In Vitro Activity of the New Fluoroketolide Solithromycin (CEM-101) against a Large Collection of Clinical Neisseria gonorrhoeae Isolates and International Reference Strains,Including Those with High-Level Antimicrobial Resistance: Potential Treatment Option for Gonorrhea?

Gonorrhea may become untreatable, and new treatment options are essential. We investigated the in vitro activity of the first fluoroketolide, solithromycin. Clinical Neisseria gonorrhoeae isolates and reference strains (n = 246), including the two extensively drug-resistant strains H041 and F89 and additional isolates with clinical cephalosporin resistance and multidrug resistance, were examined. The activity of solithromycin was mainly superior to that of other antimicrobials (n = 10) currently or previously recommended for gonorrhea treatment. Solithromycin might be an effective treatment option for gonorrhea.     

In Vitro Activity of β-Lactams in Combination with β-Lactamase Inhibitors against Multidrug-Resistant Mycobacterium tuberculosis Isolates

The combination of β-lactams and β-lactamase inhibitors has been shown to have potent in vitro activity against multidrug-resistant tuberculosis (MDR-TB) isolates. In order to identify the most potent β-lactam–β-lactamase inhibitor combination against MDR-TB, we selected nine β-lactams and three β-lactamase inhibitors, which belong to different subgroups. A total of 121 MDR-TB strains were included in this study. Out of the β-lactams used herein, biapenem was the most effective against MDR-TB and had an MIC₅₀ value of 8 μg/ml. However, after the addition of clavulanate or sulbactam, meropenem exhibited the most potent anti-MDR-TB activity with an MIC₅₀ value of 4 μg/ml. For meropenem, 76 (62.8%), 41 (33.9%), and 22 (18.2%) of the 121 MDR-TB strains were subjected to a synergistic effect when the drug was combined with sulbactam, tazobactam, or clavulanate, respectively. Further statistical analysis revealed that significantly more strains experienced a synergistic effect when exposed to the combination of meropenem with sulbactam than when exposed to meropenem in combination with tazobactam or clavulanate, respectively (P < 0.01). In addition, a total of 10.7% (13/121) of isolates harbored mutations in the blaC gene, with two different nucleotide substitutions: AGT333AGG and ATC786ATT. For the strains with a Ser111Arg substitution in BlaC, a better synergistic effect was observed in the meropenem-clavulanate and in the amoxicillin-clavulanate combinations than that in a synonymous single nucleotide polymorphism (SNP) group. In conclusion, our findings demonstrate that the combination of meropenem and sulbactam shows the most potent activity against MDR-TB isolates. In addition, the Ser111Arg substitution of BlaC may be associated with an increased susceptibility of MDR-TB isolates to meropenem and amoxicillin in the presence of clavulanate.     

In Vitro Activity of Ceftolozane Alone and in Combination with Tazobactam against Extended-Spectrum-β-Lactamase-Harboring Enterobacteriaceae

Ceftolozane, formally CXA-101, is a new antipseudomonal cephalosporin that is also active in vitro against Enterobacteriaceae but is vulnerable to extended-spectrum β-lactamases (ESBLs). The addition of tazobactam is intended to broaden coverage to most ESBL-producing Escherichia coli and Klebsiella pneumonia as well as other Enterobacteriaceae. The in vitro activities of ceftolozane-tazobactam combinations against 67 clinically and molecularly characterized ESBL-producing isolates were examined by checkerboard MIC testing to evaluate their potential clinical feasibility and to assess the optimal tazobactam concentrations to be used in MIC determinations of ceftolozane. Isolates included those from E. coli (n = 32), K. pneumoniae (n = 19), Enterobacter cloacae (n = 15), and Citrobacter freundii (n = 1). Checkerboard experiments were performed to study interactions over the range of 0.008 to 64 mg/liter ceftolozane and 0.063 to 32 mg/liter tazobactam using 2-fold-dilution series. The MIC₅₀ and MIC₉₀ of ceftolozane alone for all isolates were 16 and ≥64 mg/liter, respectively. Increasing concentrations of tazobactam resulted in decreasing MICs of ceftolozane. The 50th and 90th percentile concentrations of tazobactam required to reduce the MIC of ceftolozane to 8 mg/liter for all organisms in this ESBL collection were 0.5 and 4 mg/liter, respectively. For E. coli, K. pneumoniae, and E. cloacae, these values were 0.5 and 2, 1 and 16, and 0.5 and 4 mg/liter, respectively. When combined with a fixed amount of 4 mg/liter tazobactam (current CLSI concentration used for susceptibility testing), 90% of the isolates would have an MIC of ≤4 mg/liter. The combination ceftolozane-tazobactam is a promising alternative option for treating infections due to ESBL-harboring isolates.     

Does the Activity of the Combination of Imipenem and Colistin In Vitro Exceed the Problem of Resistance in Metallo-β-Lactamase-Producing Klebsiella pneumoniae Isolates?

Using time-kill methodology, we investigated the interactions of an imipenem-colistin combination against 42 genetically distinct Klebsiella pneumoniae clinical isolates carrying a bla_VIM-1-type gene. Irrespective of the imipenem MIC, the combination was synergistic (50%) or indifferent (50%) against colistin-susceptible strains, while it was antagonistic (55.6%) and rarely synergistic (11%) against non-colistin-susceptible strains (with synergy being observed only against strains with colistin MICs of 3 to 4 μg/ml). The combination showed improved bactericidal activity against isolates susceptible either to both agents or to colistin.During the past decade, VIM metallo-β-lactamases (MBLs) have spread rapidly among Enterobacteriaceae (4). MBL producers commonly exhibit a multiple-drug resistance phenotype as a result of combined chromosomally encoded or plasmid-mediated resistance mechanisms. Frequently, colistin and tigecycline remain the only therapeutic choices. Tigecycline has demonstrated in vitro activity against MBL producers (18), but evidence of in vivo efficacy against a variety of clinical infections (i.e., bacteremia or pneumonia) is still limited. On the other hand, randomized controlled trials supporting the use of colistin as a single-drug regimen, as well as studies on its pharmacokinetic/pharmacodynamic properties, are lacking (11). Recently, the emergence of colistin resistance among Klebsiella pneumoniae isolates further jeopardized the already limited treatment options in the intensive care unit setting (2). For all these reasons, combination therapies are frequently used in clinical practice, especially in hospitals with high rates of infections by MBL producers.(Some of these data were presented at the 45th Infectious Disease Society Annual Meeting, 2007 [15a].)We investigated the in vitro activities of imipenem and colistin alone and in combination against 42 unique clinical isolates of MBL-producing K. pneumoniae isolated in Greek hospitals from February 2004 to September 2006. MICs were determined by Etest (AB Biodisc, Solna, Sweden) and interpreted according to CLSI breakpoints for imipenem (3) and EUCAST breakpoints for colistin (7). The presence of a bla_VIM gene was confirmed by PCR (15). On the basis of PCR-restriction fragment length polymorphism analysis (9), all isolates carried a bla_VIM-1-type gene. Extended-spectrum β-lactamase production was detected with a modified CLSI confirmatory test (8). Genetic relatedness among studied isolates was evaluated with repetitive extragenic palindromic PCR methods (10). Patterns that differed by more than one amplification band were characterized as different. In vitro interactions between imipenem and colistin were tested using time-kill methodology. Antibiotic concentrations used were 10 μg/ml for imipenem (Merck, Rahway, NJ) and 5 μg/ml for colistin sulfate (Sigma, St. Louis, MO) because these concentrations represent the steady state achievable in human serum during treatment (12, 16) and thus are clinically relevant. For susceptible strains, if 4× MIC was not higher than 10 or 5 μg/ml for imipenem or colistin, respectively, this concentration was also tested.Synergy was defined as a ≥2-log₁₀ decrease in CFU/ml between the combination and the most active single agent at the different time points, with the number of surviving organisms in the presence of the combination being ≥2 log₁₀ CFU/ml below the number of organisms in the starting inoculum. Antagonism was defined as a ≥2-log₁₀ increase in CFU/ml between the combination and the most active single agent. All other interactions were characterized as indifferent. Bactericidal activity of single antibiotics or combinations was defined as a ≥3-log₁₀ reduction in the CFU/ml of the initial inoculum after 24 h of incubation (1, 6). The lower limit of detection was 1.6 log₁₀ CFU/ml. For analysis of the results, isolates were classified into four groups according to susceptibility to imipenem and colistin. The chi-square test was used to compare proportions of killing activity or synergy between groups by using Yates continuity correction in two-by-two tables. P values of <0.05 were considered statistically significant.The results are shown in Table ​Table1.1. The imipenem-colistin combination exhibited synergy against 12 of 24 (50%) colistin-susceptible MBL-producing K. pneumoniae isolates tested, but it was antagonistic against 10 of 18 (55.6%) non-colistin-susceptible isolates. Of note, isolates showing colistin MICs of 3 to 4 μg/ml behaved more like colistin-susceptible isolates, since in two of them (50%) a synergistic interaction was noted after exposure to the combination.

TABLE 1.

MICs (μg/ml) of imipenem and colistin against bla_VIM-1-type MBL-producing K. pneumoniae isolates and in vitro interaction of the combination

Is Neisseria gonorrhoeae Initiating a Future Era of Untreatable Gonorrhea?: Detailed Characterization of the First Strain with High-Level Resistance to Ceftriaxone

Recently, the first Neisseria gonorrhoeae strain (H041) that is highly resistant to the extended-spectrum cephalosporin (ESC) ceftriaxone, the last remaining option for empirical first-line treatment, was isolated. We performed a detailed characterization of H041, phenotypically and genetically, to confirm the finding, examine its antimicrobial resistance (AMR), and elucidate the resistance mechanisms. H041 was examined using seven species-confirmatory tests, antibiograms (30 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of ESC resistance determinants (penA, mtrR, penB, ponA, and pilQ). Transformation, using appropriate recipient strains, was performed to confirm the ESC resistance determinants. H041 was assigned to serovar Bpyust, MLST sequence type (ST) ST7363, and the new NG-MAST ST4220. H041 proved highly resistant to ceftriaxone (2 to 4 μg/ml, which is 4- to 8-fold higher than any previously described isolate) and all other cephalosporins, as well as most other antimicrobials tested. A new penA mosaic allele caused the ceftriaxone resistance. In conclusion, N. gonorrhoeae has now shown its ability to also develop ceftriaxone resistance. Although the biological fitness of ceftriaxone resistance in N. gonorrhoeae remains unknown, N. gonorrhoeae may soon become a true superbug, causing untreatable gonorrhea. A reduction in the global gonorrhea burden by enhanced disease control activities, combined with wider strategies for general AMR control and enhanced understanding of the mechanisms of emergence and spread of AMR, which need to be monitored globally, and public health response plans for global (and national) perspectives are important. Ultimately, the development of new drugs for efficacious gonorrhea treatment is necessary.     

In Vitro Activity of Ceftaroline Alone and in Combination against Clinical Isolates of Resistant Gram-Negative Pathogens,Including β-Lactamase-Producing Enterobacteriaceae and Pseudomonas aeruginosa

Ceftaroline is a novel broad-spectrum cephalosporin that exhibits bactericidal activity against many gram-positive and -negative pathogens. However, the activity of ceftaroline cannot be solely relied upon for eradication of multidrug-resistant gram-negative isolates, such as Pseudomonas aeruginosa and extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, which represent a current clinical concern. As drug combinations might be beneficial by potential synergy, we evaluated the in vitro activity of ceftaroline combined with meropenem, aztreonam, cefepime, tazobactam, amikacin, levofloxacin, and tigecycline. Susceptibility testing was performed for 20 clinical P. aeruginosa isolates, 10 ESBL-producing Escherichia coli isolates, 10 ESBL-producing Klebsiella pneumoniae isolates, and 10 AmpC-derepressed Enterobacter cloacae isolates. Time-kill experiments were performed for 10 isolates using antimicrobials at one-fourth the MIC. Ceftaroline exhibited a MIC range of 0.125 to 1,024 μg/ml and was reduced 2- to 512-fold by combination with tazobactam (4 μg/ml) for ESBL-producing strains. In time-kill experiments, ceftaroline plus amikacin was synergistic against 90% of the isolates (and indifferent for one P. aeruginosa isolate). Ceftaroline plus tazobactam was indifferent for E. cloacae and P. aeruginosa strains but synergistic against 100% of E. coli and K. pneumoniae isolates. Combinations of ceftaroline plus meropenem or aztreonam were also synergistic for all E. coli and E. cloacae isolates, respectively, but indifferent against 90% of the other isolates. Finally, combinations of ceftaroline plus either tigecycline, levofloxacin, or cefepime were indifferent for 100% of the isolates. No antagonism was observed with any combination. Ceftaroline plus amikacin appeared as the most likely synergistic combination. This represents a promising therapeutic option, and further studies are warranted to elucidate the clinical value of ceftaroline combinations against resistant gram-negative pathogens.Infections due to multidrug-resistant (MDR) gram-negative pathogens affect both immunocompetent and immunocompromised patients and represent a current and important clinical concern. Over the last decade the incidence of these infections has increased throughout the world, leading to an alarming deficit in effective antimicrobial agents (18, 21). Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae as well as Pseudomonas aeruginosa are among the most important and frequent nosocomial pathogens and are also resistant to many classes of antibiotics (3, 32). The anti-infective agents currently available to treat Enterobacteriaceae infections include fluoroquinolones and β-lactams, for which the activity has been markedly compromised by the emergence of ESBL enzymes and the spread of plasmid-mediated fluoroquinolone resistance (25). For infections caused by P. aeruginosa, which displays a high rate of multidrug resistance, empirical therapy often requires combination therapy (2, 14). Although it may not always prevent the emergence of resistance, antimicrobial combinations may nevertheless enhance the killing effect and the likelihood of cure, by extending the spectra of activities of drugs active against MDR organisms (3, 5, 24).Combinations of a β-lactam with an aminoglycoside or a β-lactam inhibitor are the most common and have demonstrated greater efficiencies than monotherapy with a β-lactam in serious infections, including gram-negative sepsis or bacteremia (14, 31). Due to the potential toxicity of aminoglycosides, other combinations, such as a β-lactam plus a fluoroquinolone or double β-lactam combinations, have also been investigated and have demonstrated promising results both in vivo and in vitro (13, 28). Mechanisms responsible for the synergistic effect observed with some of these combinations have been investigated. For example, it has been suggested that penetration of aminoglycosides is increased in the presence of a β-lactam, and the degradation of β-lactams may be considerably reduced in the presence of a β-lactamase inhibitor (19).Several reviews recently reported anti-infective agents either currently available or in development to treat MDR gram-negative infections, but these reviews also emphasized the urgent need for new therapeutic strategies (3, 21, 22). Ceftaroline (formerly referred to as PPI-0903 M or T-91825) is a novel semisynthetic cephalosporin, discovered and initially synthesized by Takeda Chemical Industries, Ltd., Japan (11). Currently in phase III development by Forest Laboratories, ceftaroline exhibits a broad spectrum of activity, covering most of the resistant gram-positive pathogens as well as many common gram-negative organisms (9, 12, 26, 27). The unique biological activity of this cephalosporin results from its higher affinity for the altered penicillin-binding protein 2, PBP2′ (or PBP2a), which is predominantly expressed in methicillin-resistant Staphylococcus aureus, including strains with reduced susceptibility to glycopeptides (23). Ceftaroline also exhibits excellent activity against Streptococcus pneumoniae isolates, and a clinical trial for community-acquired pneumonia is currently under way (http://clinicaltrials.gov). Like other β-lactams, ceftaroline activity against gram-negative species is limited by its affinity for the PBPs and its susceptibility to β-lactamases, especially the ESBL enzymes and cephalosporinases of Enterobacteriaceae and P. aeruginosa strains (23, 27). Although minimum to no activity was reported against MDR gram-negative bacilli, ceftaroline represents a potential candidate for combination therapy, which may extend its spectrum of activity as well as offer a novel and unique therapeutic option to cover mixed infections due to methicillin-resistant S. aureus and MDR gram-negative organisms (27).The objective of this study was to evaluate the in vitro activity of ceftaroline against clinical MDR gram-negative isolates and to investigate its potential for synergy in combination with a large panel of antimicrobials, including β-lactams (aztreonam, meropenem, and cefepime), an aminoglycoside (amikacin), a β-lactamase inhibitor (tazobactam), fluoroquinolone (levofloxacin), and glycylcycline (tigecycline), which potentially may offer synergistic combinations.(A portion of this work was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy and the 46th Annual Meeting of the Infectious Diseases Society of America, Washington, DC, in 2008.)     

In Vitro and In Vivo Antifungal Activity of Amphotericin B Lipid Complex: Are Phospholipases Important?

Amphotericin B lipid complex for injection (ABLC) is a suspension of amphotericin B complexed with the lipids l-α-dimyristoylphosphatidylcholine (DMPC) and l-α-dimyristoylphosphatidylglycerol. ABLC is less toxic than amphotericin B deoxycholate (AmB-d), while it maintains the antifungal activity of AmB-d. Active amphotericin B can be released from ABLC by exogenously added (snake venom, bacteria, or Candida-derived) phospholipases or by phospholipases derived from activated mammalian vascular tissue (rat arteries). Such extracellular phospholipases are capable of hydrolyzing the major lipid in ABLC. Mutants of C. albicans that were resistant to ABLC but not AmB-d in vitro were deficient in extracellular phospholipase activity, as measured on egg yolk agar or as measured by their ability to hydrolyze DMPC in ABLC. ABLC was nevertheless effective in the treatment of experimental murine infections produced by these mutants. Isolates of Aspergillus species, apparently resistant to ABLC in vitro (but susceptible to AmB-d), were also susceptible to ABLC in vivo. We suggest that routine in vitro susceptibility tests with ABLC itself as the test material may not accurately predict the in vivo activity of ABLC and that the enhanced therapeutic index of ABLC relative to that of AmB-d in vivo may be due, in part, to the selective release of active amphotericin B from the complex at sites of fungal infection through the action of fungal or host cell-derived phospholipases.Amphotericin B has been the agent of choice for the treatment of serious fungal infections for more than 35 years. However, administration of the most common preparation of amphotericin B (a sodium deoxycholate colloidal suspension) is associated with severe, dose-limiting acute and chronic toxicities, particularly nephrotoxicity.Amphotericin B lipid complex for injection (ABLC) is a suspension of amphotericin B complexed with the lipids l-α-dimyristoylphosphatidylcholine (DMPC) and l-α-dimyristoylphosphatidylglycerol (DMPG) (11). The safety and efficacy of ABLC have been extensively evaluated in laboratory (4–6, 14) and clinical (7, 24, 25) studies. Those studies have shown that ABLC is, in general, markedly less toxic than amphotericin B deoxycholate (AmB-d) and has antifungal activity at least comparable to and sometimes enhanced over that of AmB-d.Complexation with lipids appears to stabilize amphotericin B in a self-associated state so that it is not available to interact with cellular membranes (the presumed major site of its antifungal activity and its mammalian toxicity) (12, 17). It was previously demonstrated that ABLC is more than 1,000-fold less hemolytic to erythrocytes in vitro than AmB-d and that active (hemolytic) amphotericin B can be released from ABLC by a heat-labile, extracellular fungal product (lipase) (17). It has also been demonstrated that in vitro the MICs of ABLC for certain phospholipase-deficient non-Candida albicans Candida species and phospholipase-deficient mutants of C. albicans are higher than those of AmB-d (13). In the present study we further evaluated the role of phospholipases in the in vitro and in vivo antifungal activity of ABLC.     

In Vitro Activity of Nifuratel on Vaginal Bacteria: Could It Be a Good Candidate for the Treatment of Bacterial Vaginosis?

Bacterial vaginosis is characterized by a shift of the physiological flora to a diverse spectrum of bacteria, where Gardnerella vaginalis and Atopobium vaginae are the most important markers. In this study, the antimicrobial activity of nifuratel against G. vaginalis, A. vaginae, and lactobacilli was compared with that of the two currently used antibiotics metronidazole and clindamycin. Results suggest that nifuratel has a better spectrum of activity, being highly active against G. vaginalis and A. vaginae without affecting lactobacilli.     

In Vitro Activity of Daptomycin in Combination with β-Lactams,Gentamicin, Rifampin,and Tigecycline against Daptomycin-Nonsusceptible Enterococci

Enterococci that are nonsusceptible (NS; MIC > 4 μg/ml) to daptomycin are an emerging clinical concern. The synergistic combination of daptomycin plus beta-lactams has been shown to be effective against vancomycin-resistant Enterococcus (VRE) species in vitro. This study systematically evaluated by in vitro time-kill studies the effect of daptomycin in combination with ampicillin, cefazolin, ceftriaxone, ceftaroline, ertapenem, gentamicin, tigecycline, and rifampin, for a collection of 9 daptomycin-NS enterococci that exhibited a broad range of MICs and different resistance-conferring mutations. We found that ampicillin plus daptomycin yielded the most consistent synergy but did so only for isolates with mutations to the liaFSR system. Daptomycin binding was found to be enhanced by ampicillin in a representative isolate with such mutations but not for an isolate with mutation to the yycFGHIJ system. In contrast, ampicillin enhanced the killing of the LL-37 human antimicrobial peptide against daptomycin-NS E. faecium with either the liaFSR or yycFGHIJ mutation. Antagonism was noted only for rifampin and tigecycline and only for 2 or 3 isolates. These data add support to the growing body of evidence indicating that therapy combining daptomycin and ampicillin may be helpful in eradicating refractory VRE infections.     

In Vitro Activity and the Efficacy of Arbekacin,Cefminox, Fosfomycin,Biapenem Against Gram-Negative Organisms: New Treatment Options?

In recent years bacteria have shown much more potential to develop antimicrobial resistance. This mandates not only to the development of new drugs but also to the re-evaluation of already existing ones. Present study is sought to evaluate the in vitro activity of a few newer antibiotics which are not marketed in many countries at present. This study evaluates the in vitro activity of newer antibiotics i. e arbekacin, cefminox, fosfomycin and biapenem against clinical isolates Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. The minimum inhibition concentration of antibiotics was determined by the agar dilution method. The main finding of the present study is that fosfomycin was active against both E. coli and K. pneumoniae where as cefminox exhibited an insignificant intermediate level activity against both E. coli and K. pneumoniae. Biapenem was more active on E. coli than other tested organisms. Colistin showed inhibition for the entire organisms in present study. The present study suggests that colistin and fosfomycin are promising antibiotics for future use; however clinical trials are needed to confirm these findings and to evaluate potency of these four antibiotics.     

In Vitro Activities of 21 Antimicrobial Agents Alone and in Combination with Aminoglycosides or Fluoroquinolones against Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates Causing Bacteremia

We evaluated the in vitro activity of various antimicrobials alone and in combination against 291 extended-spectrum-β-lactamase-producing Escherichia coli (ESBL-EC) isolates causing bacteremia in South Korean hospitals. Ceftazidime, cefepime, and piperacillin-tazobactam in combination with amikacin showed greater activity than found in combination with ciprofloxacin. In settings with a high prevalence of ESBL-producing pathogens, combination aminoglycoside antimicrobial therapy, especially with amikacin, may be considered for empirical therapy against suspected Gram-negative sepsis as a carbapenem-saving strategy.     

Effects of Klebsiella pneumoniae Carbapenemase Subtypes,Extended-Spectrum β-Lactamases,and Porin Mutations on the In Vitro Activity of Ceftazidime-Avibactam against Carbapenem-Resistant K. pneumoniae

Avibactam is a novel β-lactamase inhibitor with affinity for Klebsiella pneumoniae carbapenemases (KPCs). In combination with ceftazidime, the agent demonstrates activity against KPC-producing K. pneumoniae (KPC-Kp). KPC-Kp strains are genetically diverse and harbor multiple resistance determinants, including defects in outer membrane proteins and extended-spectrum β-lactamases (ESBLs). Mutations in porin gene ompK36 confer high-level carbapenem resistance to KPC-Kp strains. Whether specific mechanisms of antimicrobial resistance also influence the activity of ceftazidime-avibactam is unknown. We defined the effects of ceftazidime-avibactam against 72 KPC-Kp strains with diverse mechanisms of resistance, including various combinations of KPC subtypes and ESBL and ompK36 mutations. Ceftazidime MICs ranged from 64 to 4,096 μg/ml and were lowered by a median of 512-fold with the addition of avibactam. All strains exhibited ceftazidime-avibactam MICs at or below the CLSI breakpoint for ceftazidime (≤4 μg/ml; range, 0.25 to 4). However, the MICs were within two 2-fold dilutions of the CLSI breakpoint against 24% of the strains, and those strains would be classified as nonsusceptible to ceftazidime by EUCAST criteria (MIC > 1 μg/ml). Median ceftazidime-avibactam MICs were higher against KPC-3 than KPC-2 variants (P = 0.02). Among KPC-2-Kp strains, the presence of both ESBL and porin mutations was associated with higher drug MICs compared to those seen with either factor alone (P = 0.003 and P = 0.02, respectively). In conclusion, ceftazidime-avibactam displays activity against genetically diverse KPC-Kp strains. Strains with higher-level drug MICs provide a reason for caution. Judicious use of ceftazidime-avibactam alone or in combination with other agents will be important to prevent the emergence of resistance.     

Activity of a New Cephalosporin,CXA-101 (FR264205), against β-Lactam-Resistant Pseudomonas aeruginosa Mutants Selected In Vitro and after Antipseudomonal Treatment of Intensive Care Unit Patients

CXA-101, previously designated {"type":"entrez-nucleotide","attrs":{"text":"FR264205","term_id":"258272820","term_text":"FR264205"}}FR264205, is a new antipseudomonal cephalosporin. We evaluated the activity of CXA-101 against a highly challenging collection of β-lactam-resistant Pseudomonas aeruginosa mutants selected in vitro and after antipseudomonal treatment of intensive care unit (ICU) patients. The in vitro mutants investigated included strains with multiple combinations of mutations leading to several degrees of AmpC overexpression (ampD, ampDh2, ampDh3, and dacB [PBP4]) and porin loss (oprD). CXA-101 remained active against even the AmpD-PBP4 double mutant (MIC = 2 μg/ml), which shows extremely high levels of AmpC expression. Indeed, this mutant showed high-level resistance to all tested β-lactams, except carbapenems, including piperacillin-tazobactam (PTZ), aztreonam (ATM), ceftazidime (CAZ), and cefepime (FEP), a cephalosporin considered to be relatively stable against hydrolysis by AmpC. Moreover, CXA-101 was the only β-lactam tested (including the carbapenems imipenem [IMP] and meropenem [MER]) that remained fully active against the OprD-AmpD and OprD-PBP4 double mutants (MIC = 0.5 μg/ml). Additionally, we tested a collection of 50 sequential isolates that were susceptible or resistant to penicillicins, cephalosporins, carbapenems, or fluoroquinolones that emerged during treatment of ICU patients. All of the mutants resistant to CAZ, FEP, PTZ, IMP, MER, or ciprofloxacin showed relatively low CXA-101 MICs (range, 0.12 to 4 μg/ml; mean, 1 to 2 μg/ml). CXA-101 MICs of pan-β-lactam-resistant strains ranged from 1 to 4 μg/ml (mean, 2.5 μg/ml). As described for the in vitro mutants, CXA-101 retained activity against the natural AmpD-PBP4 double mutants, even when these exhibited additional overexpression of the MexAB-OprM efflux pump. Therefore, clinical trials are needed to evaluate the usefulness of CXA-101 for the treatment of P. aeruginosa nosocomial infections, particularly those caused by multidrug-resistant isolates that emerge during antipseudomonal treatments.The growing threat of Pseudomonas aeruginosa antimicrobial resistance results from, on the one hand, the extraordinary capacity of this microorganism for developing resistance to almost every available antibiotic by the selection of mutations in chromosomal genes and, on the other hand, the increasing prevalence of transferable resistance determinants, particularly those encoding class B carbapenemases (or metallo-β-lactamases [MBLs]) or extended-spectrum β-lactamases (ESBLs), frequently cotransferred with genes encoding aminoglycoside-modifying enzymes (16, 19).Particularly noteworthy among the mutation-mediated resistance mechanisms are those leading to the repression or inactivation of the porin OprD, conferring resistance to carbapenems (5, 7, 24, 26), or those leading to the hyperproduction of the chromosomal cephalosporinase AmpC, such as AmpD or PBP4 inactivation (11, 20), causing resistance to penicillins and cephalosporins. In addition, mutations leading to the upregulation of one of several efflux pumps encoded in the P. aeruginosa genome may confer resistance or reduced susceptibility to multiple agents, including almost all β-lactams, fluoroquinolones, and aminoglycosides (3, 18, 25). Furthermore, the accumulation of these chromosomal mutations can lead to the emergence of multidrug-resistant (MDR) (or even pan-antibiotic-resistant) strains which eventually may be responsible for outbreaks in the hospital setting (4). Indeed, sequential development of resistance to almost all available antipseudomonal agents is a not uncommon outcome of the treatment of severe P. aeruginosa infections, frequently occurring in intensive care unit (ICU) patients or patients with hematological disease (2, 10).Unfortunately, over the last 2 decades there has been very limited progress in developing novel antipseudomonal agents which can overcome MDR in P. aeruginosa (19). CXA-101 (previously designated {"type":"entrez-nucleotide","attrs":{"text":"FR264205","term_id":"258272820","term_text":"FR264205"}}FR264205), a new cephalosporin with promising characteristics for the treatment of P. aeruginosa infections, appears stable against the most common resistance mechanisms driven by mutation in this species (27, 28).The objective of this study was to investigate the activity of CXA-101 against β-lactam-resistant P. aeruginosa mutants selected in vitro and after antipseudomonal treatment of ICU patients. The in vitro mutants investigated included a highly challenging collection of strains with multiple combinations of mutations leading to various levels of AmpC overexpression (ampD, ampDh2, ampDh3, and dacB [PBP4]) and porin loss (oprD). Additionally, a well-characterized collection of 50 sequential isolates susceptible or resistant to penicillicins, cephalosporins, carbapenems, or fluoroquinolones (first-line antipseudomonal agents) that emerged during treatment of ICU patients was tested.     

In Vitro Resistance Studies with Bacteria That Exhibit Low Mutation Frequencies: Prediction of “Antimutant” Linezolid Concentrations Using a Mixed Inoculum Containing both Susceptible and Resistant Staphylococcus aureus

Bacterial resistance studies using in vitro dynamic models are highly dependent on the starting inoculum that might or might not contain spontaneously resistant mutants (RMs). To delineate concentration-resistance relationships with linezolid-exposed Staphylococcus aureus, a mixed inoculum containing both susceptible cells and RMs was used. An RM selected after the 9th passage of the parent strain (MIC, 2 μg/ml) on antibiotic-containing media (RM9; MIC, 8 μg/ml) was chosen for the pharmacodynamic studies, because the mutant prevention concentration (MPC) of linezolid against the parent strain in the presence of RM9 at 10² (but not at 10⁴) CFU/ml did not differ from the MPC value determined in the absence of the RMs. Five-day treatments with twice-daily linezolid doses were simulated at concentrations either between the MIC and MPC or above the MPC. S. aureus RMs (resistant to 2× and 4× MIC but not 8× and 16× MIC) were enriched at ratios of the 24-h area under the concentration-time curve (AUC₂₄) to the MIC that provide linezolid concentrations between the MIC and MPC for 100% (AUC₂₄/MIC, 60 h) and 86% (AUC₂₄/MIC, 120 h) of the dosing interval. No such enrichment occurred when linezolid concentrations were above the MIC and below the MPC for a shorter time (37% of the dosing interval; AUC₂₄/MIC, 240 h) or when concentrations were consistently above the MPC (AUC₂₄/MIC, 480 h). These findings obtained using linezolid-susceptible staphylococci supplemented with RMs support the mutant selection window hypothesis. This method provides an option to delineate antibiotic concentration-resistance relationships with bacteria that exhibit low mutation frequencies.     

Enterococci with Glycopeptide Resistance in Turkeys, Turkey Farmers, Turkey Slaughterers, and (Sub)Urban Residents in the South of The Netherlands: Evidence for Transmission of Vancomycin Resistance from Animals to Humans?

The number of vancomycin-resistant enterococci (VRE) relative to the total number of enterococci was determined in fecal samples from turkeys and three human populations in 1996, each with a different level of contact with turkeys, i.e., turkey farmers, turkey slaughterers, and (sub)urban residents. The percentage of VRE relative to the total enterococcal population (i.e., the degree of resistance) was low (2 to 4%) in all groups (except in six samples). No difference was observed between farmers who used avoparcin and those who did not. The pulsed-field gel electrophoresis (PFGE) patterns of the VRE isolates from the different populations were quite heterogeneous, but isolates with the same PFGE pattern were found among animal and human isolates, in addition to the isolates which were described previously (A. E. van den Bogaard, L. B. Jensen, and E. E. Stobberingh, N. Engl. J. Med. 337:1558-1559, 1997). Detailed molecular characterization of vanA-containing transposons from different isolates showed, that in addition to a previously reported strain, similar transposons were present in VRE isolates from turkeys and turkey farmers. Moreover, similar VanA elements were found not only in isolates with the same PFGE pattern but also in other strains from both humans and animals.