inhibitor of the S. aureus low-affinity protein PBP 2a (6, 8). A previous study reported that ceftobiprole had poor inhibitory activity against β-lactam-resistant E. faecium and that ceftobiprole did not bind to the mutated, low-affinity PBP 5fm protein isolated from such a strain (10). To better understand the difference between these two low-affinity PBPs with respect to ceftobiprole, we have characterized the interaction between an unmutated form of PBP 5fm and ceftobiprole.To perform this study, we used the penicillin-sensitive Enterococcus faecium strain D63 (benzylpenicillin MIC = 5 μg/ml and ampicillin MIC = 15 μg/ml) and its laboratory-derived penicillin-resistant strain D63r (benzylpenicillin MIC = 70 μg/ml and ampicillin MIC = 125 μg/ml) (21). The PBP profile of the latter strain exhibits a 6-fold increase in quantity of PBP 5fm (21). The ceftobiprole MICs determined by the microdilution method (4) for E. faecium D63r and D63 were 8 and 2 μg/ml, respectively. These values contrasted with those reported by other workers, who have found that most ampicillin-resistant E. faecium clinical isolates were resistant to ceftobiprole (13). They concluded that ceftobiprole was ineffective against ampicillin-resistant enterococcal strains.To further study the killing effects of benzylpenicillin and ceftobiprole on E. faecium D63r, we exposed exponentially growing cultures of both the sensitive and the resistant strains to increasing concentrations of antibiotic corresponding to 1 and 4 times the respective MICs (Fig. ​(Fig.1)1) (18). For concentrations higher than their MICs, benzylpenicillin and ceftobiprole show killing effects (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Time-kill curves for resistant E. faecium D63r and susceptible E. faecium D63 in the presence of benzylpenicillin or ceftobiprole. The MICs for D63r were 70 μg/ml for benzylpenicillin and 8 μg/ml for ceftobiprole. The MICs for D63 were 5 μg/ml for benzylpenicillin and 2 μg/ml for ceftobiprole. The surviving bacteria were counted after 0, 4, and 24 h of incubation at 37°C by subculturing serial dilutions (at least 10-fold, to minimize drug carryover).Our study was completed by the determination of the 50% inhibitory concentration (IC₅₀) values for ceftobiprole, benzylpenicillin, cefepime, and ceftazidime for the different PBPs of E. faecium D63r by using purified membrane preparations and fluorescent ampicillin (20, 21). Membrane preparations (300 μg of proteins) were first incubated (20 min at 37°C) with increasing concentrations of ceftobiprole and next incubated (with a saturating concentration of 25 μM fluorescent ampicillin) for an hour at 37°C. The titration of the PBPs by ceftobiprole (Fig. ​(Fig.22 and Table ​Table1)1) revealed that at a 2-fold MIC, all high-molecular-mass PBPs are inhibited by the antibiotic (Fig. ​(Fig.2)2) and that the low-molecular-mass protein PBP 6, which acts as a d,d-carboxypeptidase (7), is not affected. The high-molecular-mass protein PBP 2 is the most sensitive to ceftobiprole (IC₅₀ = 0.2 μg/ml). PBPs 1 and 3 show similar IC₅₀s (<1 μg/ml), whereas the low-affinity proteins PBP 5fm, PBP 4, and PBP 5* possess slightly higher values (≥1 μg/ml). This pattern of inhibition is completely different from that obtained with benzylpenicillin, for which the resistant protein PBP 5fm is the most insensitive PBP. The IC₅₀ for ceftobiprole on purified soluble PBP 5fm (sPBP 5fm) gives results similar to those observed for the membrane preparations (0.7 μg/ml). Note that PBP 4 and PBP 5* exhibit very similar IC₅₀ profiles (Table ​(Table1).1). A similar observation was made for PBP 4 and PBP 4* in Enterococcus hirae. PBP 4* was shown to be produced by a proteolytic cleavage of the 60 N-terminal amino acid residues of PBP 4 (11). It is very likely that, in the E. faecium membranes used here, PBP 5* was the result of an N-terminal truncation of PBP 4.Open in a separate windowFIG. 2.Relative affinities of ceftobiprole (A) and benzylpenicillin (B) for E. faecium D63r PBPs. The affinities of ceftobiprole and other β-lactams for PBPs were analyzed by a competition assay using fluorescent ampicillin. Membrane proteins were prepared from D63r and D63. Nonlabeled antibiotics were incubated with membrane proteins at 37°C for 20 min, followed by the addition of fluorescent ampicillin during 1-h incubation. The membrane fractions were subjected to SDS-PAGE and fluorography.

TABLE 1.

Inhibition of PBPs from E. faecium D63r and D63

Genetic Context of Plasmid-Carried blaCMY-2-Like Genes in Enterobacteriaceae

Analysis of 15 European clinical Enterobacteriaceae isolates showed that differences in the genetic context of bla_CMY-2-like genes reflected the replicon type, usually IncA/C or IncI1. These bla_CMY-2 loci may originate from the same ISEcp1-mediated mobilization from the Citrobacter freundii chromosome as structures described in earlier studies.Among plasmid-mediated AmpC enzymes, CMY-2 is the most prevalent both in France and worldwide, especially in Salmonella species (1, 17). Previous studies in the United States revealed that large plasmids carrying bla_CMY-2 have been transmitted between different genera of bacteria, and notably from enteric Salmonella strains to pathogenic Escherichia coli strains (22). American studies of plasmids encoding CMY-2 using mixed-plasmid microarrays showed that the plasmids fell into two clusters (3); likewise, replicon-typing experiments revealed two replicon types, I1 and A/C (11). Similar studies in Taiwan confirmed that plasmids from diverse isolates carrying bla_CMY-2 genes have common restriction patterns, evidence of interspecies spread (23). Further investigations of Taiwanese isolates allowed the identification of a specific transposon-like element responsible for the spread of bla_CMY-2 among members of the family Enterobacteriaceae (6, 19). In the American and Taiwanese studies, ISEcp1 has been presumed to be involved both in the mobilization of bla_CMY-2 from the Citrobacter freundii chromosome and in the expression of ampC lacking chromosomal ampR (6, 10, 12, 14, 15, 19). Poirel et al. have shown the involvement of ISEcp1B, an ISEcp1-like element, in the expression and mobilization of the β-lactamase gene bla_CTX-M-19 (18).We characterized 15 CMY-type-producing isolates, including eight E. coli isolates, four Klebsiella pneumoniae isolates, two Proteus mirabilis isolates, and one Salmonella enterica isolate. We describe the genetic organization of bla_CMY-2-like genes in these European isolates and compare our findings with those from the United States and Taiwan.Table ​Table11 lists the 15 clinical isolates and their sources. Six isolates were previously reported (4, 7, 9, 13, 21). Total DNA was extracted by using a QIAamp DNA Mini kit (Qiagen, Courtaboeuf, France). Using repetitive-element PCR, we showed that three of the eight E. coli clinical isolates were clonally related (profile A: TN2106, TN38148, TN386) according to the interpretation criteria of van Belkum et al. (Table ​(Table1)1) (16, 20). Enterobacterial repetitive intergenic consensus PCR of the four K. pneumoniae isolates gave different amplification patterns (Table ​(Table1)1) (16).

TABLE 1.

Clinical isolates, origins, epidemiological features, and exploration by PCR mapping

Antifungal Activity of Micafungin in Serum

We have evaluated the antifungal activity of micafungin in serum by using the disk diffusion method with serum-free and serum-added micafungin standard curves. Serum samples from micafungin-treated patients have been shown to exhibit adequate antifungal activity, which was in proportion to both the applied dose and the actual concentration of micafungin measured by high-performance liquid chromatography. The antifungal activity of micafungin in serum was also confirmed with the broth microdilution method.Micafungin has been shown to bind to serum proteins at a level of 99.8% (13). If the unbound drug contributes to its pharmacological activity (the free-drug hypothesis), only 0.2% of total micafungin would be available to exert antifungal activity in the presence of serum, and the MIC for micafungin in vitro would increase 500-fold. However, several studies have shown that this ratio varies from 4- to 267-fold (6, 7, 11), indicating that the antifungal activities of micafungin in serum may not follow the free-drug hypothesis; instead, observed activities are mostly superior to those predicted. Furthermore, it remains unclear whether these results can be applied to micafungin in a patient''s serum. To address this issue, we collected serum samples from micafungin-treated patients and examined the relationship between micafungin concentration and its in vitro antifungal activity in serum.This study was approved by the institutional review board, and informed consent was obtained from each patient. Patients with hematologic malignancies, admitted into Osaka University Medical Hospital, were administered micafungin at a dose of 50 to 300 mg/body once daily. The efficacy of prophylaxis was defined as the absence of proven, probable (EORTC-IFICG/NIAID-MSG) (1), or suspected (unexplained persistent fever and clinical findings) (10) fungal infection, through the end of therapy. The efficacy of the drug for suspected fungal infections was indicated by improvement of persistent fever and clinical findings.Blood samples were collected from patients just before (trough) and after (peak) micafungin infusion, at least 4 days after initiating treatment (steady state) (2). Micafungin concentration in serum was measured by high-performance liquid chromatography (HPLC) (9, 12). The disk diffusion method was performed according to National Committee for Clinical Laboratory Standards (NCCLS) M44-A guidelines (5). To obtain standard curves, we prepared two types of serial dilution disks impregnated with micafungin standard solution, one in RPMI 1640 (serum-free standard) and the other in heat-inactivated serum from volunteers (serum-added standard). Disks were applied to Sabouraud dextrose agar plates inoculated with Candida albicans FP633, a clinical isolate kindly provided by Astellas Pharma Inc., Tokyo, Japan. The diameter of the area of complete growth inhibition (inhibitory zone) was measured. Similarly, disks were impregnated with serum samples collected from patients, and the inhibitory zones were measured. The determination of antifungal activity of micafungin in a patient''s serum was based on two standard curves, as described above. To determine the inhibitory titer in a patient''s serum, we utilized the broth microdilution method based on the guidelines in NCCLS M27-A2 (4). Serum from a patient was serially diluted twofold with serum from a volunteer, supplemented with 20 mM HEPES, and inoculated with C. albicans FP633. MIC was defined as the lowest concentration where no visible growth was observed. Serum inhibitory titers were defined as the highest dilution of serum that completely inhibited fungal growth.In all seven patients, micafungin was effective for prophylaxis or treatment against fungal infections (Table ​(Table1).1). Serum peak concentrations (C_max) of micafungin (measured by HPLC) ranged from 5.59 to 37.1 μg/ml at a dose of 50 to 300 mg/body and closely correlated with both daily dose and dosage in terms of body weight (Table ​(Table2).2). Standard curves were prepared from both serum-free and serum-added micafungin standard disks (Fig. ​(Fig.1).1). The antifungal activity of micafungin remained intact in serum: 20 to 50% (by measured value) or 25 to 30% (by standard curve).Open in a separate windowFIG. 1.Estimation of micafungin concentration in serum samples from patient no. 5, using the disk diffusion method. (A) Concentration measured using HPLC, 16.4 μg/ml. (B) Concentration estimated from the serum-free micafungin standard curve, 6.0 μg/ml. (C) Concentration estimated from the serum-added micafungin standard curve, 22.1 μg/ml. Ratio of concentration B to concentration A (%) = 6.0/16.4 × 100 = 37. Ratio of concentration C to concentration A (%) = 22.1/16.4 = 134.8.

TABLE 1.

Patient background

Genetic and Functional Variability of AmpC-Type β-Lactamases from Acinetobacter baumannii

The incidence of naturally occurring AmpC β-lactamases with extended activities toward several cephalosporins was evaluated among 17 ceftazidime (CAZ)-resistant Acinetobacter baumannii isolates. Five AmpC β-lactamases (named ADC β-lactamases) were identified, among which those possessing the Val208Ala (inside the omega-loop) or Asn283Ser (helix H-10) substitution conferred higher levels of resistance (4- to 64-fold higher) to CAZ and to cefotaxime in Escherichia coli. This study demonstrates that peculiar AmpCs playing a role in resistance to broad-spectrum cephalosporins in A. baumannii may be identified.Acinetobacter baumannii is commonly associated with serious nosocomial infections (4, 10, 14). A growing number of β-lactamases conferring resistance to broad-spectrum cephalosporins in Acinetobacter spp. have been identified. Even though resistance to oxyiminocephalosporins (ceftazidime and cefotaxime) may be related to production of extended-spectrum β-lactamases (ESBLs), it is usually associated with overproduction of an AmpC-type β-lactamase (1, 2, 9). Overexpression of the bla_AmpC gene of A. baumannii may occur as a result of an insertion sequence, ISAba1, providing strong promoter sequences (5, 8, 13), being located upstream.Most AmpC-type β-lactamases naturally produced by Gram-negative bacteria hydrolyze amino- and ureidopenicillins, cephamycins (cefoxitin and cefotetan), and, at a low level, oxyiminocephalosporins, such as ceftazidime, cefotaxime, and ceftriaxone, and monobactams, such as aztreonam (3). AmpCs possessing a broad substrate activity have been reported in Enterobacteriaceae and Pseudomonas aeruginosa (11, 12, 16, 19, 20). These AmpCs (or extended-spectrum AmpCs [ESACs]) with activities against several cephalosporins may confer reduced susceptibility to those molecules (6, 11, 12, 19). They differ from “regular” cephalosporinases by amino acid substitutions or insertions/deletions in four specific regions that are all located in the vicinity of the active site: the Ω loop, the H-10 helix, the H-2 helix, and the C-terminal extremity of the protein (15).Recently, we have identified the first ESAC (named ADC-33, according to the numbering of AmpC β-lactamases from A. baumannii [9]) from a single A. baumannii isolate (21). ADC-33, possessing a Pro210Arg substitution together with a duplication of an Ala residue at position 215 (inside the Ω loop), hydrolyzed ceftazidime, cefepime, and aztreonam at high levels (21).The present study aimed to evaluate the extent of the variability of ADC β-lactamases in A. baumannii. Seventeen nonrepetitive A. baumannii clinical isolates that were recovered in 2007-2008 from patients hospitalized at the Bicêtre Hospital and were resistant to ceftazidime were included in this study. Genotyping was performed by using pulsed-field gel electrophoresis (PFGE) according to the instructions of the manufacturer (Bio-Rad, Marnes-la Coquette, France) as previously described (17, 22), and it identified eight distinct clones among the 17 isolates (Table ​(Table11).

TABLE 1.

Features of the A. baumannii isolates studied^d

In Vitro Activities of Echinocandins against Candida krusei Determined by Three Methods: MIC and Minimal Fungicidal Concentration Measurements and Time-Kill Studies

We evaluated the in vitro activities of anidulafungin, micafungin, and caspofungin against Candida krusei by determining MIC and minimum fungicidal concentration (MFC) measurements and by the time-kill method. The geometric mean (GM)-MIC/GM-MFC values were 0.1/0.34, 0.25/0.44, and 1/2.29, respectively. The mean times to reach 99.9% growth reduction were 19.1 ± 18.2 h (mean ± standard deviation) for 2 mg/liter anidulafungin, 37.4 ± 8.8 h for 2 mg/liter caspofungin, and 30.7 ± 12.2 h for 1 mg/liter micafungin. Anidulafungin exhibited the highest time-kill rate, followed by micafungin. The three echinocandins showed fungicidal activity at concentrations reached in serum.Candida krusei is isolated mainly from immunocompromised patients and those who have received fluconazole treatment. The isolation (2 to 60%) and mortality (30 to 60%) rates depend on the institution and on the unit to which the patient is admitted (10, 13, 14, 17, 20). Treatment of invasive C. krusei infections can be difficult due to its intrinsic resistance to fluconazole and its reduced susceptibility to amphotericin B and flucytosine (2, 15). In immunocompromised patients, fungicidal agents may be required to eradicate infection since the major host defense (phagocytic killing of neutrophils and monocytes/macrophages) is reduced (9).Anidulafungin, caspofungin, and micafungin are novel antifungals that share the same spectrum of activity but differ in their MICs. Although the fungicidal activities and killing rates of echinocandins have been previously evaluated (3, 7, 8, 16), none of these studies performed a head-to-head comparison. However, these agents also may differ in their fungicidal activities. This study aimed to determine their relative antifungal activities against C. krusei by three measures: (i) MICs, (ii) minimal fungicidal concentrations (MFCs), and (iii) time-kill curves.Anidulafungin (Pfizer España, Madrid, Spain), caspofungin (Merck Sharpe & Dome, Madrid, Spain), and micafungin (Fugisawa Pharmaceutical Company, Japan) were provided by their manufacturers. Caspofungin and micafungin were dissolved in water and anidulafungin in dimethyl sulfoxide; further dilutions were prepared in standard RPMI 1640 medium (Sigma-Aldrich, Madrid, Spain). Final anidulafungin and micafungin drug concentrations ranged from 0.03 to 16 mg/liter, and that of caspofungin from 0.12 to 64 mg/liter.MIC and MFC values were obtained for 20 C. krusei bloodstream isolates, the caspofungin-resistant strain CY-118 (MIC > 8 mg/liter) (11), and C. krusei ATCC 6258. MICs were determined by following the CLSI M27-A3 microdilution method (4). MIC₂ and MIC₀ (minimum concentrations that produce ≥50 and 100% growth reduction, respectively) were determined at 24 and 48 h. As previously described but using a larger inoculum size (1), MFCs were obtained by plating 0.1 ml from clear MIC wells following 48 h of incubation onto Sabouraud dextrose agar plates (SDA) (1). The MFC was the lowest drug concentration that resulted in ≤1 colony (≈99% killing). C. krusei ATCC 6258 was included in each batch of experiments (5).Time-kill studies were performed for six blood isolates (randomly selected from the 20 blood isolates), CY-118, and ATCC 6258. The antifungal carryover effect and the time-kill curve for each agent were determined as previously described (RPMI 1640 medium, 1 × 10⁶ to 5 × 10⁶-CFU/ml inoculum, and 5-ml volume) (2). The anidulafungin and caspofungin drug concentrations evaluated were 0.03, 0.12, 0.5, 2, and 8 mg/liter and, in addition, 32 mg/liter for caspofungin. The micafungin concentrations assessed were 0.06, 0.25, 1, 8, and 16 mg/liter. These concentrations are within the range achieved clinically (6, 18, 19). At 0, 2, 4, 6, 12, 24, and 48 h, aliquots of 0.1 ml were removed to determine the number of CFU/ml. The lowest limit of accurate and reproducible detectable colony counts was 100. All experiments were performed twice with three replicates for every dilution of each time point.To our knowledge, this is the first study comparing the killing activities of anidulafungin, caspofungin, and micafungin head to head with the same strains.The geometric mean (GM)-MIC₂, GM-MIC₀, and GM-MFC and the concentrations that inhibited and killed 90% of isolates (MIC₉₀ and MFC₉₀, respectively) were calculated. Time-kill data were fitted to the exponential equation N_t = N₀ × e^−kt, where N_t is the number of viable cells at time t, N₀ is the starting inoculum, k is the kill rate, and t is the incubation time. The goodness of fit for each isolate/drug was assessed by the R² value. The times (hours) to achieve 50, 90, 99, and 99.9% reductions in growth compared to the starting inoculum size were calculated from the kill rate value as described elsewhere (2).The MICs for C. krusei ATCC 6258 were within the established ranges (5); the anidulafungin, micafungin, and caspofungin MFCs for this strain were 0.25, 0.5, and 2 mg/liter, respectively. Table ​Table11 summarizes the MIC and MFC determinations for the 20 blood isolates. Anidulafungin was the most active, followed by micafungin and caspofungin. The MFC/MIC₀ ratios were ≤2 for 90, 90, and 100% of isolates, respectively. The anidulafungin and micafungin MFCs were 1 to 7 dilutions lower than those of caspofungin, while the anidulafungin MFCs were only 1 to 2 dilutions lower than those of micafungin.

TABLE 1.

In vitro activities^a of echinocandin against 20 C. krusei bloodstream isolates

OXA-134, a Naturally Occurring Carbapenem-Hydrolyzing Class D β-Lactamase from Acinetobacter lwoffii

Acinetobacter lwoffii, a species whose natural habitat is the human skin, intrinsically possesses a chromosomal gene encoding a carbapenem-hydrolyzing class D β-lactamase, OXA-134. This species may therefore constitute a reservoir for carbapenemase genes that may spread among other Acinetobacter species.Acinetobacter baumannii, the most common Acinetobacter species isolated from humans, is an opportunistic pathogen for which resistance to carbapenems is increasing worldwide (13-15). Carbapenem resistance in A. baumannii is associated mostly with acquired carbapenem-hydrolyzing class D β-lactamases (CHDLs) (19). Four groups of acquired CHDLs in A. baumannii, OXA-23, OXA-40, OXA-58, and OXA-143, have been identified (9, 18). In addition, A. baumannii possesses a naturally occurring bla_OXA-51 or bla_OXA-69 CHDL-encoding gene that reduces the efficacy of carbapenems when it is overexpressed (4, 5, 7, 22). Identification of the sources of acquired and clinically relevant CHDLs is important to better understand the way and the reason why these resistance determinants are spreading. Acinetobacter radioresistens has recently been identified as the natural carrier of bla_OXA-23, a gene encoding one of the most commonly acquired CHDLs in A. baumannii (16). However, the progenitors of the other acquired CHDLs identified in Acinetobacter species remain unknown. Our study aimed to evaluate whether other Acinetobacter species may represent additional reservoirs of CHDL-encoding genes.The screening panel included strains belonging to 23 Acinetobacter species, including A. junii, A. johnsonii, A. haemolyticus, A. baylyi, A. lwoffii, A. radioresistens, A. schindleri, A. ursingii, A. calcoaceticus, A. gerneri, A. tjernbergiae, A. bouvetii, A. tandoii, A. grimontii, A. towneri, A. parvus, and Acinetobacter genomospecies 3, 6, 9, 10, 13, 15, 16, and 17. Acinetobacter genomospecies 9 is now classified as A. lwoffii (15). Strains were identified at the species level by using 16S rRNA sequencing (3). Susceptibility testing was analyzed by the disk diffusion method in accordance with the guidelines of the Clinical and Laboratory Standards Institute (1), and MICs were determined by using Etest strips (AB bioMérieux, Solna, Sweden).Screening for the known CHDL-encoding bla_OXA-23, bla_OXA-40, bla_OXA-58, and bla_OXA-143 genes was performed by PCR using internal primers (8, 9). This screening was positive only for the bla_OXA-23 gene and only for A. radioresistens strain 3 and A. lwoffii strain 1. After sequencing, A. radioresistens strain 3 was found to possess the bla_OXA-23 gene, in accordance with previous results (16). Sequencing of the amplicon obtained from A. lwoffii strain 1 identified a gene encoding a novel OXA-type β-lactamase. Thermal asymmetric interlaced (TAIL) PCR experiments were performed in order to obtain the entire sequence of this gene (11, 12). It encoded a 273-amino-acid protein named OXA-134 that shared 63, 58, 57, and 53% amino acid identity with OXA-23, OXA-40, OXA-51, and OXA-58, respectively. OXA-134 possessed the typical features of a class D β-lactamase, including the STFK tetrad at positions 70 to 73 according to class D β-lactamase (DBL) numbering (Fig. ​(Fig.1)1) (2). Also, as observed for other CHDLs (except for OXA-58), an FGN motif at DBL positions 144 to 146 replaced the usual YGN motif of classical class D β-lactamases (18). Finally, a KSG element was identified at DBL positions 216 to 218, as observed in the CHDLs OXA-40 and OXA-51, whereas a KTG motif is present in most class D β-lactamases, including the CHDLs OXA-23 and OXA-58 (18). A phylogenetic analysis showed that OXA-134-like β-lactamases were constituting a separate subgroup of CHDLs but that this subgroup was more closely related to the identified class D β-lactamases from Acinetobacter spp. than to other known CHDLs (Fig. ​(Fig.2)2) .Open in a separate windowFIG. 1.Amino acid alignment of the seven OXA-134-like class D β-lactamases identified in this study. Dashes indicate amino acids identical to those in the OXA-187 sequence. Amino acid motifs which are well conserved (even if possibly variable) among class D β-lactamases are shaded in gray. Numbering is according to DBL numbering (2).Open in a separate windowFIG. 2.Dendrogram obtained for 32 class D β-lactamases by neighbor-joining analysis. The alignment used for tree calculation was performed with ClustalX. Branch lengths are drawn to scale and are proportional to the number of amino acid changes. The distance along the vertical axis has no significance. The different clusters identified allowed the identification of nine main groups, considering that proteins from the same group have more than 80% amino acid identity. The class D β-lactamases which are considered to be naturally occurring are indicated together with the names of the corresponding species. R. pickettii, Ralstonia pickettii; B. pseudomallei, Burkholderia pseudomallei; A. xylosoxidans, Alcaligenes xylosoxidans; A. jandaei, Aeromonas jandaei; L. gormanii, Legionella gormanii; P. aeruginosa, Pseudomonas aeruginosa; P. pnomenusa, Pandoraea pnomenusa; C. jejuni, Campylobacter jejuni; B. pilosicoli, Brachyspira pilosicoli; S. oneidensis, Shewanella oneidensis; S. algae, Shewanella algae.A. lwoffii is a commensal organism found on human skin, the perineum, and the oropharynx. It has been associated with catheter-related bloodstream infections in immunocompromised patients and with bacteremia associated with community-acquired gastroenteritis and gastritis (20, 21). All the Acinetobacter genomospecies 9/A. lwoffii isolates we included in our study were fully susceptible to all antibiotics tested, including penicillins, imipenem, and meropenem. It is therefore likely that the bla_OXA-134-like genes were not expressed (or were expressed at an insignificant level) in these hosts.In order to study the biochemical properties of OXA-134, cloning of the bla_OXA-134 gene into the kanamycin-resistant plasmid pCR-BluntII-TOPO (Invitrogen, Life Technologies, Cergy-Pontoise, France) was performed using PCR products generated with primers PreOXA-134A (5′-GAAAAATGACCAAAATTTGATCG-3′) and PreOXA-134B (5′-TATTTGCATCATCCTTCAGC-3′) as described previously (16). Escherichia coli TOP10(pOXA-134) showed reduced susceptibility to imipenem and meropenem and resistance to most penicillins that was not inhibited by β-lactamase inhibitors (Table ​(Table11).

TABLE 1.

MICs of β-lactams for the different A. lwoffii isolates, E. coli TOP10 harboring recombinant plasmid pOXA-134, and the E. coli TOP10 reference strain

ACHN-490, a Neoglycoside with Potent In Vitro Activity against Multidrug-Resistant Klebsiella pneumoniae Isolates

The in vitro activity of ACHN-490, a novel aminoglycoside (“neoglycoside”), was evaluated against 102 multidrug-resistant (MDR) Klebsiella pneumoniae strains, including a subset of 25 strains producing the KPC carbapenemase. MIC₅₀ values for gentamicin, tobramycin, and amikacin were 8 μg/ml, 32 μg/ml, and 2 μg/ml, respectively; MIC₉₀ values for the same antimicrobials were ≥64 μg/ml, ≥64 μg/ml, and 32 μg/ml, respectively. ACHN-490 showed an MIC₅₀ of 0.5 μg/ml and an MIC₉₀ of 1 μg/ml, which are significantly lower than those of comparator aminoglycosides. ACHN-490 represents a promising aminoglycoside for the treatment of MDR K. pneumoniae isolates, including those producing KPC β-lactamase.The spread of Klebsiella pneumoniae isolates producing extended-spectrum β-lactamases (ESBLs) represents a serious threat to our therapeutic armamentarium (21). These isolates are also frequently resistant to other classes of antibiotics, such as β-lactam/β-lactamase inhibitor combinations, quinolones, and aminoglycosides (8, 9), thereby limiting our choice to carbapenems for the treatment of serious infections (21).Unfortunately, there is growing concern regarding the emergence of carbapenem-resistant K. pneumoniae isolates (20). In particular, K. pneumoniae isolates producing KPC carbapenemases (KPC-Kp) are spreading at an alarming rate in North and South America, the Caribbean, Europe, Israel, and Asia (6, 7, 15, 17, 18). Like ESBL producers, KPC-Kp are often resistant to quinolones and aminoglycosides (6). Therefore, our therapeutic options against KPC-Kp are limited to tigecycline and colistin. However, tigecycline may not reach desired serum levels to treat bloodstream infections (19), leaving colistin as the “last choice” against infections caused by KPC-Kp (13). Unfortunately, colistin-resistant KPC-Kp isolates are also reported in the United States (1, 12). As a result of this therapeutic dilemma, new antimicrobial agents with potent activity against multidrug-resistant (MDR) K. pneumoniae need to be developed.Recently, there has been an increased interest in developing novel aminoglycosides. This new attention is due to (i) the potent bactericidal activity of aminoglycosides against a wide spectrum of aerobic gram-positive and gram-negative pathogens, (ii) the more gradual decline in susceptibility to aminoglycosides among gram-negative bacteria than that in susceptibility to other antimicrobials, and (iii) the ability of novel aminoglycosides to bypass common mechanisms of resistance that have gradually decreased the susceptibility to clinically used aminoglycosides (e.g., gentamicin, tobramycin, and amikacin) (11, 14, 16).ACHN-490 (Achaogen, San Francisco, CA) is a “neoglycoside,” a next-generation aminoglycoside, currently in early clinical development (FDA, http://clinicaltrials.gov/), which has never been reported previously in the literature. The chemical structure of ACHN-490 is presented in Fig. ​Fig.11.Open in a separate windowFIG. 1.Chemical structure of ACHN-490 [6′-(hydroxylethyl)-1-(haba)-sisomicin].In the present work, we analyzed the in vitro activity of ACHN-490 against a collection of 102 K. pneumoniae clinical isolates collected from January 2006 to October 2007 at the University of Pittsburgh Medical Center, and three Cleveland institutions, including University Hospitals Case Medical Center, the Cleveland Clinic, and the Louis Stokes Department of Veterans Affairs Medical Center.The 102 K. pneumoniae isolates were selected based on an MDR phenotype (i.e., resistance to ≥3 antibiotic classes). Twenty-five isolates were KPC-Kp and were part of a previous study in which the β-lactamase background and clonality were characterized (6). The remaining 77 MDR K. pneumoniae isolates were ESBL producers, according to the phenotypic results (see below).MICs were determined by a microdilution method using cation-adjusted Mueller-Hinton broth, according to the Clinical and Laboratory Standards Institute (CLSI) criteria (2). Specific panels containing the following antibiotics were customized by Trek Diagnostics (Cleveland, OH): cefotaxime, cefotaxime-clavulanate (constant concentration of 4 mg/liter), ceftazidime, ceftazidime-clavulanate (constant concentration of 4 mg/liter), piperacillin-tazobactam, imipenem, ciprofloxacin, tigecycline, gentamicin, tobramycin, amikacin, arbekacin, neomycin, and ACHN-490. The following ATCC control strains were used: Escherichia coli 25922, Pseudomonas aeruginosa 27853, and K. pneumoniae 700603. Susceptibility results were interpreted according to the guidelines recommended by CLSI (3). Tigecycline MICs were interpreted according to the U.S. FDA criteria (i.e., susceptible at an MIC of ≤2 μg/ml). According to the CLSI criteria, isolates were defined as ESBL producers when they showed a ≥3 twofold concentration decrease in MICs for ceftazidime or cefotaxime when tested in combination with clavulanate versus their MICs when tested alone (3).The 25 KPC-Kp isolates were analyzed by PCR for the presence of 16S rRNA methylase genes (i.e., armA, rmtA, rmtB, rmtC, rmtD, and npmA), using primers and conditions previously reported (4, 23). In addition, these strains were examined by PCR and sequencing for the presence of the most common aminoglycoside-modifying enzymes (AMEs) in gram-negative pathogens (22). In particular, the following genes were analyzed: aac(6′)-Ib, aac(6′)-Ic, aac(6′)-Id, ant(3")-Ia, ant(2")-Ia, aac(3)-Ia, aac(3)-Ib, aac(3)-IIc, aph(3′)-VIa, and aph(3′)-VIb, using primers previously reported (5, 10).As shown in Table ​Table1,1, MDR K. pneumoniae isolates were highly resistant to ceftazidime and piperacillin-tazobactam (each MIC₉₀, >32 μg/ml). Two-thirds of the isolates were resistant to ciprofloxacin, whereas approximately 75% and 90% of strains were still susceptible to imipenem and tigecycline, respectively. Almost all KPC-Kp isolates were resistant to β-lactams and quinolones, whereas tigecycline frequently remained active in vitro (Table ​(Table1).1). All of these 25 isolates were colistin susceptible, as previously reported (6).

TABLE 1.

Susceptibility results of MDR K. pneumoniae isolates, including those producing KPC enzymes

Assessment of Oritavancin Serum Protein Binding across Species

Biophysical methods to study the binding of oritavancin, a lipoglycopeptide, to serum protein are confounded by nonspecific drug adsorption to labware surfaces. We assessed oritavancin binding to serum from mouse, rat, dog, and human by a microbiological growth-based method under conditions that allow near-quantitative drug recovery. Protein binding was similar across species, ranging from 81.9% in human serum to 87.1% in dog serum. These estimates support the translation of oritavancin exposure from nonclinical studies to humans.Estimates of serum protein binding are essential to translate drug exposure from nonclinical species to humans during assessments of toxicology, pharmacokinetics, and pharmacodynamics since the free fraction dictates drug activity (3, 7, 17, 18). Recent evidence supports the concept of an “active fraction” that offers insight into the pharmacodynamic behavior of highly protein-bound drugs, such as daptomycin (20).Oritavancin is a late-stage investigational lipoglycopeptide under study for treatment of serious Gram-positive infections (6). Nonspecific binding of oritavancin to labware surfaces (1, 2) and to dialysis membranes has called into question the accuracy of previous oritavancin human serum binding estimates (85.7% to 89.9% [16]). We therefore used conditions that minimize nonspecific oritavancin binding (4, 5) to estimate its binding to serum by a single in vitro methodology for three nonclinical species (mouse, rat, and dog) and humans. Protein binding estimates were derived from serum-induced increases in oritavancin MICs (9, 10, 21). To control for any impact of serum components on bacterial growth and antibiotic activity, oritavancin activity in serum was compared to its activity in serum ultrafiltrate, which is devoid of albumin, the protein responsible for the majority of oritavancin serum binding (23). The method was benchmarked using daptomycin and ceftriaxone (8, 18, 22). (Part of this work was previously presented at the 19th European Congress of Clinical Microbiology and Infectious Diseases as a poster [12].)Pooled serum from humans, mice, and rats was from Equitech-Bio (Kerrville, TX); pooled serum from beagle dogs was from Bioreclamation (Liverpool, NY). Serum ultrafiltrate was prepared using Centricon Plus-50 ultrafilters (Millipore, Billerica, MA), whose molecular mass cutoff (50 kDa) excludes albumin. MICs against Staphylococcus aureus ATCC 29213 were determined by broth microdilution (4) using arithmetic drug dilutions in 95% serum or 95% serum ultrafiltrate, each supplemented with 5% cation-adjusted Mueller-Hinton broth (CAMHB). Serum protein binding for each drug was calculated using the following formula: % bound = (1 − [mean MIC in serum ultrafiltrate/mean MIC in serum]) × 100.The MICs for each condition, serum source, and test agent were precise (Table ​(Table1),1), with a mean coefficient of variation of 17%. MICs as determined under CLSI M7-A8 conditions (Table ​(Table1)1) (4) were within the quality control ranges (5).

TABLE 1.

Oritavancin, ceftriaxone, and daptomycin MICs against S. aureus ATCC 29213 in cation-adjusted Mueller-Hinton broth and 95% serum ultrafiltrate and 95% serum from human, mouse, rat, and dog

In Vitro Activity of Azithromycin against Nontyphoidal Salmonella enterica

The in vitro activity of azithromycin against 1,237 nontyphoidal Salmonella enterica isolates collected from Finnish patients between 2003 and 2008 was investigated. Only 24 (1.9%) of the isolates tested and 15 (5.1%) of the 294 isolates with reduced fluoroquinolone susceptibility had azithromycin MICs of ≥32 μg/ml. These data show that azithromycin has good in vitro activity against nontyphoidal S. enterica, and thus, it may be a good candidate for clinical treatment studies of salmonellosis.Salmonella is one of the most common causes of food-borne illnesses and a major cause of human infections all over the world (23). Salmonella infections are usually treated with fluoroquinolones or extended-spectrum cephalosporins. Unfortunately, excessive use of fluoroquinolones both in human and in veterinary medicine has led to increasing numbers of resistant isolates, including nontyphoidal strains of Salmonella enterica (18, 19, 28). In addition, the nonclassical quinolone resistance phenotype (the Qnr phenotype), showing reduced susceptibility to ciprofloxacin (MIC of ≥0.125 μg/ml) but susceptibility or only low-level resistance to nalidixic acid (MIC of ≤32 μg/ml), has become more common (6, 14, 19, 20, 22). Extended-spectrum β-lactamase (ESBL) producers have emerged in Enterobacteriaceae and in Salmonella, and there are reports of the coappearance of ESBL and qnr genes in the same transferable genetic elements (5, 10, 21, 27). These resistance problems may jeopardize the treatment of severe Salmonella infections. Thus, alternative antibiotics for the treatment of Salmonella infections are needed.Salmonella isolates are intrinsically resistant to erythromycin via active efflux (2) but naturally susceptible to azithromycin (29), which is a 15-membered erythromycin derivative. Resistance to macrolides is usually conferred by mutations in nucleotides A2058 and A2059 of the 23S rRNA, according to the Escherichia coli numbering (26). Also, the alteration of the 50S ribosomal subunit proteins L4 (rlpD) and L22 (rlpV) may lead to macrolide resistance (4).The purpose of the present study was to determine the in vitro activity of azithromycin against nontyphoidal Salmonella isolates collected between 2003 and 2008 from Finnish patients. Special attention was paid to isolates with reduced fluoroquinolone susceptibility or showing the Qnr phenotype. In addition, mutations in the 23S rRNA and in the L4 and L22 ribosomal proteins were investigated.(This work was presented in part at the 19th European Congress of Clinical Microbiology and Infectious Diseases [ECCMID], Helsinki, Finland, 2009 [P1043].)A total of 1,237 nontyphoidal Salmonella isolates (638 domestic and 599 foreign) collected from Finnish patients between 2003 and 2008 were included in this study. Starting in January each year, we collected the first 100 domestic and the first 100 foreign, i.e., collected from Finnish travelers returning from abroad, Salmonella isolates. The strains were serotyped at the National Salmonella Reference Centre in Finland.The MICs of antimicrobial agents for the isolates were determined by the agar dilution method according to the CLSI guidelines (8). Mueller-Hinton II agar (Becton Dickinson, Cockeysville, MD) was used as the culture medium. All 1,237 Salmonella isolates were tested for susceptibility to ciprofloxacin, nalidixic acid, and azithromycin (all from Sigma, Steinheim, Germany). We tested 809 selected isolates for erythromycin (Sigma) and 635 for telithromycin (Sanofi Aventis, Paris, France) susceptibility. Control strains for susceptibility testing were as described previously (14). On the basis of earlier publications (1, 16, 17), the MIC breakpoint chosen for reduced ciprofloxacin susceptibility was ≥0.125 μg/ml. For nalidixic acid, CLSI breakpoints were used (9). Based on the EUCAST recommendation for S. enterica serovar Typhi (www.eucast.org) and a previous publication (3), the epidemiological cutoff value chosen for azithromycin was ≥32 μg/ml. The susceptibility data were analyzed by using the WHONET 5.4 computer program.Pyrosequencing was used to detect macrolide resistance causing point mutations in the ribosomal target sites A2058 and A2059 (E. coli numbering) of the 23S rRNA in 22 isolates with azithromycin MICs of ≥32 μg/ml, 44 isolates belonging to the Qnr phenotype, and 73 isolates showing erythromycin MICs of ≥32 μg/ml. Pyrosequencing was performed with previously described primers and protocols (15) using a PSQ 96MA pyrosequencer.The 50S ribosomal proteins L4 and L22 were amplified, and mutations were screened in 24 isolates having azithromycin MICs of ≥32 μg/ml, of which 6 were of the Qnr phenotype. In addition, 13 isolates having only erythromycin MICs of ≥32 μg/ml and showing the Qnr phenotype were tested. DNA was prepared as described above. The specific primers used for amplification of the complete L4 and L22 genes rlpD and rlpV were Salm_L4_f (5′-TGAAGGCGTAAGGGGATAGCA-3′) and Salm_L4_r (5′-TCAGCAGA CGTTCTTCACGAA-3′) and Salm_L22_f (5′-GAAATAAGGTAG GAGGAAGAG-3′) and Salm_L22_r (5′-CCATTGCTAGTCTCCAGAGTC-3′). The PCR conditions were as follows: 94°C for 10 min, 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s for 33 cycles. Any L4- or L22-positive results were confirmed by direct sequencing of both strands of amplicons using specific PCR primers as previously described (24). Amino acid sequences were then compared with the known rlpD and rlpV genes by a BLAST search through the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/blast/).Two different populations of S. enterica were detected regarding the azithromycin MIC distribution. The majority of the isolates had azithromycin MICs of 4 to 8 μg/ml, i.e., representing the wild-type population, whereas a minority of the isolates had MICs of 32 to ≥128 μg/ml, i.e., over the epidemiological cutoff value. Between 2003 and 2008, 24 (1.9%) of the 1,237 isolates had azithromycin MICs of ≥32 μg/ml (Fig. ​(Fig.1;1; Table ​Table1).1). Nine (1.4%) of the 638 domestic isolates and 15 (2.1%) of the 599 foreign isolates had azithromycin MICs over the epidemiological cutoff value.Open in a separate windowFIG. 1.Histograms of azithromycin (n = 1,237), erythromycin (n = 809), and telithromycin (n = 635) MICs for S. enterica isolates collected from Finnish travelers between 2003 and 2008. The vertical line represents the resistance breakpoint.

TABLE 1.

Twenty-four S. enterica isolates showing azithromycin MICs of ≥32 μg/ml

Prevalence of Fosfomycin Resistance among CTX-M-Producing Escherichia coli Clinical Isolates in Japan and Identification of Novel Plasmid-Mediated Fosfomycin-Modifying Enzymes

We evaluated the in vitro activity of fosfomycin against a total of 192 CTX-M β-lactamase-producing Escherichia coli strains isolated in 70 Japanese clinical settings. Most of the isolates (96.4%) were found to be susceptible to fosfomycin. On the other hand, some of the resistant isolates were confirmed to harbor the novel transferable fosfomycin resistance determinants named FosA3 and FosC2, which efficaciously inactivate fosfomycin through glutathione S-transferase activity.Clinical efficacy of an old antibiotic, fosfomycin, is being reassessed owing to its in vitro high potent activity against multidrug-resistant Gram-negative bacilli belonging to the family Enterobacteriaceae (5, 6, 8). In the present study, we investigated the prevalence of fosfomycin resistance among CTX-M extended-spectrum β-lactamase (ESBL)-producing Escherichia coli clinical isolates in Japan and clarified the molecular mechanisms underlying the fosfomycin resistance, with special focus on exogenous resistance determinants, like the FosA^TN protein (9).A total of 192 CTX-M ESBL-producing E. coli isolates, which were collected from 70 medical facilities throughout Japan between 2002 and 2007, were retrospectively subjected to fosfomycin susceptibility testing with the agar dilution method according to the CLSI guideline (4). The result is shown in Fig. ​Fig.1.1. Most of the strains (96.4%) investigated were susceptible to fosfomycin (MIC, ≤64 μg/ml), while seven isolates (3.6%) showed nonsusceptibility to fosfomycin (MIC, ≥128 μg/ml). It seems likely that CTX-M-producing E. coli isolates that have acquired fosfomycin resistance are infrequent in Japan, and these data suggest the probable clinical efficacy of fosfomycin for the treatment of infectious diseases, like urinary tract infections (UTIs), caused by CTX-M-producing E. coli to some extent.Open in a separate windowFIG. 1.Distribution of fosfomycin MICs for the 192 CTX-M-producing E. coli isolates.We evaluated the fosfomycin resistance mechanism of the 10 isolates and found reduced susceptibility to fosfomycin (MIC, ≥64 μg/ml) (Fig. ​(Fig.11 and Table ​Table1).1). The transmissibility of the fosfomycin resistance determinant in the 10 isolates was investigated, and it was found that the nature of fosfomycin resistance of three strains, 08-642, 06-607, and C316, was successfully transferred to a recipient E. coli strain. The cefotaxime resistance phenotype was cotransferred to a recipient strain with the fosfomycin resistance (Table ​(Table11).

TABLE 1.

Characteristics of E. coli strains used in the study

Comparison of In Vitro Activities of Fluoroquinolone-Like 2,4- and 1,3-Diones

Bacterial resistance presents a difficult issue for fluoroquinolone treatment of bacterial infections. In previous work, we reported that 8-methoxy-quinazoline-2,4-diones are active against quinolone-resistant mutants of Escherichia coli. Here, we demonstrate the activity of a representative 8-methoxy-quinazoline-2,4-dione against quinolone-resistant gyrases. Furthermore, 8-methoxy-quinazoline-2,4-dione and other diones are shown to inhibit Staphylococcus aureus gyrase and topoisomerase IV with similar degrees of efficacy, suggesting that the diones might act as dual-targeting agents against S. aureus.Antibiotic resistance is among the most difficult problems we currently face during the treatment of bacterial infections (10, 32). The fluoroquinolones are among the antibacterials affected by resistance, which can severely limit their clinical use (1, 6, 29). Thus, there is an urgent need to develop antimicrobial agents that are effective against drug-resistant pathogens. Two properties of quinolone-like compounds are likely to be useful for finding effective derivatives: (i) activity against quinolone-resistant mutants already present (12) and (ii) equal effectiveness against the two quinolone targets, DNA gyrase and topoisomerase IV (Topo IV) (dual-targeting agents; dual-targeting agents are expected to slow the emergence of drug-resistant mutants [4, 19, 23, 26, 31]). The presence of an 8-methoxy group (5, 22, 33) and changing the quinolone core structure to either quinazoline-2,4-dione (2, 8, 18) or pyrido[1,2-c]pyrimidine-1,3-dione (UI7) improve the activity of fluoroquinolone-like compounds against fluoroquinolone-resistant bacteria. We recently identified 8-methoxy-quinazoline-2,4-diones that show little increase in MIC due to gyrA or gyrB quinolone resistance mutations of Escherichia coli (12). To establish that this activity against mutant bacteria is due to improved activity against gyrase, we examined the activity of a representative 8-methoxy-quinazoline-2,4-dione (UING5-207; 8-methoxy 2,4-dione) with purified gyrase. We also assessed its effect on the activity of purified Topo IV to determine whether the in vivo results were likely due to a target switch. Furthermore, to better understand how dione structure influences target selection, we compared the effectiveness of diones for inhibition of catalytic activities of Staphylococcus aureus and E. coli topoisomerases.Based on MIC values determined in previous work (12), we selected three mutant gyrases, GyrA S83W gyrase, GyrA G81C gyrase, and GyrA A67S gyrase, as examples exhibiting high, moderate, and low levels of quinolone resistance in vivo. Mutations were introduced into the E. coli gyrA gene using the overlap extension PCR technique (17), and subunits of E. coli and S. aureus gyrases and Topo IVs were expressed and purified. The active enzymes were reconstituted as described previously (13-16, 28). In vitro activities of the 8-methoxy 2,4-dione were compared with those of a cognate 8-methyl-quinazoline-2,4-dione (UIJR1-048; 8-methyl 2,4-dione), 5-methoxypyrido[1,2-c]pyrimidine-1,3-dione (UIJR1-100; 5-methoxy 1,3-dione), 8-methoxy fluoroquinolone (UING5-249), and ciprofloxacin, a clinically important fluoroquinolone. Synthesis of the diones and 8-methoxy fluoroquinolone was achieved using methods described previously (7, 12, 30); their structures are shown in Fig. ​Fig.1.1. A DNA supercoiling assay (for example, see Fig. ​Fig.2)2) was employed to examine the effects of these compounds on the catalytic activities of wild-type and mutant gyrases; a decatenation assay was used with Topo IV (Table ​(Table1).1). The abilities of these compounds to poison the E. coli topoisomerases were assessed using a DNA cleavage assay (Table ​(Table2).2). These assays were conducted as described previously (24). The activity against mutant enzymes was expressed as the ratio of either the 50% inhibitory concentration (IC₅₀) or the CC₃ value (the concentration required to triple the level of DNA cleavage from the background level in the absence of drug) for a mutant gyrase relative to that of wild-type gyrase for catalytic inhibition and poisoning assays, respectively. The CC₃ value was used to minimize bias due to multiple cleavage events in a single DNA molecule.Open in a separate windowFIG. 1.Structures of the diones and fluoroquinolones used in this study. The structures of 8-methoxy 2,4-dione (UING5-207), 8-methyl 2,4-dione (UIJR1-048), 5-methoxy 1,3-dione (UIJR1-100), 8-methoxy fluoroquinolone (UING5-249), and ciprofloxacin are shown.Open in a separate windowFIG. 2.Representative results of supercoiling assays. The supercoiling assay was conducted using wild-type gyrase and GyrA S83W gyrase to examine the effect of either ciprofloxacin (A) or 8-methoxy 2,4-dione (B). wt, wild-type gyrase; S83W, GyrA S83W gyrase.

TABLE 1.

Inhibition of the catalytic activities of E. coli gyrase and Topo IV