Potential of high-dose cefepime/tazobactam against multiresistant Gram-negative pathogens.

BACKGROUND: Early ß-lactamase inhibitors were combined with established penicillins, but different combinations may be more appropriate to counter current ß-lactamase threats, with development facilitated by the US Generating Antibiotic Incentives Now (GAIN) Act. Cefepime/tazobactam is especially attractive, combining an AmpC-stable cephalosporin with a clinically established inhibitor, active against ESBLs and suitable for high-dose administration. METHODS: Organisms (n = 563) were clinical isolates submitted to the UK national reference laboratory. MICs were determined by CLSI agar dilution with tazobactam at 4 mg/L and, for a subset, at 8 mg/L. RESULTS: Cefepime/tazobactam 8 + 4 mg/L achieved coverage of 96%-100% of Enterobacteriaceae with penicillinases, AmpC, ESBL, K1 or OXA-48 ß-lactamases. Even at 1 + 4 mg/L, the combination inhibited >94% of isolates with penicillinases, AmpC enzymes or ESBLs. Most Enterobacteriaceae with KPC and NDM carbapenemase were resistant at current cefepime breakpoints but 80% of those with VIM types were susceptible at 8 + 4 mg/L. Tazobactam did little to potentiate cefepime against non-fermenter groups, though gains were seen against AmpC-producing Acinetobacter spp. and Stenotrophomonas maltophilia. Increasing the tazobactam concentration to 8 mg/L gave further small increases in activity against Enterobacteriaceae groups. CONCLUSIONS: High-dose cefepime/tazobactam, justifying an 8 + 4 or 8 + 8 mg/L breakpoint, can achieve a carbapenem-like spectrum, with some additional coverage of OXA-48 (and maybe VIM) Enterobacteriaceae. Clinical evaluation is warranted.

In vitro activity of cefepime/zidebactam (WCK 5222) against Gram-negative bacteria.

Objectives: Diazabicyclooctanes (DBOs) inhibit class A, class C and some class D ß-lactamases. A few also bind PBP2, conferring direct antibacterial activity and a ß-lactamase-independent 'enhancer' effect, potentiating ß-lactams targeting PBP3. We tested a novel DBO, zidebactam, combined with cefepime. Methods: CLSI agar dilution MICs were determined with cefepime/zidebactam in a chequerboard format. Bactericidal activity was also measured. Results: Zidebactam MICs were ≤2 mg/L (mostly 0.12-0.5 mg/L) for most Escherichia coli , Klebsiella , Citrobacter and Enterobacter spp., but were >32 mg/L for Proteeae, most Serratia and a few E. coli , Klebsiella and Enterobacter/Citrobacter . The antibacterial activity of zidebactam dominated chequerboard studies for Enterobacteriaceae, but potentiation of cefepime was apparent for zidebactam-resistant isolates with class A and C enzymes, illustrating ß-lactamase inhibition. Overall, cefepime/zidebactam inhibited almost all Enterobacteriaceae with AmpC, ESBL, K1, KPC and OXA-48-like ß-lactamases at 1 + 1 mg/L and also 29 of 35 isolates with metallo-carbapenemases, including several resistant to zidebactam alone. Zidebactam MICs for 36 of 50 Pseudomonas aeruginosa were 4-16 mg/L, and the majority of AmpC, metallo-ß-lactamase-producing and cystic fibrosis isolates were susceptible to cefepime/zidebactam at 8 + 8 mg/L. Zidebactam MICs for Acinetobacter baumannii and Stenotrophomonas maltophilia were >32 mg/L; potentiation of cefepime was frequent for S. maltophilia , but minimal for A. baumannii . Kill curve results largely supported MICs. Conclusions: Zidebactam represents a second triple-action DBO following RG6080, with lower MICs for Enterobacteriaceae and P. aeruginosa . Clinical evaluation of cefepime/zidebactam must critically evaluate the reliance that can be placed on this direct antibacterial activity and on the enhancer effect as well as ß-lactamase inhibition.

In Vitro Activity of Eravacycline against Carbapenem-Resistant Enterobacteriaceae and Acinetobacter baumannii.

Eravacycline and comparators were tested against carbapenem- and tigecycline-resistant Enterobacteriaceae and Acinetobacter isolates received at the United Kingdom's national reference laboratory. Eravacycline MICs correlated closely with those of tigecycline but mostly were around 2-fold lower; both molecules retained full activity against isolates with high-level tetracycline and minocycline resistance. MIC90s of eravacycline and tigecycline were raised ca. 2-fold for carbapenem-resistant Enterobacteriaceae compared with carbapenem-susceptible controls, probably reflecting subsets of isolates with increased efflux.

Interactions of OP0595, a Novel Triple-Action Diazabicyclooctane, with ß-Lactams against OP0595-Resistant Enterobacteriaceae Mutants.

OP0595 is a novel diazabicyclooctane which, like avibactam, inhibits class A and C ß-lactamases. In addition, unlike avibactam, it has antibacterial activity, with MICs of 0.5 to 4 µg/ml for most members of the family Enterobacteriaceae, owing to inhibition of PBP2; moreover, it acts synergistically with PBP3-active ß-lactams independently of ß-lactamase inhibition, via an "enhancer effect." Enterobacteriaceae mutants stably resistant to 16 µg/ml OP0595 were selected on agar at frequencies of approximately 10(-7). Unsurprisingly, OP0595 continued to potentiate substrate ß-lactams against mutants derived from Enterobacteriaceae with OP0595-inhibited class A and C ß-lactamases. Weaker potentiation of partners, especially aztreonam, cefepime, and piperacillin--less so meropenem--remained frequent for OP0595-resistant Enterobacteriaceae mutants lacking ß-lactamases or with OP0595-resistant metallo-ß-lactamases (MBLs), indicating that the enhancer effect is substantially retained even when antibiotic activity is lost.

In vitro selection of ceftazidime-avibactam resistance in Enterobacteriaceae with KPC-3 carbapenemase.

Ceftazidime-avibactam is active against most Enterobacteriaceae isolates with KPC carbapenemases. We investigated whether this activity could be compromised by mutation. Single-step and multistep selections were attempted using ceftazidime-avibactam (avibactam fixed at 1 or 4 µg/ml) versus two strains each of Enterobacter cloacae and Klebsiella pneumoniae, all with the KPC-3 enzyme. Mutant bla KPC alleles were sequenced, and their parentage was confirmed by typing. Ceftazidime-avibactam selected mutants at up to 16× MIC, with frequencies of ca. 10(-9). This contrasted with previous experience for ceftaroline-avibactam, where mutant frequencies under similar conditions were <10(-9). The MICs of ceftazidime with 1 µg/ml avibactam for the ceftazidime-avibactam-selected mutants rose from 1 to 8 µg/ml to 16 to >256 µg/ml and those of ceftazidime with 4 µg/ml avibactam from 0.25 to 1 µg/ml to 4 to 128 µg/ml; ceftaroline-avibactam MICs rose less, typically from 0.5 to 1 µg/ml to 1 to 8 µg/ml. The MICs of carbapenems and cephalosporins except ceftazidime and piperacillin-tazobactam were reduced for many mutants. Sequencing of blaKPC revealed point and insertion changes in 12/13 mutants investigated, representing all four parents; one mutant lacked bla KPC changes and possibly had reduced permeability. Amino acid changes commonly involved Ω loop alterations or 1 to 6 amino acid insertions immediately C-terminal to this loop. The most frequent change, seen in four mutants from three strains, was Asp179Tyr, replacing a residue that ordinarily forms a salt bridge to stabilize the Ω loop. Since ceftaroline-avibactam was less affected than ceftazidime-avibactam, we postulate that these mutations increase ceftazidimase specificity rather than conferring avibactam resistance. The clinical relevance remains uncertain.

Activity of OP0595/ß-lactam combinations against Gram-negative bacteria with extended-spectrum, AmpC and carbapenem-hydrolysing ß-lactamases.

BACKGROUND: OP0595 is a diazabicyclooctane that (i) acts as a PBP2-active antibacterial, (ii) inhibits Class A and C ß-lactamases and (iii), like mecillinam, gives ß-lactamase-independent potentiation of ß-lactams targeting other PBPs. We tested its behaviour against ß-lactam-resistant Enterobacteriaceae and non-fermenters. METHODS: Organisms were UK clinical isolates; MICs were determined by CLSI agar dilution for OP0595 alone or combined at 1-4 mg/L with aztreonam, biapenem, cefepime or piperacillin. RESULTS: MICs of OP0595 for Escherichia coli, Enterobacter, Citrobacter and Klebsiella spp. were mostly 1-4 mg/L but values >4 mg/L were seen for minorities of isolates irrespective of other resistances, and for 50%-60% of those with ertapenem resistance involving porin loss plus ESBL or AmpC activity. OP0595 MICs for Serratia, Proteeae and non-fermenters mostly were >4 mg/L. When its MIC was ≤4 mg/L, OP0595's antibacterial activity dominated combination activity. For 'OP0595-resistant' (MIC >4 mg/L) isolates with Class A or C ß-lactamases OP0595 achieved strong potentiation of substrate ß-lactams, contingent on ß-lactamase inhibition. ß-Lactamase-independent potentiation was evident with aztreonam, cefepime and piperacillin-less so for biapenem-for many OP0595-resistant Enterobacteriaceae with Class B carbapenemases, which are not inhibited by OP0595. OP0595 acted solely as a ß-lactamase inhibitor for non-fermenters. CONCLUSIONS: OP0595 inhibited Enterobacteriaceae, not non-fermenters; its combinations had broad activity versus Enterobacteriaceae, largely contingent on OP0595's antibacterial activity but also on inhibition of Class A and C ß-lactamases and on the ß-lactam-enhancer effect, which allowed activity against many OP0595-resistant metallo-ß-lactamase-producing Enterobacteriaceae. For non-fermenters OP0595 acted only as a ß-lactamase inhibitor.

Pathogens of skin and skin-structure infections in the UK and their susceptibility to antibiotics, including ceftaroline.

OBJECTIVES: Bacterial skin and skin-structure infections (SSSIs) are frequent settings for antibiotic use. We surveyed their UK aetiology and pathogen susceptibility, including susceptibility to ceftaroline. METHODS: Consecutive SSSI isolates were collected at 35 UK hospitals, to a maximum of 60/site, together with 15 'supplementary' MRSA/site. Isolates were re-identified and BSAC susceptibility testing was performed, with parallel CLSI agar testing for ceftaroline. RESULTS: Isolates (n = 1908) were collected from 1756 hospitalized patients, predominantly with surgical and traumatic infections, abscesses and infected ulcers and largely from general medicine and general surgery patients. They included 1271 Staphylococcus aureus (201 MRSA), 162 ß-haemolytic streptococci, 269 Enterobacteriaceae, 138 Pseudomonas aeruginosa and 37 enterococci. Most (944/1756) patients had monomicrobial MSSA infections. Rates of resistance to quinolones, gentamicin and cephalosporins were <20% in Enterobacteriaceae and <10% in P. aeruginosa. MRSA rates varied greatly among hospitals and were 2.5-fold higher in general medicine than in general surgery patients. At breakpoint, ceftaroline inhibited: (i) all MSSA and 97.6% of MRSA, with MICs of 2 mg/L for the few resistant MRSA; (ii) all ß-haemolytic streptococci; and (iii) 83% of Enterobacteriaceae. High-level ceftaroline resistance in Enterobacteriaceae involved ESBLs or AmpC enzymes. Ceftaroline MICs by CLSI methodology generally equalled those by BSAC or were 2-fold higher, but this differential was 4-16-fold for P. aeruginosa. CONCLUSIONS: Irrespective of patient group, SSSIs were dominated by S. aureus. Most pathogens were susceptible, but 15.8% of S. aureus were MRSA, with locally higher prevalence.

Susceptibility testing challenges with ceftaroline, MRSA and a 1 mg/L breakpoint.

OBJECTIVES: A 1 mg/L susceptibility breakpoint for ceftaroline and staphylococci is universally agreed; EUCAST counts MIC >1 mg/L as resistant whereas CLSI and FDA count 2 mg/L as intermediate and >2 mg/L as resistant. We investigated whether routine diagnostic tests reliably distinguish MICs of 1 versus 2 mg/L. METHODS: Thirty-five UK laboratories collected Staphylococcus aureus isolates and performed tests with 5 µg (as EUCAST) or 30 µg (as CLSI) discs and either confluent growth on Mueller-Hinton agar (as EUCAST and CLSI) or semi-confluent growth on Iso-Sensitest agar (as BSAC). They also ran Etests for MRSA. Reference MICs were determined centrally by CLSI and BSAC agar dilution. RESULTS: We obtained paired local disc and central MIC results for 1607 S. aureus (33% MRSA). EUCAST's zone breakpoint recognized 56% of isolates found resistant in MIC tests, but the positive predictive value (PPV) for resistance was 11.0%; corresponding proportions by CLSI testing were 28.0% and 13.4%. The BSAC disc method detected 25% of resistant isolates, with a PPV of 18.2%. Essential agreement, ±1 dilution, of local Etests and central agar MICs was >95%, but only 20% of the isolates found non-susceptible by agar dilution were found non-susceptible by Etest and vice versa. Review for isolates with the modal MIC (0.25 mg/L) indicated that the same laboratories reported large or small zones irrespective of disc and method, implying systematic bias. CONCLUSIONS: MRSA with ceftaroline MICs of 1 and 2 mg/L were poorly discriminated by routine methods. Solutions lie in greater standardization, automation or dosages justifying a higher breakpoint.

Comparative in vitro activity of sulfametrole/trimethoprim and sulfamethoxazole/trimethoprim and other agents against multiresistant Gram-negative bacteria.

OBJECTIVES: Sulfamethoxazole/trimethoprim is standard therapy for infections caused by opportunist non-fermenters except Pseudomonas aeruginosa and Acinetobacter. Sulfametrol(e)/trimethoprim is an alternative to sulfamethoxazole/trimethoprim available in some EU countries, with possible pharmacological advantages. We compared their activities against (i) non-fermenters, (ii) multiresistant Enterobacteriaceae and (iii) reference strains with sul1 and sul2. METHODS: Test isolates were recent submissions to the reference laboratory, or were Escherichia coli previously shown to have sul1 or sul2. Identification was by MALDI-ToF, by 16S rRNA gene sequencing or with API20NE strips. MICs were determined by CLSI agar dilution. The Stenotrophomonas maltophilia and Burkholderia series were enhanced by inclusion of 25% sulfamethoxazole/trimethoprim-resistant isolates; other series were not enhanced. RESULTS: MICs of sulfametrole/trimethoprim for non-fermenters tracked those of sulfamethoxazole/trimethoprim, being equal in 97/170 cases, 2-fold higher in 57/170 cases and 2-fold lower in 12/170 cases. Despite supplementing the Burkholderia and S. maltophilia collections with sulfamethoxazole/trimethoprim-resistant organisms, the antifolate combinations retained better activity against these and other non-fermenters than did piperacillin/tazobactam, moxifloxacin, ticarcillin/clavulanate, tigecycline, cefotaxime or imipenem. By contrast, few (5%-20%) of the extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae were susceptible to the sulphonamides or their trimethoprim combinations, probably reflecting widespread co-carriage of sul1 and sul2, which both conferred resistance. CONCLUSIONS: Antifolate combinations remain the most active antimicrobials against less common non-fermenters, importantly including S. maltophilia and Burkholderia spp., but resistance is prevalent among ESBL- and carbapenemase-producing Enterobacteriaceae. Sulfametrole/trimethoprim had similar activity to sulfamethoxazole/trimethoprim against non-fermenters.

Discovery of a novel class of boron-based antibacterials with activity against gram-negative bacteria.

Gram-negative bacteria cause approximately 70% of the infections in intensive care units. A growing number of bacterial isolates responsible for these infections are resistant to currently available antibiotics and to many in development. Most agents under development are modifications of existing drug classes, which only partially overcome existing resistance mechanisms. Therefore, new classes of Gram-negative antibacterials with truly novel modes of action are needed to circumvent these existing resistance mechanisms. We have previously identified a new a way to inhibit an aminoacyl-tRNA synthetase, leucyl-tRNA synthetase (LeuRS), in fungi via the oxaborole tRNA trapping (OBORT) mechanism. Herein, we show how we have modified the OBORT mechanism using a structure-guided approach to develop a new boron-based antibiotic class, the aminomethylbenzoxaboroles, which inhibit bacterial leucyl-tRNA synthetase and have activity against Gram-negative bacteria by largely evading the main efflux mechanisms in Escherichia coli and Pseudomonas aeruginosa. The lead analogue, AN3365, is active against Gram-negative bacteria, including Enterobacteriaceae bearing NDM-1 and KPC carbapenemases, as well as P. aeruginosa. This novel boron-based antibacterial, AN3365, has good mouse pharmacokinetics and was efficacious against E. coli and P. aeruginosa in murine thigh infection models, which suggest that this novel class of antibacterials has the potential to address this unmet medical need.

Activity of MK-7655 combined with imipenem against Enterobacteriaceae and Pseudomonas aeruginosa.

OBJECTIVES: MK-7655 is a novel inhibitor of class A and C ß-lactamases. We investigated its potential to protect imipenem. METHODS: Chequerboard MICs were determined by CLSI agar dilution: (i) for Enterobacteriaceae with carbapenemases; (ii) for Enterobacteriaceae with carbapenem resistance contingent on combinations of impermeability together with an extended-spectrum ß-lactamase or AmpC enzyme; and (iii) for Pseudomonas aeruginosa and other non-fermenters. RESULTS: At a concentration of 4 mg/L, MK-7655 reduced imipenem MICs for Enterobacteriaceae with KPC carbapenemases from 16-64 mg/L to 0.12-1 mg/L. Synergy also was seen for Enterobacteriaceae with impermeability-mediated carbapenem resistance, with weaker synergy seen for isolates with the OXA-48 enzyme. On the other hand, MK-7655 failed to potentiate imipenem against Enterobacteriaceae with metallo-carbapenemases. In the case of P. aeruginosa, where endogenous AmpC confers slight protection versus imipenem, 4 mg/L MK-7655 reduced the MIC of imipenem for all isolates, except those with metallo-carbapenemases: the MICs of imipenem fell from 1-2 mg/L to 0.25-0.5 mg/L for imipenem-susceptible P. aeruginosa and from 16-64 mg/L to 1-4 mg/L for OprD-deficient strains. No potentiation was seen for chryseobacteria or for Stenotrophomonas maltophilia. CONCLUSIONS: MK-7655 potentiated imipenem against Enterobacteriaceae with KPC carbapenemases or combinations of ß-lactamase and impermeability, but not those with metallo-carbapenemases. It augmented the activity of imipenem against P. aeruginosa in general and OprD mutants in particular.

Characterization of ß-lactamase and porin mutants of Enterobacteriaceae selected with ceftaroline + avibactam (NXL104).

OBJECTIVES: Ceftaroline + avibactam (NXL104) is a novel inhibitor combination active against Enterobacteriaceae with class A and C ß-lactamases. We investigated its risk of mutational resistance. METHODS: Single- and multi-step mutants were sought and characterized from Enterobacteriaceae with extended-spectrum ß-lactamases (ESBLs), AmpC ß-lactamases and KPC ß-lactamases. RESULTS: Overgrowth occurred on agar with low MIC multiples of ceftaroline + avibactam, but frequencies for single-step mutants were <10(-9). Most mutants were unstable, with only three remaining resistant on subculture. For one, from an CTX-M-15-positive Escherichia coli, the ceftaroline + avibactam MIC was raised, but the organism had reduced resistance to ceftaroline and lost resistance to other oxyimino-cephalosporins, with this profile retained when the mutant bla(CTX-M-15) was cloned into E. coli DH5α. Sequencing identified a Lys237Gln substitution in the CTX-M-15 variant. The other two stable single-step mutants were from an AmpC-derepressed Enterobacter cloacae strain; these had unaltered or slightly reduced resistance to other ß-lactams. Both had amino acids 213-226 deleted from the Ω loop of AmpC. Further stable mutants were obtained from AmpC-inducible and -derepressed E. cloacae in multi-step selection, and these variously had reduced expression of OmpC and OmpF, and/or Asn366His/Ile substitutions in AmpC. CONCLUSIONS: Stable resistant mutants were difficult to select. Those from AmpC-derepressed E. cloacae had porin loss or AmpC changes, including Ω loop deletions. A Lys237Gln substitution in CTX-M-15 conferred resistance, but largely abolished ESBL activity.

Activities of NXL104 combinations with ceftazidime and aztreonam against carbapenemase-Producing Enterobacteriaceae.

Combinations of NXL104 with ceftazidime and aztreonam were tested against carbapenem-resistant members of the Enterobacteriaceae. Ceftazidime-NXL104 was active against strains with the OXA-48 enzyme or with combinations of impermeability and an extended-spectrum ß-lactamase (ESBL) or AmpC enzyme and also against most Klebsiella spp. with the KPC enzyme, but metallo-ß-lactamase producers were resistant. Aztreonam-NXL104 was active against all carbapenemase producers at 4 and 4 µg/ml, including those with metallo-ß-lactamases.

False extended-spectrum {beta}-lactamase phenotype in clinical isolates of Escherichia coli associated with increased expression of OXA-1 or TEM-1 penicillinases and loss of porins.

OBJECTIVES: Two clinical isolates of Escherichia coli, EC18 and EC21, were non-susceptible (MICs 4-16 mg/L) to cefpirome and cefepime, with marked synergy with clavulanate, yet were susceptible to cefotaxime and ceftazidime (MICs ≤ 1 mg/L). EC19, from the same patient as EC21, was susceptible to all four cephalosporins. We sought to characterize the molecular basis of resistance in isolates EC18 and EC21. METHODS: PFGE was used to study the genetic relationships of the isolates, and MICs were determined. ß-Lactamases were characterized by PCR, isoelectric focusing (IEF), construction of genomic libraries and sequencing. A double mutant of E. coli J53 was constructed, lacking OmpC and OmpF porins. Plasmids from clinical isolates were transformed into E. coli J53 and J53ΔompCF. Outer membrane proteins (OMPs) were analysed by SDS-PAGE and OmpA by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. Expression of omp and bla genes was analysed by RT-PCR. RESULTS: Isolates EC19 and EC21 had identical PFGE profiles, whereas EC18 was distinct. PCR and IEF confirmed ß-lactamases with pIs of 5.4 (TEM-1) in EC18 and 7.4 (OXA-1) in both EC19 and EC21. EC18 had bla(TEM-1b) with the strong promoter P5 and lacked OmpC and OmpF. RT-PCR showed stronger expression of bla(OXA-1) in EC21 versus EC19, along with diminished expression of OmpC, though with increased OmpF. Plasmids extracted from EC18 and EC21 conferred increased MICs of cefpirome and cefepime, although susceptibility to cefotaxime and ceftazidime was retained. CONCLUSIONS: The 'cefpiromase' or 'cefepimase' ESBL phenotype of the clinical isolates non-susceptible to cefpirome and cefepime resulted from high expression of TEM-1 or OXA-1 ß-lactamases combined with loss of porins.

Efflux pumps, OprD porin, AmpC beta-lactamase, and multiresistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Expression of ampC, oprD, mexA, mexC, mexE, and mexX was studied in 25 Pseudomonas aeruginosa isolates from cystic fibrosis patients, including 14 isolates of the Liverpool epidemic strain. Overexpressed mexA or ampC and reduced oprD were associated with beta-lactam resistance. A specific combination of mexR, nalC, and nalD mutations occurred in 11 Liverpool strain isolates, including 7 with upregulated mexA.

In vitro activity of ceftazidime+NXL104 against Pseudomonas aeruginosa and other non-fermenters.

BACKGROUND: NXL104 potentiates ceftazidime and ceftaroline against Enterobacteriaceae with extended-spectrum, AmpC, KPC and OXA ß-lactamases. We examined whether similar potentiation was obtained against non-fermenters, which are less permeable than Enterobacteriaceae and have more potent efflux. METHODS: MICs of ceftazidime+NXL104 (with NXL104 at 4 mg/L) and comparators were determined by CLSI agar dilution for: (i) Pseudomonas aeruginosa AmpC mutants and extended-spectrum ß-lactamase (ESBL)-producing transconjugants; (ii) clinical P. aeruginosa isolates with AmpC enzymes, ESBLs or up-regulated efflux; (iii) P. aeruginosa and Burkholderia cepacia complex isolates from cystic fibrosis patients; and (iv) Acinetobacter baumannii with OXA carbapenemases, which also compromise ceftazidime. RESULTS: NXL104 reversed AmpC-mediated ceftazidime resistance in P. aeruginosa, reducing MICs for fully derepressed mutants and isolates to ≤ 8 mg/L. NXL104 also reversed ceftazidime resistance caused by the ESBL PER-1, but not that due to OXA ESBLs or VEB-1 enzyme. Efflux-mediated resistance was unaffected. Resistance to ceftazidime in isolates of P. aeruginosa and the B. cepacia complex from patients with cystic fibrosis was variably overcome, generally to greater effect for B. cepacia. NXL104 had little effect on MICs of ceftazidime for A. baumannii isolates with OXA carbapenemases. CONCLUSIONS: The potentiation of ceftazidime against many ß-lactamase-producing P. aeruginosa and B. cepacia complex strains confirms that NXL104 penetrates these organisms. The utility of the combination against these pathogens will depend on the local prevalence of strains with ß-lactamase- versus efflux-mediated resistance. The lack of potentiation against A. baumannii may reflect failure of NXL104 to penetrate these bacteria to inhibit relevant (OXA-23, -40, -51 and -58) carbapenemases.

Activity of BAL30376 (monobactam BAL19764 + BAL29880 + clavulanate) versus Gram-negative bacteria with characterized resistance mechanisms.

BACKGROUND: BAL30376 combines the siderophore monobactam BAL19764 (Syn/PTX 2416) with the bridged monobactam BAL29880 to inhibit AmpC enzymes and with clavulanate to inhibit extended-spectrum ß-lactamases (ESBLs). We tested BAL30376 and its components versus isolates and laboratory strains of Enterobacteriaceae and non-fermenters. METHODS: MICs were determined on Mueller-Hinton agar supplemented with 2,2'-bipyridyl to chelate Fe(3+) and induce TonB-mediated uptake. RESULTS: Unprotected BAL19764 had MICs  ≤  1 mg/L for most cephalosporin-susceptible Enterobacteriaceae, but values for a few isolates ranged up to 8 mg/L; its MICs were substantially raised for isolates with AmpC ß-lactamases and ESBLs. Those of BAL30376 were ≤ 1 mg/L for 84% of ESBL producers and ≤ 4 mg/L for 85% of AmpC producers, excluding isolates with exceptional impermeability. Laboratory transformants with metallo- or OXA-48 carbapenemases were susceptible to unprotected BAL19764, but many clinical isolates with these enzymes were resistant, probably having additional mechanisms; BAL30376, by contrast, was active at 4 mg/L versus 31/35 metallo-ß-lactamase producers and 14/19 with OXA-48, although those with KPC carbapenemases were resistant. AmpC-mediated resistance to BAL19764 in Pseudomonas aeruginosa was overcome by BAL30376, as was that due to PER-1 enzyme; but MICs > 16 mg/L were frequent for cystic fibrosis isolates. Many Burkholderia cepacia and carbapenemase-producing Acinetobacter baumannii were susceptible to BAL19764 and BAL30376 at ≤ 4 mg/L, but others were highly resistant, with MICs  ≥  128 mg/L. CONCLUSIONS: BAL30376 overcomes most AmpC-, ESBL- and carbapenemase-mediated resistance in Enterobacteriaceae, though strains with KPC carbapenemases are resistant. It was active against many problem non-fermenters, though resistance was common in P. aeruginosa from cystic fibrosis. Raised MICs for some isolates were independent of ß-lactamase.

Activity of the siderophore monobactam BAL30072 against multiresistant non-fermenters.

BACKGROUND: We tested the activity of BAL30072, a novel siderophore monobactam, against multiresistant clinical isolates of Pseudomonas aeruginosa, Burkholderia cepacia group and Acinetobacter spp. and against laboratory P. aeruginosa strains with defined resistance mechanisms. METHODS: MICs were determined on Mueller-Hinton agar supplemented with 2,2' bipyridyl to induce iron transport; comparators were aztreonam, imipenem, meropenem and piperacillin/tazobactam. RESULTS: BAL30072 was strikingly active against Acinetobacter baumannii, with 73% of 200 carbapenemase-producing isolates, most of them belonging to the UK-dominant OXA-23 clone 1 and SE clone lineages, susceptible at 1 mg/L and 89% at 8 mg/L. Resistance nevertheless was seen in a few representatives of these clones and appeared commoner among isolates representing other A. baumannii clones. Sixty-eight per cent of 50 B. cepacia complex isolates from cystic fibrosis (CF) were susceptible to BAL30072 at 1 mg/L and 78% at 8 mg/L, compared with only 22% susceptible to aztreonam at 8 mg/L. Activity against P. aeruginosa was good, though less dramatic, with 36% of 50 (mostly multiresistant) CF isolates susceptible at 8 mg/L, compared with 12% susceptible to aztreonam at 8 mg/L. BAL30072 was active against 11/19 metallo-beta-lactamase-producing P. aeruginosa at 8 mg/L compared with 3/19 for aztreonam (12/19 versus 8/19 at 16 mg/L). Studies on P. aeruginosa mutants, isolates and transconjugants showed that BAL30072 was affected by efflux, AmpC and by a few uncommon acquired beta-lactamases, including some extended-spectrum OXA types and PER-1. CONCLUSIONS: BAL30072 displayed impressive activity against many carbapenemase-producing A. baumannii, particularly against the two clones most prevalent in the UK, and also against B. cepacia complex isolates from CF; it was more active than aztreonam against P. aeruginosa.

Polyclonal multiply antibiotic-resistant methicillin-resistant Staphylococcus aureus with Panton-Valentine leucocidin in England.

OBJECTIVES: Community-associated methicillin-resistant Staphylococcus aureus (MRSA) including those encoding Panton-Valentine leucocidin (PVL) are often described as more susceptible to a range of antibiotics than their hospital-associated counterparts. Recent scattered reports of the emergence of multiresistant PVL-MRSA have highlighted the potential for resistance to emerge. Here we detail polyclonal multiply antibiotic-resistant PVL-MRSA occurring in England. METHODS: PVL-MRSA from community-based and hospitalized patients located across England were identified by PCR. Isolates were characterized via MIC determinations, toxin gene profiling, PFGE, SCCmec, spa and agr typing. Multilocus sequence typing (MLST) was performed on selected isolates. Patient demographic and available disease data were retained for analysis. RESULTS: Seventy-six PVL-MRSA isolates resistant to three further classes of antibiotic were identified between 2005 and 2008 from centres in each of the Health Protection Agency's geographic regions in England. Patient demographics were typical for PVL-MRSA, and some travel associations were identified along with clonal spread. One instance of familial transmission in the community was detected. PVL-MRSA belonging to MLST clonal complex (CC) 1 (sequence type 772) were consistently highly resistant; multiply antibiotic-resistant representatives of CCs 5, 8, 22, 59 and 80 were also identified. Ciprofloxacin resistance was common amongst the study isolates (51 of 76 isolates). CONCLUSIONS: Genetically diverse multiply antibiotic-resistant PVL-MRSA were identified, and included representatives of a recently emerged multiresistant clone (dubbed the Bengal Bay clone). Risk factors and disease presentations were typical for PVL-MRSA infections. This work highlights the diminishing utility of ciprofloxacin susceptibility for putative identification of PVL-MRSA.

Performance of the Oxoid M.I.C.Evaluator Strips compared with the Etest assay and BSAC agar dilution.

OBJECTIVES: The Oxoid M.I.C.Evaluator (M.I.C.E; Thermo Fisher Scientific) comprises an antibiotic gradient on a plastic support. We compared its performance with Etest--a similar product--using BSAC agar dilution as a reference. METHODS: Parallel MIC tests were performed by M.I.C.Evaluator, Etest and agar dilution on Iso-Sensitest agar. In total, 9354 organism/strip combinations were tested by each method, using 1017 bacteria representing clinically important fastidious and non-fastidious species. RESULTS: Essential agreement of strip MIC values (+/-1 doubling dilution) with the agar dilution reference, with off-scale results excluded, was 89.9% for M.I.C.Evaluator versus 89.5% for Etest (P > 0.05). These proportions were similar, at 89.5% and 89.3% (P > 0.05), respectively, if off-scale values were counted as agreeing if they could agree (e.g. a strip MIC >32 mg/L and an agar dilution MIC of 128 mg/L). For both strips, agreement with agar dilution was best for non-fastidious genera, Moraxella, Listeria, Pasteurella and Campylobacter spp. and weaker for streptococci, anaerobes, Neisseria spp. and, especially, Haemophilus influenzae. Many 'disagreements', especially for H. influenzae, concerned organisms unequivocally resistant by all methods (e.g. ampicillin MIC 256 mg/L by agar dilution, 16 or 32 mg/L by both strips); nevertheless both strips underestimated imipenem MICs for Proteus. There was no difference between the two strip types in the proportion of agreements with agar dilution (P > 0.05); nevertheless their results agreed better with each other than with agar dilution (P < 0.01). CONCLUSIONS: The M.I.C.Evaluator performed almost identically to the Etest, giving good agreement with BSAC agar dilution.