Abstract
Ceftazidime-avibactam and comparator antibiotics were tested by the broth microdilution method against 200 Enterobacteriaceae and 25 Pseudomonas aeruginosa strains resistant to fluoroquinolones (including strains with the extended-spectrum β-lactamase [ESBL] phenotype and ceftazidime-resistant strains) collected from our institution. The MICs and mechanisms of resistance to fluoroquinolone were also studied. Ninety-nine percent of fluoroquinolone-resistant Enterobacteriaceae strains were inhibited at a ceftazidime-avibactam MIC of ≤4 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference). Ceftazidime-avibactam was very active against ESBL Escherichia coli (MIC90 of 0.25 mg/liter), ESBL Klebsiella pneumoniae (MIC90 of 0.5 mg/liter), ceftazidime-resistant AmpC-producing species (MIC90 of 1 mg/liter), non-ESBL E. coli (MIC90 of ≤0.125 mg/liter), non-ESBL K. pneumoniae (MIC90 of 0.25 mg/liter), and ceftazidime-nonresistant AmpC-producing species (MIC90 of ≤0.5 mg/liter). Ninety-six percent of fluoroquinolone-resistant P. aeruginosa strains were inhibited at a ceftazidime-avibactam MIC of ≤8 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference), with a MIC90 of 8 mg/liter. Additionally, fluoroquinolone-resistant mutants from each species tested were obtained in vitro from two strains, one susceptible to ceftazidime and the other a β-lactamase producer with a high MIC against ceftazidime but susceptible to ceftazidime-avibactam. Thereby, the impact of fluoroquinolone resistance on the activity of ceftazidime-avibactam could be assessed. The MIC90 values of ceftazidime-avibactam for the fluoroquinolone-resistant mutant strains of Enterobacteriaceae and P. aeruginosa were ≤4 mg/liter and ≤8 mg/liter, respectively. We conclude that the presence of fluoroquinolone resistance does not affect Enterobacteriaceae and P. aeruginosa susceptibility to ceftazidime-avibactam; that is, there is no cross-resistance.
INTRODUCTION
Avibactam is a new broad-spectrum non-β-lactam β-lactamase inhibitor with activity against clinically relevant enzymes belonging to Ambler classes A, C, and some D (e.g., extended-spectrum β-lactamase [ESBL], Klebsiella pneumoniae carbapenemase [KPC], AmpC, and OXA-48) but not to class B β-lactamases (1–3). Strains that harbor such β-lactamases are increasing in prevalence worldwide, are among the most important and frequently isolated nosocomial pathogens, and are often additionally resistant to many classes of antibiotics, such as the fluoroquinolones (4). It is known, mainly in Pseudomonas aeruginosa but also in Enterobacteriaceae, that the overexpression of some efflux pumps that confer resistance to fluoroquinolones, often associated with other mechanisms, such as mutations in genes encoding DNA gyrase and topoisomerase IV or acquisition of some plasmid-mediated quinolone-resistance genes, may increase the MIC of β-lactam antibiotics, such as ceftazidime (5). The aim of this study was to evaluate the in vitro activity of the combination ceftazidime-avibactam at a fixed avibactam concentration of 4 mg/liter (19) compared with other β-lactam antibiotics, such as ceftaroline, which is the active compound of ceftaroline fosamil, ceftazidime, piperacillin, piperacillin-tazobactam, aztreonam, imipenem, and meropenem, against fluoroquinolone-resistant Enterobacteriaceae and P. aeruginosa clinical isolates and against laboratory-generated fluoroquinolone-resistant mutants of the aforementioned microorganisms with a well-defined mechanism of resistance to quinolones.
MATERIALS AND METHODS
Bacterial strains.
A series of 200 Enterobacteriaceae and 25 P. aeruginosa strains resistant to fluoroquinolones (mainly from urinary tract infections and bacteremia occurring in different patients) were collected as consecutive clinical isolates in our institution (Hospital Clinic, Barcelona, Spain). Enterobacteriaceae and P. aeruginosa isolates were considered resistant to fluoroquinolones according to EUCAST breakpoints for ciprofloxacin. Some Enterobacteriaceae isolates showing a MIC above the epidemiological cutoff but considered to be susceptible according EUCAST rules (0.064 to ≤0.5 mg/liter) and with any mechanism of resistance to fluoroquinolones were also studied. Species identification was performed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Bremen, Germany). An ESBL screen-positive phenotype was defined according to CLSI guidelines (6). Enterobacteriaceae and P. aeruginosa were considered resistant to ceftazidime using the CLSI breakpoints for ceftazidime (>16 mg/liter and >32 mg/liter, respectively). The collection included Escherichia coli (n = 60; 50% ESBL), K. pneumoniae (n = 40; 50% ESBL), Enterobacter cloacae (n = 25; 20% ceftazidime resistant), Citrobacter freundii (n = 25; 68% ceftazidime resistant), Serratia marcescens (n = 25; 20% ceftazidime resistant), Proteus mirabilis (n = 25; 28% ceftazidime resistant), and P. aeruginosa (n = 25; 24% ceftazidime resistant). For each species, additional quinolone-resistant mutants were generated in vitro from two different clinical isolates, one from an isolate with a low MIC against ceftazidime and one from a β-lactamase producer with a high MIC for ceftazidime but susceptible to ceftazidime-avibactam.
In vitro susceptibility test methods.
The MIC values of imipenem, piperacillin, aztreonam, piperacillin-tazobactam, ciprofloxacin, norfloxacin, levofloxacin, nalidixic acid (Sigma-Aldrich Co., St. Louis, MO, USA), meropenem, ceftaroline, ceftazidime, and ceftazidime-avibactam (AstraZeneca) (avibactam was used at a fixed concentration of 4 mg/liter in combination with ceftazidime) were determined in Mueller-Hinton broth by the microdilution method according to CLSI guidelines (Oxoid Ltd., Basingstoke, United Kingdom) (6). The EUCAST breakpoints for ciprofloxacin were used to assess fluoroquinolone resistance, and some isolates showing a ciprofloxacin MIC of ≤0.5 mg/liter but possessing a mechanism of resistance to fluoroquinolones were also included in the study. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 strains were used as reference strains for quality control of in vitro susceptibility testing.
Detection of β-lactamases.
Isolates with ESBL phenotypes were characterized by PCR and sequencing, as described before (7). Ceftazidime-resistant species showing an AmpC-overproduction phenotype or a plasmid-mediated AmpC phenotype were confirmed using a ceftazidime or cefotaxime-boronic acid synergy test with 30 μg ceftazidime or cefotaxime discs (Becton Dickinson, Sparks, MD, USA) together with 400 μg phenylboronic acid (Sigma-Aldrich Co., St. Louis, MO, USA) (8).
Detection of the mechanisms of resistance to quinolones.
Detection of mutations in the DNA gyrase and topoisomerase IV genes were studied by amplifying and sequencing the quinolone resistance-determining regions (QRDRs) of the gyrA, gyrB, parC, and parE genes by PCR, using primers described before (9–13). Investigation of plasmid-mediated quinolone resistance (PMQR) genes was performed via screening by PCR of qnrA, qnrB, qnrS, aac(6′)Ib-cr, and qepA genes. The aac(6′)Ib-cr gene was sequenced in all isolates. Overexpression of efflux pumps was investigated by performing ciprofloxacin, norfloxacin, and nalidixic acid MIC measurements using Etest strips (AB bioMérieux, Solna, Sweden) in Mueller-Hinton agar (Becton Dickinson, Sparks, MD, USA) in the absence and presence of 20 mg/liter phenylalanine-arginine-β-naphthylamide (PAβN; Becton Dickinson, Sparks, MD, USA), an efflux pump inhibitor.
Selection of mutants.
Mutants were selected by following either a single- or multiple-step selection method. To perform a single-step selection method, an inoculum (109 CFU/ml) from an overnight broth culture was spread on Mueller-Hinton agar (Becton Dickinson, Sparks, MD, USA) supplemented with ciprofloxacin at 2 to 4× the MICs previously found. After overnight incubation, mutants with MICs that were ≥2-fold the MIC of ciprofloxacin were retained.
In the multiple-step selection method, several passages were performed. An inoculum (108 CFU) was added to 10-ml aliquots of nutrient broth containing ciprofloxacin at the MICs found previously and incubated for up to 48 h. This was repeated daily, each time doubling the ciprofloxacin concentration. Daily subculturing was performed until mutants with MICs that were ≥2-fold the MIC of ciprofloxacin were streaked to single colonies and retained.
RESULTS
Using the CLSI breakpoints for ceftazidime (≤4 mg/liter for susceptibility), ceftazidime-avibactam was among the most active agents tested against the panel of fluoroquinolone-resistant Enterobacteriaceae (ciprofloxacin MIC of ≥0.5 mg/liter) studied (Table 1), with 99.7% susceptibility. Similarly, 96% of fluoroquinolone-resistant P. aeruginosa strains were inhibited by ≤8 mg/liter of ceftazidime-avibactam (using the CLSI susceptibility breakpoint for ceftazidime alone as a reference).
TABLE 1.
Activity of ceftazidime-avibactam and comparators against fluoroquinolone-resistant Enterobacteriaceae and P. aeruginosa isolates
| Microorganism (no. of isolates tested) | Activity (mg/liter) for: |
|||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Meropenem |
Imipenem |
Piperacillin |
Piperacillin-tazobactama |
Ceftaroline |
Aztreonam |
Ceftazidime |
Ceftazidime-avibactamb |
|||||||||
| MIC90 | Range | MIC90 | Range | MIC90 | Range | MIC90 | Range | MIC90 | Range | MIC90 | Range | MIC90 | Range | MIC90 | Range | |
| Enterobacteriaceae (200) | ||||||||||||||||
| Non-ESBL E. coli (30) | <0.06 | <0.06 | 0.125 | <0.06 to 0.25 | >128 | <0.125 to >128 | 16 | 0.125 to >128 | 0.5 | <0.125 to 1 | 0.25 | <0.125 to 1 | 0.25 | <0.125 to 0.5 | <0.125 | <0.125 to 0.25 |
| ESBL E. coli (30) | <0.06 | <0.06 | 0.125 | <0.06 to 0.5 | >128 | >128 | 128 | 1 to >128 | >128 | 4 to >128 | 128 | 4 to >128 | 64 | 0.25 to >128 | 0.25 | <0.125 to 0.25 |
| Non-ESBL K. pneumoniae (20) | 0.125 | <0.06 to 2 | 0.5 | <0.06 to 0.5 | >128 | 8 to >128 | 64 | <0.125 to >128 | 0.5 | <0.125 to 64 | 0.5 | <0.125 to 0.5 | 0.5 | <0.125 to 1 | 0.25 | 0.03 to 1 |
| ESBL K. pneumoniae (20) | <0.06 | <0.06 to 4 | 0.25 | <0.06 to 1 | >128 | >128 | >128 | 4 to >128 | >128 | 32 to >128 | >128 | 128 to >128 | >128 | 8 to >128 | 0.5 | <0.125 to 1 |
| Other CAZ-S Enterobacteriaceaec (66) | <0.06 | <0.06 to 1 | 4 | <0.06 to 8 | >128 | 0.25 to >128 | 128 | <0.125 to >128 | >128 | <0.125 to >128 | 4 | <0.125 to >128 | 2 | <0.125 to 4 | 0.5 | <0.015 to 1 |
| Other CAZ-R Enterobacteriaceaed (34) | 1 | <0.06 to 2 | 4 | 0.125 to 8 | >128 | 8 to >128 | >128 | 0.25 to >128 | >128 | 8 to >128 | >128 | <0.125 to >128 | >128 | 2 to >128 | 1 | <0.125 to 128e |
| P. aeruginosa (25) | 16 | 0.125 to 64 | 32 | 0.5 to 64 | >128 | 2 to >128 | 128 | 1 to >128 | >128 | 2 to >128 | 64 | 0.25 to 128 | 64 | 0.25 to 128 | 8 | 0.25 to 16 |
Tazobactam at 4 mg/liter.
Avibactam at 4 mg/liter.
CAZ-S, ceftazidime-susceptible Enterobacteriaceae isolates: P. mirabilis, E. cloacae, C. freundii, and S. marcescens.
CAZ-R, ceftazidime-resistant isolates: P. mirabilis, E. cloacae, C. freundii, and S. marcescens due to AmpC overproduction (n = 23), plasmid-mediated AmpC (n = 7), or ESBL-producing strains (n = 4).
MIC of 128 mg/liter for a single AmpC-overproducing S. marcescens isolate with a MIC of 0.25 mg/liter versus meropenem. Otherwise, the range is <0.125 to 2 mg/liter.
All fluoroquinolone-resistant E. coli isolates were inhibited by ceftazidime-avibactam below the ceftazidime breakpoint (MIC90 of ≤0.25 mg/liter), including ESBL-producing isolates (highest MIC detected, 0.5 mg/liter). The ESBLs carried by the E. coli isolates were mainly CTX-M-14, CTX-M-15, CTX-M-9, and SHV-12 (Table 2).
TABLE 2.
Ceftazidime resistance mechanisms studied and associated CAZ-AVIa MIC ranges
| Microorganism (no. of strains tested) | CTX-M-14 |
CTX-M-15 |
CTX-M-9 |
CTX-M-1 |
SHV-12 |
Plasmidic AmpC producers |
AmpC overproduction |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. of strains | CAZ-AVI MIC range | No. of strains | CAZ-AVI MIC range | No. of strains | CAZ-AVI MIC range | No. of strains | CAZ-AVI MIC range | No. of strains | CAZ-AVI MIC range | No. of strains | CAZ-AVI MIC range | No. of strains | CAZ-AVI MIC range | |
| E. coli (30) | 16 | <0.125 | 6 | <0.125 to 0.5 | 4 | <0.125 | 4 | <0.125 to 0.25 | ||||||
| K. pneumoniae (20) | 19 | <0.125 to 1 | 1 | 0.5 | 1b | 0.5 | ||||||||
| E. cloacae (5) | 5 | <0.125 to 0.5 | ||||||||||||
| C. freundii (17) | 1 | <0.25 | 3 | <0.125 to 0.5 | 13 | <0.125 to 1 | ||||||||
| P. mirabilis (7) | 7 | <0.125 to 1 | ||||||||||||
| S. marcescens (5) | 5 | <0.125 to 128 | ||||||||||||
CAZ-AVI, ceftazidime-avibactam; avibactam at 4 mg/liter.
Detected in a strain together with CTX-M-15.
When tested against fluoroquinolone-resistant ESBL-producing (mainly CTX-M-15, CTX-M-1, and SHV-12) (Table 2) and non-ESBL-producing K. pneumoniae isolates, ceftazidime-avibactam inhibited all isolates below the ceftazidime breakpoint (MIC90 of 0.25 mg/liter), and the highest MIC was 1 mg/liter.
Of the 25 fluoroquinolone-resistant C. freundii isolates, 17 were ceftazidime resistant; 4 of these were carrying ESBL enzymes, and 13 showed AmpC overproduction. All C. freundii isolates had ceftazidime-avibactam MIC values below the susceptibility breakpoint of ceftazidime alone. All ceftazidime-susceptible strains were inhibited by ≤0.125 mg/ml ceftazidime-avibactam, and ceftazidime-avibactam was active against all ceftazidime-resistant strains (MIC90 of 1 mg/liter).
Ceftazidime-avibactam was active against 25 fluoroquinolone-resistant E. cloacae strains (MIC90 of 0.5 mg/liter), including 5 ceftazidime-resistant strains that showed AmpC overproduction; the highest MIC detected was 1 mg/liter. In addition, ceftazidime-avibactam inhibited all strains of fluoroquinolone-resistant P. mirabilis (MIC90 of 0.5 mg/liter), including seven ceftazidime-resistant strains showing a plasmid-AmpC production phenotype. The highest MIC observed in fluoroquinolone-resistant P. mirabilis isolates was, again, 1 mg/liter.
Twenty-five fluoroquinolone-resistant S. marcescens isolates, including 5 with ceftazidime resistance, were tested. All strains but one were inhibited by ≤4 mg/liter ceftazidime-avibactam (MIC90 of 1 mg/liter). One ceftazidime-resistant strain that showed an AmpC overproduction phenotype was not inhibited by ceftazidime-avibactam (MIC of 128 mg/liter).
Finally, MIC90 values of ceftazidime-avibactam for 25 fluoroquinolone-resistant P. aeruginosa clinical isolates are also shown in Table 1. Ninety-six percent of isolates were inhibited by ≤8 mg/liter ceftazidime-avibactam (MIC90 of 8 mg/liter). For the six isolates that were ceftazidime resistant, avibactam reversed ceftazidime resistance in 5 instances, while the remaining isolate was inhibited by 16 mg/liter ceftazidime-avibactam. Remarkably, ceftazidime-avibactam showed very good activity against meropenem-nonsusceptible P. aeruginosa (16 isolates), with 90% inhibited by ≤8 mg/liter ceftazidime-avibactam. This may be due to a loss of porins, mainly OprD, that affect carbapenems but not ceftazidime; however, further investigation is necessary to confirm this hypothesis.
The activity of quinolones against fluoroquinolone-resistant Enterobacteriaceae is presented in Table 3. Mechanisms of fluoroquinolone resistance detected and the associated ceftazidime-avibactam MIC ranges are shown in Table 4. Most of the fluoroquinolone-resistant strains studied (n = 200; 88.9%) showed mutations in gyrA and/or parC, gyrB, or parE QRDR regions. Twenty-five percent of the isolates did not have mutations in QRDR regions. PMQR genes were detected in 20 strains (K. pneumoniae, n = 9; E. cloacae, n = 8; C. freundii, n = 3); PMQR genes and overexpression of an efflux pump were observed in 3 strains (S. marcescens, n = 2; K. pneumoniae, n = 1), and 2 K. pneumoniae strains showed only an efflux pump overexpression. The ceftazidime-avibactam MIC ranges were not noticeably affected by the detected fluoroquinolone-resistance mechanisms, except in one S. marcescens strain that showed mutations in the QRDR regions.
TABLE 3.
Activity of quinolones against fluoroquinolone-resistant Enterobacteriaceae and P. aeruginosa isolates
| Microorganism (no. of isolates tested) | Activity (mg/liter) for: |
|||||||
|---|---|---|---|---|---|---|---|---|
| Ciprofloxacin |
Levofloxacin |
Norfloxacin |
Nalidixic acid |
|||||
| MIC90 | Range | MIC90 | Range | MIC90 | Range | MIC90 | Range | |
| Enterobacteriaceae (200) | ||||||||
| ESBL E. coli (30) | >128 | 8 to >128 | 64 | 4 to 64 | >128 | 16 to >128 | >1,024 | >1,024 to >1,024 |
| ESBL K. pneumoniae (20) | >128 | 2 to >128 | 64 | 0.5 to >128 | >128 | 4 to >128 | >1,024 | 4 to >1,024 |
| Other CAZ-R Enterobacteriaceaea (34) | >128 | 0.25 to >128 | >128 | 0.25 to >128 | >128 | 1 to >128 | >1,024 | 8 to >1,024 |
| Non-ESBL E. coli (30) | 128 | <0.125 to >128 | 32 | <0.125 to 64 | >128 | 0.25 to >128 | >1,024 | 256 to >1,024 |
| Non-ESBL K. pneumoniae (20) | 128 | 0.25 to >128 | 64 | 0.5 to >128 | >128 | 1 to >128 | >1,024 | 8 to >1,024 |
| Other CAZ-S Enterobacteriaceaeb (66) | 128 | 0.25 to >128 | 64 | 0.25 to 128 | >128 | 0.5 to >128 | >1,024 | 2 to >1,024 |
| P. aeruginosa (25) | >128 | 8 to >128 | >128 | 8 to >128 | >128 | 8 to >128 | >1,024 | >1,024 to >1,024 |
CAZ-R, ceftazidime-resistant isolates: P. mirabilis, E. cloacae, C. freundii, and S. marcescens due to AmpC overproduction (n = 23), plasmid-mediated AmpC (n = 7), or ESBL-producing strains (n = 4).
CAZ-S, ceftazidime-susceptible Enterobacteriaceae isolates: P. mirabilis, E. cloacae, C. freundii, and S. marcescens.
TABLE 4.
| Organism (no. of strains tested) | QRDR |
PMQR |
EP |
QRDR + PMQR |
QRDR + EP |
PMQR + EP |
||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. (%) of strains | CAZ-AVI MIC range | No. (%) of strains | CAZ-AVI MIC range | No. (%) of strains | CAZ-AVI MIC range | No. (%) of strains | CAZ-AVI MIC range | No. (%) of strains | CAZ-AVI MIC range | No. (%) of strains | CAZ-AVI MIC range | |
| Non-ESBL E. coli (30) | 26 (86.7) | <0.125 to 0.25 | 1 (3.3) | <0.125 | 3 (10) | <0.125 to 0.25 | ||||||
| ESBL E. coli (30) | 26 (86.7) | <0.125 to 0.25 | 4 (13.3) | <0.125 to 0.5 | ||||||||
| Non-ESBL K. pneumoniae (20) | 7 (35) | <0.125 to 1 | 5 (25) | <0.125 | 2 (10) | <0.125 to 0.25 | 5 (25) | <0.25 to 0.5 | 1 (5) | <0.125 | ||
| ESBL K. pneumoniae (20) | 3 (15) | 0.25 to 0.5 | 4 (20) | <0.125 | 13 (65) | <0.125 to 1 | ||||||
| E. cloacae (25) | 8 (32) | <0.25 to 1 | 17 (68) | <0.25 to 0.5 | ||||||||
| C. freundii (25) | 17 (68) | <0.25 to 1 | 3 (12) | <0.125 | 5 (20) | <0.125 to 1 | ||||||
| P. mirabilis (25) | 20 (80) | <0.125 to 0.25 | 5 (20) | <0.125 to 1 | ||||||||
| S. marcescens (25) | 20 (80) | <0.125 to 128 | 1 (4) | <0.125 | 2 (8) | 0.25 to 1 | 2 (8) | <0.125 to 1 | ||||
| P. aeruginosa (25) | 20 (80) | 0.25 to 16 | 5 (20) | 1 to 8 | ||||||||
QRDR, quinolone resistance-determining region mutations, found in gyrA and/or gyrB, parC, or parE; PMQR, plasmid-mediated quinolone resistance, qnrA, qnrB, qnrS, aac(6′)-Ib-cr, or qepA; EP, efflux pump overproduction.
CAZ-AVI, ceftazidime-avibactam; avibactam at 4 mg/liter.
The ceftazidime mechanisms of resistance in Enterobacteriaceae and associated ceftazidime MIC ranges are shown in Table 2. No changes in the ranges of the ceftazidime-avibactam MIC and ceftazidime resistance mechanisms were detected, except in one S. marcescens strain that showed AmpC overproduction.
When mutant strains resistant to fluoroquinolones were generated by selection in vitro, all Enterobacteriaceae and P. aeruginosa strains tested were inhibited by ≤4 and ≤8 mg/liter of ceftazidime-avibactam, respectively (Table 5). For the large majority of the strains, no significant increases in the ceftazidime-avibactam MICs were observed. Among 13 fluoroquinolone-resistant mutant strains, one 4-fold ceftazidime-avibactam MIC increase (0.25 mg/liter to 1 mg/liter) was observed in the strain S. marcescens 243 and its mutant strain, S. marcescens 272, which was inhibited by 8 mg/liter of ceftazidime due to AmpC overproduction. In this strain, the fluoroquinolone resistance mechanism detected was efflux pump overexpression.
TABLE 5.
Activity of ceftazidime-avibactam against fluoroquinolone-resistant mutant strains obtained in vitro
| Strain (mutation type)a | Mechanism | MIC (mg/liter)b |
||
|---|---|---|---|---|
| CIPc | CAZ | CAZ-AVI | ||
| E. coli 1 (parent) | gyrA (Ser83Leu) | 0.047 | 0.125 | <0.125 |
| E. coli 2 (MUT-SP) | gyrA (Ser83Leu); parC (Ser80Arg); acrAB-tolC++e | 0.25 | 0.125 | 0.25 |
| E. coli 242 (parent) | NDd | 0.047 | 16 | <0.125 |
| E. coli 271 (MUT-SP) | Efflux pump | 0.25 | 16 | <0.125 |
| K. pneumoniae 229 (parent) | ND | 0.023 | <0.125 | <0.125 |
| K. pneumoniae 255 (MUT-SS) | gyrA (Ser83Ile) | 0.25 | <0.125 | <0.125 |
| K. pneumoniae 146 (parent) | qnrB | 0.25 | >128 | <0.125 |
| K. pneumoniae 251 (MUT-SS) | qnrB + efflux pump | 16 | >128 | <0.125 |
| P. mirabilis 127 (parent) | ND | 0.032 | <0.125 | <0.015 |
| P. mirabilis 267 (MUT-SP) | Efflux pump | 0.38 | <0.125 | <0.125 |
| E. cloacae 131 (parent) | ND | 0.032 | 32 | 0.25 |
| E. cloacae 264 (MUT-SP) | Efflux pump | 0.5 | >128 | 0.5 |
| E. cloacae 123 (parent) | ND | 0.016 | 0.125 | 0.125 |
| E. cloacae 263 (MUT-SP) | Efflux pump | 0.19 | 0.25 | 0.125 |
| S. marcescens 147 (parent) | ND | 0.064 | <0.125 | <0.125 |
| S. marcescens 261 (MUT-SS) | Efflux pump | 0.75 | <0.125 | <0.125 |
| S. marcescens 243 (parent) | ND | 0.38 | 8 | 0.25 |
| S. marcescens 272 (MUT-SP) | Efflux pump | 8 | 8 | 1 |
| C. freundii 145 (parent) | qnrB | 0.125 | >128 | 0.25 |
| C. freundii 253 (MUT-SS) | qnrB + gyrA (Ser83Ile) | 2 | >128 | <0.125 |
| C. freundii 137 (parent) | ND | 0.047 | 0.125 | 0.125 |
| C. freundii 266 (MUT-SP) | Efflux pump | 0.5 | 0.5 | 0.25 |
| P. aeruginosa 51 (parent) | ND | 0.25 | 64 | 8 |
| P. aeruginosa 260 (MUT-SS) | Efflux pump | 2 | 64 | 8 |
| P. aeruginosa 124 (parent) | ND | 0.125 | 1 | 1 |
| P. aeruginosa 262 (MUT-SP) | Efflux pump | 0.5 | 1 | 1 |
MUT-SS, mutant strain obtained by a single step; MUT-SP, mutant strain obtained by serial passage.
CIP, ciprofloxacin; CAZ, ceftazidime; CAZ-AVI, ceftazidime-avibactam.
EUCAST susceptibility breakpoint for ciprofloxacin against Enterobacteriaceae and P. aeruginosa: ciprofloxacin MIC of ≤0.5 mg/liter determined by Etest.
ND, not detected.
++, overexpression of acrAB-tolC.
DISCUSSION
The purpose of this study was to examine the activity of ceftazidime-avibactam against fluoroquinolone-resistant clinical isolates of P. aeruginosa and Enterobacteriaceae, in both those with and without ESBLs. Our results showed that, as demonstrated by Lagacé-Wiens et al. (14), ceftazidime-avibactam is active against a wide variety of clinical ESBL-carrying Enterobacteriaceae. Moreover, in our study, all strains were fluoroquinolone resistant, and ceftazidime-avibactam retained excellent potency (MIC90 of 0.25 mg/liter), as shown in Table 1, against fluoroquinolone-resistant ESBL-carrying strains, inhibiting all isolates at <1 mg/liter. In addition to studying E. coli and K. pneumoniae that are ESBL producers, we also studied a group of ceftazidime-resistant (MIC of >16 mg/liter) C. freundii, E. cloacae, P. mirabilis, and S. marcescens strains that have stably derepressed chromosomal AmpC. The MIC90 of ceftazidime-avibactam for these clinical isolates was 1 mg/liter. For isolates of Enterobacteriaceae that were nonsusceptible to ceftazidime, the addition of 4 mg/liter avibactam in ceftazidime-avibactam greatly increased the activity of ceftazidime against all species, as was found previously in a study performed in the United States on clinical isolates from bloodstream, pneumonia, intra-abdominal, and urinary tract infections (15). One Serratia marcescens strain inhibited by >128 mg/liter of ceftazidime was not inhibited by the combination of ceftazidime-avibactam (MIC of 128 mg/liter), despite susceptibility to imipenem and meropenem. The mechanism of resistance may be associated with porin loss, thus restricting ceftazidime and/or avibactam entry, but further studies would be needed to elucidate this hypothesis.
As seen in other studies (2, 15–17), avibactam increased the activity of ceftazidime against ceftazidime-resistant P. aeruginosa strains. Moreover, ceftazidime-avibactam was highly active against meropenem-nonsusceptible strains (MIC90 of 8 mg/liter), as also shown by Sader et al. (18). Although resistance to ceftazidime in this microorganism is usually considered multifactorial, most of the strains showed overexpression of the chromosomally encoded AmpC β-lactamase and, occasionally, acquisition of ESBLs such as PER-1, which may explain this inhibition.
Fluoroquinolone-resistant strains were also selected in vitro from parent strains that were both susceptible and resistant to ceftazidime alone (Table 5). These newly fluoroquinolone-resistant Enterobacteriaceae strains exhibited MICs against ceftazidime ranging from <0.125 mg/liter to >128 mg/liter. However, ceftazidime-avibactam retained potency with MICs in a range of <0.125 mg/liter to 1 mg/liter. When fluoroquinolone-resistant mutant strains of P. aeruginosa were obtained in a similar manner, a significant increase in the ceftazidime-avibactam MIC was not observed. When characterized, most of the mutant strains showed efflux pump overexpression as their mechanism of fluoroquinolone resistance. This and other target-specific resistance mechanisms had no effect on the activity of ceftazidime-avibactam.
In conclusion, ceftazidime-avibactam demonstrated potent in vitro activity against a collection of 200 Enterobacteriaceae and 25 P. aeruginosa strains resistant to fluoroquinolones, including ESBL-carrying and AmpC-overproducing strains, collected as consecutive clinical isolates in our institution. The specific resistance mechanisms detected were mutations in gyrA, gyrB, parC, and parE genes, in PMQR genes qnrA, qnrB, qnrS, aac(6′)Ib-cr, and qepA, and in efflux pumps. This work shows that resistance to fluoroquinolones has minimal effects on the potent activity of ceftazidime-avibactam against both ESBL and non-EBSL strains. The combination ceftazidime-avibactam thus may provide an excellent alternative to carbapenems in infections caused by ESBL-carrying and AmpC-overproducing fluoroquinolone-resistant strains that are endemic worldwide.
ACKNOWLEDGMENTS
This study was funded in part by Forest Laboratories Inc., a former subsidiary of Actavis plc, and in part by AstraZeneca. This study also was supported by grant 2014SGR0653 from the Departament de Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya, by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, cofinanced by European Regional Development Fund (ERDF), “A Way to Achieve Europe,” the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015).
W.W.N. is an employee of AstraZeneca. T.A.K. is a former employee of AstraZeneca.
REFERENCES
- 1.Endimiani A, Choudhary Y, Bonomo RA. 2009. In vitro activity of NXL104 in combination with beta-lactams against Klebsiella pneumoniae isolates producing KPC carbapenemases. Antimicrob Agents Chemother 53:3599–3601. doi: 10.1128/AAC.00641-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Aktaş Z, Kayacan C, Oncul O. 2012. In vitro activity of avibactam (NXL104) in combination with beta-lactams against Gram-negative bacteria, including OXA-48 β-lactamase-producing-Klebsiella pneumoniae. Int J Antimicrob Agents 39:86–89. doi: 10.1016/j.ijantimicag.2011.09.012. [DOI] [PubMed] [Google Scholar]
- 3.Zhanel GG, Lawson CD, Adam H, Schweizer F, Zelenitsky S, Lagacé-Wiens PRS, Denisuik A, Rubinstein E, Gin AS, Hoban DJ, Lynch JP, Karlowsky JA. 2013. Ceftazidime-avibactam: a novel cephalosporin/β-lactamase inhibitor combination. Drugs 73:159–177. doi: 10.1007/s40265-013-0013-7. [DOI] [PubMed] [Google Scholar]
- 4.Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M, Cornaglia G, Garau J, Gniadkowski M, Hayden MK, Kumarasamy K, Livermore DM, Maya JJ, Nordmann P, Patel JB, Paterson DL, Pitout J, Villegas MV, Wang H, Woodford N, Quinn JP. 2013. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis 13:785–796. doi: 10.1016/S1473-3099(13)70190-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Livermore DM. 2001. Of Pseudomonas, porins, pumps and carbapenems. J Antimicrob Chemother 47:247–250. doi: 10.1093/jac/47.3.247. [DOI] [PubMed] [Google Scholar]
- 6.Clinical and Laboratory Standards Institute. 2012. Performance standards for antimicrobial susceptibility testing; 22nd informational supplement. CLSI M100-S22. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 7.Bradford PA, Bratu S, Urban C, Visalli M, Mariano N, Landman D, Rahal JJ, Brooks S, Cebular S, Quale J. 2004. Emergence of carbapenem-resistant Klebsiella species possessing the class A carbapenem-hydrolyzing KPC-2 and inhibitor-resistant TEM-30 beta-lactamases in New York City. Clin Infect Dis 39:55–60. doi: 10.1086/421495. [DOI] [PubMed] [Google Scholar]
- 8.Tsakris A, Themeli-Digalaki K, Poulou A, Vrioni G, Voulgari E, Koumaki V, Agodi A, Pournaras S, Sofianou D. 2011. Comparative evaluation of combined-disk tests using different boronic acid compounds for detection of Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae clinical isolates. J Clin Microbiol 49:2804–2809. doi: 10.1128/JCM.00666-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Navia MM, Ruiz J, Ribera A, de Anta MT, Vila J. 1999. Analysis of the mechanisms of quinolone resistance in clinical isolates of Citrobacter freundii. J Antimicrob Chemother 44:743–748. doi: 10.1093/jac/44.6.743. [DOI] [PubMed] [Google Scholar]
- 10.Vila J, Ruiz J, Marco F, Barcelo A, Goñi P, Giralt E, Jimenez de Anta T. 1994. Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs. Antimicrob Agents Chemother 38:2477–2479. doi: 10.1128/AAC.38.10.2477. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Vila J, Ruiz J, Goñi P, de Anta MT. 1996. Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli. Antimicrob Agents Chemother 40:491–493. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Vila J, Ruiz J, Goñi P, Marcos A, Jimenez de Anta T. 1995. Mutation in the gyrA gene of quinolone-resistant clinical isolates of Acinetobacter baumannii. Antimicrob Agents Chemother 39:1201–1203. doi: 10.1128/AAC.39.5.1201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Fàbrega A, Merle du L, Le Bouguénec C, Jiménez de Anta MT, Vila J. 2009. Repression of invasion genes and decreased invasion in a high-level fluoroquinolone-resistant Salmonella Typhimurium mutant. PLoS One 4:e8029. doi: 10.1371/journal.pone.0008029. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Lagacé-Wiens PRS, Tailor F, Simner P, DeCorby M, Karlowsky JA, Walkty A, Hoban DJ, Zhanel GG. 2011. Activity of NXL104 in combination with beta-lactams against genetically characterized Escherichia coli and Klebsiella pneumoniae isolates producing class A extended-spectrum beta-lactamases and class C beta-lactamases. Antimicrob Agents Chemother 55:2434–2437. doi: 10.1128/AAC.01722-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Flamm RK, Farrell DJ, Sader HS, Jones RN. 2014. Ceftazidime/avibactam activity tested against Gram-negative bacteria isolated from bloodstream, pneumonia, intraabdominal and urinary tract infections in U.S. medical centers (2012). J Antimicrob Chemother 69:1589–1598. doi: 10.1093/jac/dku025. [DOI] [PubMed] [Google Scholar]
- 16.Flamm RK, Sader HS, Farrell DJ, Jones RN. 2014. Ceftazidime-avibactam activity and comparator agents tested against urinary tract isolates from a global surveillance program (2011). Diagn Microbiol Infect Dis 80:233–238. doi: 10.1016/j.diagmicrobio.2014.07.005. [DOI] [PubMed] [Google Scholar]
- 17.Mushtaq S, Warner M, Livermore DM. 2010. In vitro activity of ceftazidime+NXL104 against Pseudomonas aeruginosa and other nonfermenters. J Antimicrob Chemother 65:2376–2381. doi: 10.1093/jac/dkq306. [DOI] [PubMed] [Google Scholar]
- 18.Sader HS, Castanheira M, Flamm RK, Farrell DJ, Jones RN. 2014. Antimicrobial activity of ceftazidime-avibactam against Gram-negative organisms collected from U.S. medical centers in 2012. Antimicrob Agents Chemother 58:1684–1692. doi: 10.1128/AAC.02429-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Livermore DM, Mushtaq S, Warner M, Zhang J, Maharjan S, Doumith M, Woodford N. 2011. Activities of NXL104 combinations with ceftazidime and aztreonam against carbapenemase-producing Enterobacteriaceae. Antimicrob Agents Chemother 55:390–394. doi: 10.1128/AAC.00756-10. [DOI] [PMC free article] [PubMed] [Google Scholar]