P. J. Petersen, H. C. Jones, A. M. Venkatesan, P. A. Bradford, T. S. Mansour;
Wyeth Res., Pearl River, ny. Background



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F1-1487

Comparative In Vitro Evaluation of Pipetacillin-BLI-489 Determined by Disk Diffusion and MIC against a Predictor Panel of β-Lactamase Producing Organisms

P. J. PETERSEN, H. C. JONES, A. M. VENKATESAN, P. A. BRADFORD, T. S. MANSOUR;
Wyeth Res., Pearl River, NY.

Background: The β-lactamase inhibitor, BLI-489, has shown in vitro activity against molecular Class A, D and C enzymes and extended spectrum β-lactamases (ESBL). In this study we determined the optimal disk concentration of BLI-489 to employ for susceptibility testing. Three different disk concentration of BLI-489, in combination with piperacillin (PIP), were evaluated and zone diameters compared to MIC data for a predictor panel of β-lactamase producing isolates (N=122). Methods: The MICs determined by broth microdilution as recommended by the CLSI. The activity of PIP:BLI-489 by MIC testing was determined with a constant 4 μg/ml of BLI-489. The zone diameters of three experimental disks of PIP:BLI-489 (100:5, 100:10 and 100:20 μg/disk) were determined using CLSI recommended methodology. The PIP interpretive criteria was applied to the PIP:BLI-489 results to perform error-rate bound analysis comparing the disk diameter results to the MIC. Results: Overall, the three disk concentrations provided good separation between susceptible and resistant isolates. Importantly, there were no very major errors observed for any of the experimental disks. The PIP:BLI-489 100:5 μg disk had 3 major errors (results categorized as resistant by disk and susceptible by MIC), for a major error rate of 2.7% and was the only disk concentration that resulted in major errors. There were an increasing number of minor errors corresponding to decreasing concentration of BLI-489 in the disk. The number and percent of minor error were as follows: 20 (17.7%) for the 100:5 μg disk, 11 (9.7%) for the 100:10 μg/ml and, 4 (3.5%) for the 100:20 disks. The majority of minor errors were results categorized as susceptible by MIC and intermediate by zone diameter. Conclusions: Based on this analysis, either the PIP:BLI-489 100:10 or 100:20 μg/ml disks would seem appropriate for disk diffusion susceptibility testing and warrant further evaluation.

F1-1488


Preliminary Quality Control Parameters and Stability of Piperacillin-BLI-489 Evaluated with MIC Testing Methodology

P. J. PETERSEN, H. C. JONES, A. M. VENKATESAN, P. A. BRADFORD, T. S. MANSOUR;
Wyeth Res., Pearl River, NY.

Background: The bicyclic penem inhibitor, BLI-489 has demonstrated activity as an inhibitor of Class A, (including ESBLs) Class D and Class C β-lactamase enzymes. The combination of piperacillin (PIP) and BLI-489 is currently undergoing pre-clinical evaluations. The current study was performed to provide preliminary quality control parameters for the ATCC strains and determine stability of drug at -70ºC. Methods: The MICs of the antibiotics were determined by broth microdilution as recommended by the CLSI. For quality control evaluation, the plates were prepared on the day of test. For stability testing, plates containing the antimicrobial agent were prepared and frozen at -70°C until evaluated. Results: The results for 34 replicates with PIP:BLI-489 against E. coli ATCC 25922 demonstrated a single median, mode and mean value of 2 μg/ml. Consistent results were also demonstrated for E. coli ATCC 35218: 33 replicates showed a range of MICs of 1 to 2 μg/ml with a mean of 1.02 μg/ ml and a standard deviation of 0.17. A tight data set was also obtained for P. aeruginosa ATCC 27853 (18 replicates) with a range of MICs of 2 to 4 μg/ml, a mean of 3.43 μg/ ml and a standard deviation of 0.86. PIP:BLI-489 demonstrated reproducible results against S. aureus ATCC 29213: 14 experimental results showed a range of MICs 0.25 to 0.5 μg/ml with a mean of 0.28 and standard deviation of 0.09. The results for 14 replicates with PIP:BLI-489 against E. faecalis ATCC 29212 demonstrated identical results with a single median, mode and mean value of 2 μg/ml. The results obtained for PIP:BLI-489 were very similar to the CLSI acceptable limits for PIP:tazobactam (PIP:TZB). Results for frozen plates were consistent over 9 months of testing. Conclusions: These results indicate that the same control limits as used by PIP:TZB for the ATCC strains can be used for PIP:BLI-489 until a M23 study is completed. In addition, PIP:BLI-489 was stable up to 9 months when stored at -70°C.

F1-1489


In Vitro Activities of Piperacillin-BLI-489 and Comparative Agents against Recent Isolates from Urinary Tract Infection

P. J. PETERSEN, H. C. JONES, A. M. VENKATESAN, P. A. BRADFORD, T. S. MANSOUR;
Wyeth Res., Pearl River, NY.

Background: The penem bicyclic β-lactamase inhibitor BLI-489 has demonstrated activity and efficacy as an inhibitor of Class A (including ESBLs), Class D and Class C β-lactamases enzymes. The combination of piperacillin (PIP) and BLI-489 is currently undergoing pre-clinical evaluations. One of the clinical indications proposed for PIP-BLI-489 is for hospitalized and complicated urinary tract infections (cUTI). This study was performed to evaluate PIP-BLI-489 and eleven comparative agents against a collection of recent cUTI isolates collected from five U.S. medical centers. Methods: The MICs of the antibiotics were determined by broth microdilution as recommended by the CLSI. The activity of the combination of PIP:BLI-489 was determined with a constant 4 μg/ml of BLI-489. Etest® ESBL test strips were used according to the manufacturer’s instructions for confirmation of the presence of an ESBL. Results: The combination of PIP:BLI-489 demonstrated similar potent activity against both the E. coli (N=50) and K. pneumoniae (N=50) UTI isolates tested (MIC90s 8 and 16 μg/ml, respectively). The PIP:BLI-489 combination inhibited 100% of the E. coli and 94 % of the K. pneumoniae at a concentration of <16 μg/ml. The more commonly used antibiotics for UTI therapy: SXT, amoxicillin-clavulanic acid, gentamicin, doxycycline and levofloxacin, showed high resistance rates to both the E. coli (70%, 40%, 22%, 54%, 42%, respectively) and K. pneumoniae (52%, 24%, 38%, 46%, 26%, respectively) isolates tested. Approximately 18% of the E. coli and 42% of the K. pneumonia isolates were ESBL positive. PIP:BLI-489 inhibited 100% of the ESBL E. coli and 86% of the ESBL K. pneumoniae at a concentration of <16 μg/ml. Conclusions: The in vitro activities of PIP-BLI-489 against UTI isolates including ESBL and multidrug-resistant strains make it a strong candidates for further development for treatment of hospital associated and complicated urinary tract infections

F1-1490


Evaluation of the Development of Resistance with the Novel Penem β-Lactamase (BLA) Inhibitor BLI-489 by Single Step and Serial Passage

A. RUZIN, P. J. PETERSEN, C. H. JONES, A. M. VENKATESAN, T. S. MANSOUR;
Wyeth Res., Pearl River, NY.

Background: BLI-489 is a novel bicyclic penem inhibitor with demonstrated activity against class A, C and D BLAs. This study evaluated resistance development to the piperacillin-BLI-489 (PIP-BLI) combination using single step and serial passage selection methodologies. Methods: Spontaneous mutation frequency was measured for 11 strains on PIP-BLI-containing agar plates at 4, 8, and 16 times the MIC. Further, development of resistance was determined by the macrodilution broth method. Five BLA producing strains were exposed to a serial dilution of PIP-BLI and the highest concentration allowing growth used to inoculate subsequent serial passage for 10 days. Mutation stability was monitored in drug free media for 10 days. Results: E. coli (encoding OXA-3, OXA-7, ACT-1 or SHV-1), S. enterica ser. Typhimurium (CTX-M-5), K. pneumoniae (SHV-1 and SHV-5), E. cloacae (AmpC) and a BLA negative E. coli had a spontaneous mutation frequency of ≤1.0 x 10-9. Two AmpC-producing P. aeruginosa strains had a spontaneous mutation frequency of 6.52 x 10-6 and 1.0 x 10-7; in contrast a BLA negative P. aeruginosa strain had a mutation frequency of 2.68 x 10-8. The mutant prevention concentration (MPC) for these strains was ≤32/4 μg/ml. During the serial passage experiments, the MIC values increased 64- and 128-fold for S. enterica ser. Typhimurium (CTX-M-5) and E. cloacae (AmpC), respectively. These MIC values reverted to susceptible levels after serial passages in drug free media. The MICs increased only 4-fold for K. pneumoniae (SHV-1 and SHV-5), E. coli (OXA-3) and E. coli (SHV-1). Conclusions: PIP-BLI demonstrated a low probability of spontaneous resistance development in vitro for all of the strains tested with the exception of P. aeruginosa. The MPC value for all test strains was ≤32/4 μg/ml. Resistance developed during serial passage for two of the five strains tested, however this resistance phenotype was unstable and reverted to susceptibility after propagation in drug free media.

F1-1491


In Vivo Efficacy of ME1071, a Novel Metallo-β-Lactamase (MBL) Inhibitor, in Combination with Carbapenems against MBL-Producing Pseudomonas aeruguinosa in a Murine Pneumonia Model

A. MORINAKA1, K. OHKUMA 1, K. MATSUMOTO 1, T. MIKUNIYA 1, S. SAKAKIBARA 1, K. MAEBASHI 1, H. SUZUKI 1, J. D. DOCQUIER 2, G. M. ROSSOLINI 2;
1Meiji Seika Kaisha, Ltd., Yokohama, Japan, 2Univ. of Siena, Siena, Italy.

Background: At 47th ICAAC, we presented the activity of ME1071 (formerly CP3242), a novel competitive MBL inhibitor, against MBL-producing Gram-negative bacteria including P. aeruginosa in combination with β-lactam antibiotics. The aim of this study was to investigate the efficacy of ME1071 in combination with carbapenems against MBL-producing P. aeruginosa in a pulmonary infection model in mice. Methods: Infections were induced by intranasal injection in neutropenic ICR male mice (n=4~9) with clinically isolated P. aeruginosa strains MSC00771 producing IMP-1 and MSC18745 producing VIM-2. ME1071 in combination with biapenem or imipenem/cilastatin was administered S.C. at 4, 6 and 8 h after infection. Viable cells in the lung were counted 24 h after infection. Results: The therapeutic efficacy of ME1071/biapenem (50/25 mg/kg) or ME1071/imipenem/cilastatin (50/50/50 mg/kg) administered 3 times was demonstrated in the murine pneumonia model with P. aeruginosa MSC00771 (inoculum size: 4.9 log CFU/lung) by lowering the viable cell counts in the lung: saline (mean±SD: 8.1±0.5 log CFU/lung), biapenem ([alone]: 8.1±0.4 to [coadministration with ME1071]: 2.7±1.0, P<0.05 vs biapenem alone), imipenem/cilastatin (4.5±2.0 to 1.8±0.3, P<0.05 vs imipenem/cilastatin alone). Similarly, the coadministration of ME1071/biapenem reduced the viable cell counts in the lung with P. aeruginosa MSC18745 (inoculum size: 5.3 log CFU/lung) more than biapenem alone: saline (8.8±0.4), biapenem alone (5.1±1.3), biapenem with ME1071 (2.4±0.4, P<0.05 vs biapenem alone). Conclusions: We conclude that ME1071 coadministered with carbapenems would be a promising candidate in treatment of infection with IMP-1- or VIM-2-producing P. aeruginosa that could not be treated by carbapenem alone.

F1-1492


Spectrum and Activity of Ceftaroline Combined with NXL-104 Tested against a Challenge Collection of Pathogens with Well Characterized Resistances

M. CASTANHEIRA1, H. S. SADER 1, I. CRITCHLEY 2, G. WILLIAMS 2, R. N. JONES 1;
1JMI Lab., North Liberty, IA, 2Cerexa Inc., Oakland, CA.

Background: Ceftaroline (CPT) is a broad-spectrum cephalosporin with activity against Gram-negative and -positive (including MRSA), but limited activity against ESBL- and AmpC-producing strains. NXL104 (NXL) is a novel β-lactamase (βL) inhibitor that inhibits AmpC, ESBL and KPC. Methods: CPT, NXL, various CPT/NXL combinations (fixed 2 and 4 μg/ml and 1:1, 2:1, 4:1 and 8:1 ratios) and comparators were tested for susceptibility (S) by CLSI broth microdilution methods against 178 clinical strains, including Enterobacteriaceae (ENT) producing CTX-M (22 strains), CMY (10), FOX (5), KPC (20), SME (5) and metallo-βL (MβL; 5), P. aeruginosa (PSA; 15), A. baumannii (ACB; 15), MRSA (50; SSCmec types I-IV), MSSA (10), S. pneumoniae (SPN; 16) and E. faecalis (EF; 5). Results: The greatest NXL effect was obtained at a fixed of 4 µg/ml concentration. 93% of ENT were inhibited at ≤2/4 μg/ml of CPT/NXL while only 45 and 63% were S to cefepime and imipenem, respectively. Among ENT, only MβL-producing strains had a CPT/NXL MIC, >2/4 μg/ml. CPT/NXL was very active against MRSA (MIC90, 2/4 μg/ml), MSSA (MIC90, 0.25/4 μg/ml), SPN (highest MIC, 0.25/4 μg/ml) and ampicillin-S EF (MIC range, 1/4-4/4 μg/ml). CPT showed only marginal anti-PSA activity, but significant enhanced effect with NXL against wildtype (WT) isolates. CPT/NXL exhibited good activity against WT ACB (MIC range, 0.5/4-4/4 μg/ml), but limited activity against OXA- producing ACB.

 

No. of strains (cumulative %) inhibited at MIC (µg/ml of):

Organism (no. tested)

≤0.12

0.25

0.5

1

2

4

8

16

>16

Enterobacteriaceae (67)

 

 

 

 

 

 

 

 

 

CPT/NXLa

24 (36)

13 (55)

18 (82)

5 (90)

2 (93)

-

-

-

5 (100)

Cefepime

5 (8)

4 (13)

5 (21)

2 (24)

6 (33)

6 (42)

2 (45)

5 (55)

32 (100)

Imipenem

8 (12)

12 (31)

15 (52)

2 (55)

2 (58)

3 (63)

8 (75)

-

17 (100)

MRSA (50)

 

 

 

 

 

 

 

 

 

CPT/NXLa

-

-

3 (6)

27 (60)

18 (96)

2 (100)

-

-

-

Cefepime

-

-

-

-

-

-

4 (8)

11 (30)

35 (100)

Imipenem

4 (8)

9 (26)

1 (28)

3 (34)

2 (38)

2 (42)

2 (46)

-

27 (100)

S. pneumoniae (16)

 

 

 

 

 

 

 

 

 

CPT/NXLa

14 (88)

2 (100)

-

-

-

-

-

-

-

Cefepime

8 (50)

2 (63)

1 (69)

4 (94)

1 (100)

-

-

-

-

Imipenem

11 (69)

0 (69)

2 (81)

3 (100)

-

-

-

-

-

a. NXL at fixed 4 μg/ml

Conclusions: CPT alone was very active against Gram-positives. CPT/NXL combinations showed significant enhanced potencies compared with CPT alone against ENT strains producing CTX-M, plasmidic AmpC, and KPC βLs.

F1-1493


Bactericidal Activity of Ceftaroline Combined with NXL-104 against Critical Targeted Organisms Possessing Various Resistance Mechanisms

H. S. SADER1, G. WILLIAMS 2, I. CRICTHLEY 2, R. N. JONES 1;
1JMI Lab., North Liberty, IA, 2Cerexa Inc., Oakland, CA.

Background: Ceftaroline (CPT), a broad-spectrum cephalosporin with Gram-positive activity (including anti-MRSA), was tested in combination with NXL-104 (NXL), a potent inhibitor of AmpC, ESBL and KPC β-lactamases (βL) against a selected group of characterized Enterobacteriaceae (ENT). Methods: 6 βL-producing ENT (CMY-2, derepressed AmpC, CTX-M-15, KPC-2 and -3, and a KPC-cured with SHV-27) and 1 wildtype (WT) strain were tested. MIC and MBC were assessed according CLSI guidelines in Mueller-Hinton broth ± 10% human serum (HS). Time kill analysis (TK) used CPT, CPT/NXL combinations (fixed 4 μg/ml [CPT/NXL4] and 2:1 ratio) and NXL alone at 1X, 2X, 4X and 8X the MIC. Expression of βL genes was determined by quantitative real-time PCR. Plasmid curing was performed by culturing isolate with DNA intercalating compounds. Results: CPT and NXL MBCs were generally elevated with MBC/MIC ratios of 2 to 32 among βL-producing strains; while CPT/NXL4 had low MICs and MBC/MIC ratios at 1 or 2 (Table). 10% HS did not adversely influence CPT or CPT/NXL MIC or MBC values. βL-producing ENT had CPT/NXL4 MIC values at ≤1/4 µg/ml and MBC/MIC of 1 or 2 (1 strain). NXL showed direct antimicrobial activity (MIC, 8-16 μg/ml) against WT and 4 of 6 βL-producing strains. TK detected rapid bactericidal action of CPT/NXL combinations at ≥2X MIC, with some strains having highest enzyme expression showing regrowth at 4-12 hours at 1X MIC. KPC-cured strain was killed rapidly by CPT at ≥2X MIC.

 

Resistance

CPT

 

CPT/NXL (fixed 4)

 

CPT/NXL (2:1)

 

NXL-104

 

Species

mechanism

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

E. coli

Wild type

0.12

0.12

0.06/4

0.06/4

0.12/0.06

0.12/0.06

8

32

E. coli

CMY-2

256

>2048

0.25/4

0.5/4

2/1

2/1

8

16

E. cloacae

AmpC

1024

>2048

1/4

1/4

4/2

4/2

16

128

K. pneumoniae

CTX-M-15

2048

>2048

0.12/4

0.12/4

1/0.5

1/0.5

16

>128

K. pneumoniae

KPC-3

128

>2048

0.25/4

0.25/4

2/1

2/1

16

32

K. pneumoniae

KPC-2

1024

>2048

1/4

1/4

4/2

4/2

128

>128

K. pneumoniae

KPC-cured

4

4

0.25/4

0.5/4

1/0.5

1/0.5

>128

>128

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Conclusions: NXL demonstrated a remarkably wide and potent βL inhibitory potency against contemporary isolates producing clinically important βL. These results should be used to optimize CPT/NXL dosing regimens.

F1-1494


Activity of Chequerboard Combinations of Ceftaroline and NXL104 vs β-Lactamase Producers

S. MUSHTAQ 1, M. WARNER 1, G. WILLIAMS 2, I. CRITCHLEY 2, D. M. LIVERMORE1;
1Ctr. for Infections, London, United Kingdom, 2Cerexa Inc, Oakland, CA.



Background: Ceftaroline is a novel parenteral cephalosporin, now in Phase 3. It has similar anti-gram-negative activity to cefotaxime, but also similar lability to ESBLs, AmpC, and other potent β-lactamases. To overcome this vulnerability, it is intended to develop ceftaroline combined with NXL104, a novel β-lactamase inhibitor. We investigated the NXL104 concentrations needed for protection against key enzyme types. Methods: Chequerboard MIC titrations were set up between ceftaroline (0.008-128 mg/L) and NXL104 (0.008-16 mg/L), allowing testing of all combinations of concentrations. The test strains were clinical Enterobacteriaceae with known β-lactamase profiles, most of them highly resistant to ceftaroline alone (MIC >16 mg/L). Results: All of 60 ESBL producers were susceptible to ceftaroline-NXL104 at 1/1 mg/L; 55 were susceptible at 1/0.25 mg/L. Among 30 with high-level chromosomal AmpC, 18 were susceptible to ceftaroline-NXL104, 1/1 mg/L and 28 at 1/4 mg/L, the 2 exceptions were Enterobacter spp. inhibited by 2/4 mg/L. Among 10 isolates with plasmid AmpC, 9 were susceptible to ceftaroline-NXL104 at 1/1 mg/L and all at 1/4 mg/L. None of the 10 isolates with combinations of AmpC or ESBL and impermeability was susceptible to ceftaroline-NXL104 1/1 mg/L, but 9 were susceptible at 1/4 mg/L. Among 12 with KPC enzymes, only 2 were susceptible to ceftaroline-NXL104 1/1 mg/L, but 10 were susceptible at 1/4 mg/L; all of 8 with OXA-48 carbapenemase were susceptible at 1/1 mg/L. Among 5 Klebsiella oxytoca with high-level K1 enzyme, all were susceptible to ceftaroline-NXL104 at 1/1 mg/L. None of 5 isolates with metallo-β-lactamases was susceptible to ceftaroline-NXL104 at 1/4 mg/L or even 16/4 mg/L, although 2 producers were inhibited by NXL104 alone at 8 to 16 mg/L. Conclusions: At 1 mg/L, NXL104 protected ceftaroline against ESBLs, OXA-48, and K1 enzyme; at 4 mg/L it also protected against AmpC and KPC enzymes. Metallo-β-lactamase producers were resistant.
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