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. 2017 May 24;61(6):e00054-17. doi: 10.1128/AAC.00054-17

Antimicrobial Effects of β-Lactams on Imipenem-Resistant Ceftazidime-Susceptible Pseudomonas aeruginosa

Yu Mi Wi a, Ji-Young Choi b, Ji-Young Lee b, Cheol-In Kang c, Doo Ryeon Chung c,d, Kyong Ran Peck c, Jae-Hoon Song c,d, Kwan Soo Ko b,d,✉
PMCID: PMC5444147  PMID: 28373200

ABSTRACT

We studied the resistance mechanism and antimicrobial effects of β-lactams on imipenem-resistant Pseudomonas aeruginosa isolates that were susceptible to ceftazidime as detected by time-kill curve methods. Among 215 P. aeruginosa isolates from hospitalized patients in eight hospitals in the Republic of Korea, 18 isolates (23.4% of 77 imipenem-resistant isolates) were imipenem resistant and ceftazidime susceptible. Multilocus sequence typing revealed diverse genotypes, which indicated independent emergence. These 18 isolates were negative for carbapenemase genes. All 18 imipenem-resistant ceftazidime-susceptible isolates showed decreased mRNA expression of oprD, and overexpression of mexB was observed in 13 isolates. In contrast, overexpression of ampC, mexD, mexF, or mexY was rarely found. Time-kill curve methods were applied to three selected imipenem-resistant ceftazidime-susceptible isolates at a standard inoculum (5 × 105 CFU/ml) or at a high inoculum (5 × 107 CFU/ml) to evaluate the antimicrobial effects of β-lactams. Inoculum effects were detected for all three β-lactam antibiotics, ceftazidime, cefepime, and piperacillin-tazobactam, against all three isolates. The antibiotics had significant killing effects in the standard inoculum, but no effects in the high inoculum were observed. Our results suggest that β-lactam antibiotics should be used with caution in patients with imipenem-resistant ceftazidime-susceptible P. aeruginosa infection, especially in high-inoculum infections such as endocarditis and osteomyelitis.

KEYWORDS: Pseudomonas aeruginosa, carbapenem resistance, ceftazidime susceptible, time-kill assay, inoculum effect

INTRODUCTION

Pseudomonas aeruginosa is a major nosocomial pathogen, particularly in debilitated, ill, or immunocompromised patients (1). Although carbapenems, such as imipenem and meropenem, remain effective at treating serious multidrug-resistant P. aeruginosa infections, a rise in carbapenem resistance among isolates has been reported worldwide (2). Infections caused by carbapenem-resistant P. aeruginosa (CRPA) are a matter of concern because they are associated with a 3-fold higher mortality rate, a 9-fold higher rate of secondary bacteremia, a 2-fold increase in the duration of hospital stay, and a considerable increase in health care costs (3).

Recently, several studies have shown CRPA isolates that are susceptible to β-lactams, such as ceftazidime and cefepime (4, 5). Carbapenemase-producing CRPA clinical isolates usually show coresistance to other classes of antimicrobial agents, including β-lactams. Nevertheless, isolates with reduced sensitivity to carbapenems because of the inactivation of oprD in conjunction with other mechanisms, such as the downregulation of ampC or the overexpression of efflux pumps, sometimes show susceptibility to β-lactams (4, 5). The chromosomally encoded broad-spectrum AmpC β-lactamase contributes to the natural resistance of P. aeruginosa to many β-lactam antibiotics together with MexAB-OprM (6). In addition, a previous study showed a pronounced decrease in the rate of bacterial killing of P. aeruginosa PAO1 isolates by ceftazidime at high CFU (7); as one potential reason for the inoculum effect, increased beta-lactamase production at high CFU may occur due to the increased breakdown of the cell wall fragments of lysed bacteria (8). In a clinical setting, clinicians may consider the use of ceftazidime, cefepime, or piperacillin-tazobactam against imipenem-resistant P. aeruginosa isolates that are susceptible to ceftazidime. However, little evaluation has been done about whether the inoculum effect of β-lactams occurs for carbapenem-resistant ceftazidime-susceptible P. aeruginosa isolates.

In this study, we evaluated the carbapenem resistance mechanisms and the antimicrobial effects of ceftazidime, cefepime, and piperacillin-tazobactam on imipenem-resistant ceftazidime-susceptible P. aeruginosa clinical isolates as detected by time-kill curve methods.

RESULTS

Antimicrobial susceptibility and genotypes.

During the study period, 215 clinical isolates of P. aeruginosa were collected. Seventy-seven and 72 isolates (35.8% and 33.5%, respectively) were imipenem and meropenem resistant, respectively. Resistance rates to cefepime, ceftazidime, and piperacillin-tazobactam were 34.0%, 44.2%, and 36.7%, respectively. As many as 22.3% and 35.3% of the isolates were resistant to amikacin and ciprofloxacin, respectively, and 7.4% of isolates were colistin resistant. Among the 77 imipenem-resistant P. aeruginosa isolates, 18 isolates (23.4%) were ceftazidime susceptible. Although metallo-β-lactamase (MBL) genes were identified in 34 imipenem-resistant isolates (33 blaIMP-6 and 1 blaVIM-2) (44.2%), none of the 18 imipenem-resistant ceftazidime-susceptible isolates produced carbapenemases. Four of these imipenem-resistant P. aeruginosa isolates were susceptible to meropenem, and eight isolates were meropenem intermediate. Thus, only six isolates were resistant to both imipenem and meropenem. Among the imipenem-resistant ceftazidime-susceptible isolates, three, one, two, and three isolates were resistant to cefepime, piperacillin-tazobactam, amikacin, and ciprofloxacin, respectively (Table 1). No isolates were resistant to colistin.

TABLE 1.

Genotypes and antibiotic sensitivity of imipenem-resistant ceftazidime-susceptible P. aeruginosa isolates

Isolate ST MIC (mg/liter)a
IMP MER CAZ FEP TZP AMK CIP CST
K16-232b 1248 16 8 4 4 16/4 4 0.25 2
K16-304 508 32 32 8 8 32/4 2 2 2
K20-B046 412 8 8 8 8 32/4 16 1 2
K20-B186b 262 16 8 4 4 8/4 8 0.12 1
K20-B272 348 16 8 2 2 4/4 4 0.06 1
K20-B412 262 32 0.25 4 1 4/4 8 0.12 1
K20-B734 606 64 32 8 8 32/4 4 1 0.5
K20-B746 235 32 64 8 64 64/4 128 >64 1
K20-B784b 1154 8 0.25 1 2 1/4 8 >64 1
K21-023 298 64 8 2 2 16/4 1 0.5 1
K20-U386 233 16 16 8 8 16/4 2 2 1
K20-U422 274 8 32 8 64 64/4 16 1 0.5
K20-U705 508 16 16 2 2 4/4 2 0.06 2
K20-U754 971 16 4 2 2 4/4 4 0.12 2
K20-U888 277 32 8 8 8 8/4 8 2 2
K08-U016 244 16 8 4 8 64/4 16 2 2
K20-B923 235 16 8 4 16 16/4 128 16 2
K20-B978 641 16 4 2 32 128/4 16 1 1
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a

IMI, imipenem; MER, meropenem; CAZ, ceftazidime; FEP, cefepime; TZP, piperacillin-tazobactam; AMK, amikacin; CIP, ciprofloxacin; CST, colistin. The MIC values corresponding to resistance are underlined.

b

These isolates were used in in vitro time-kill assays.

The imipenem-resistant ceftazidime-susceptible P. aeruginosa isolates showed diverse genotypes. In the multilocus sequence typing (MLST) analysis, 16 different sequence types (STs) were identified, and only ST235 and ST262 included multiple isolates (Table 1). They tested negative for all of the carbapenemase genes analyzed.

Expression of oprD, ampC, and efflux pump genes.

All 18 imipenem-resistant ceftazidime-susceptible isolates showed a relevant decrease in oprD expression and a ≤30% decrease compared with that of reference P. aeruginosa strain PAO1 (Fig. 1A). The ampC gene overexpression was identified in only two isolates (Fig. 1B). Overexpression of efflux pumps was analyzed by means of genes mexB, mexD, mexF, and mexY. Although overexpression of mexB (≥3-fold higher than that of reference strain PAO1) was observed in 13 isolates (72.2%), mexD, mexF, or mexY overexpression (≥10-fold higher than that of reference strain PAO1) was rarely detected, with two isolates for mexD, one isolate for mexF, and one isolate for mexY (Fig. 1C to F).

FIG 1.

FIG 1

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Relative expression of oprD, ampC, and efflux pump genes in 18 imipenem-resistant ceftazidime-susceptible P. aeruginosa isolates. Black bars indicate significantly reduced expression of oprD (A) or significantly increased expression of ampC and efflux pump genes (B to F) compared with that of reference strain PAO1. Dashed lines indicate the levels of a significant decrease or increase of expression as follows: 30% (oprD) (A), 3-fold (mexB) (C), and 10-fold (ampC, mexD, mexF, and mexY, respectively) (B, D to F) compared with those of the reference strains.

Killing effects of β-lactams on imipenem-resistant ceftazidime-susceptible P. aeruginosa isolates.

Three imipenem-resistant ceftazidime-susceptible P. aeruginosa clinical isolates, K16-232, K20-B186, and K20-B784, were selected for construction of the time-kill curves against ceftazidime, cefepime, and piperacillin-tazobactam. They had the following different genotypes: ST1248, ST262, and ST1154, respectively.

Figure 2 shows the killing curves obtained with the three selected imipenem-resistant ceftazidime-susceptible P. aeruginosa isolates for standard (5 × 105 CFU/ml) and high (5 × 107 CFU/ml) inocula at ceftazidime concentrations of 0, 1, 4, 16, and 128 mg/liter. When 5 × 105 CFU/ml was inoculated, high concentrations of ceftazidime (16 and 128 mg/liter) showed significant bacterial killing effects against the three isolates after 4 h of treatment (Fig. 2A to C). Conversely, ceftazidime had no killing effects against the high inoculum of imipenem-resistant ceftazidime-susceptible isolates at any antibiotic concentration (Fig. 2D to F).

FIG 2.

FIG 2

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Time-kill curves of imipenem-resistant ceftazidime-susceptible P. aeruginosa clinical isolates exposed to ceftazidime (CAZ). (A) Isolate K16-232, inoculum of 5 × 105 CFU/ml; (B) isolate K20-B186, inoculum of 5 × 105 CFU/ml; (C) isolate K20-B784, inoculum of 5 × 105 CFU/ml; (D) isolate K16-232, inoculum of 5 × 107 CFU/ml; (E) isolate K20-B186, inoculum of 5 × 107 CFU/ml; (F) isolate K20-B784, inoculum of 5 × 107 CFU/ml. The proportion of surviving cells at each time point is shown as a ratio to live cell counts (CFU/ml) at time zero.

For cefepime, similar results were observed in comparison with those of ceftazidime (Fig. 3). Bacterial killing effects by high cefepime concentrations (16 and 128 mg/liter) were observed in two isolates, K16-232 and K20-B784, but regrowth after 8 h of treatment was shown in isolate K20-B186 (Fig. 3A to C). As with ceftazidime, no killing effects were observed for high bacterial inocula, even when a high concentration of cefepime was applied (Fig. 3D to F). When standard inocula were used with cefepime, the onset of bacterial killing showed a lag of 1 h for K20-B186 and K20-B784 (Fig. 3B and C).

FIG 3.

FIG 3

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Time-kill curves of imipenem-resistant ceftazidime-susceptible P. aeruginosa clinical isolates exposed to cefepime (CFP). (A) Isolate K16-232, inoculum of 5 × 105 CFU/ml; (B) isolate K20-B186, inoculum of 5 × 105 CFU/ml; (C) isolate K20-B784, inoculum of 5 × 105 CFU/ml; (D) isolate K16-232, inoculum of 5 × 107 CFU/ml; (E) isolate K20-B186, inoculum of 5 × 107 CFU/ml; (F) isolate K20-B784, inoculum of 5 × 107 CFU/ml. The proportion of surviving cells at each time point is shown as a ratio to live cell counts (CFU/ml) at time zero.

Figure 4 shows the killing curves for piperacillin-tazobactam with standard and high inocula. The killing activities of piperacillin-tazobactam also appeared to be time dependent for all three isolates. As with cefepime, 16/4 and 128/4 mg/liter of piperacillin-tazobactam, respectively, showed effective killing activity against the standard inocula of K16-232 and K20-B784 (Fig. 4A and C). Although killing effects by piperacillin-tazobactam were observed for K20-B784 in high inocula, no concentrations of piperacillin-tazobactam showed effective killing activities against the other two isolates (Fig. 4D to F). Even for K20-B784, regrowth was observed after 8 h of treatment (Fig. 4F).

FIG 4.

FIG 4

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Time-kill curves of imipenem-resistant ceftazidime-susceptible P. aeruginosa clinical isolates exposed to piperacillin-tazobactam (P/T). (A) Isolate K16-232, inoculum of 5 × 105 CFU/ml; (B) isolate K20-B186, inoculum of 5 × 105 CFU/ml; (C) isolate K20-B784, inoculum of 5 × 105 CFU/ml; (D) isolate K16-232, inoculum of 5 × 107 CFU/ml; (E) isolate K20-B186, inoculum of 5 × 107 CFU/ml; (F) isolate K20-B784, inoculum of 5 × 107 CFU/ml. The proportion of surviving cells at each time point is shown as a ratio to live cell counts (CFU/ml) at time zero.

Figure 5 shows the killing curves of imipenem-susceptible ceftazidime-susceptible isolate K07-089 for three antibiotics with standard and high inocula. The three antibiotics showed more effective killing effects when high concentrations were applied. However, the survival rates of the imipenem-susceptible ceftazidime-susceptible isolate were also lower for β-lactams in high inocula compared with those of imipenem-resistant ceftazidime-susceptible isolates.

FIG 5.

FIG 5

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Time-kill curves of imipenem-susceptible ceftazidime-susceptible P. aeruginosa clinical isolates (K07-089) exposed to ceftazidime (A and B), cefepime (C and D), and piperacillin-tazobactam (E and F). (A, C, and E) Inoculum of 5 × 105 CFU/ml; (B, D, and F) inoculum of 5 × 107 CFU/ml. The proportion of surviving cells at each time point is shown as a ratio to live cell counts (CFU/ml) at time zero.

DISCUSSION

In this study, among the 77 imipenem-resistant P. aeruginosa clinical isolates, 18 isolates (23.4%) were found to be susceptible to ceftazidime. These imipenem-resistant ceftazidime-susceptible isolates belong to diverse STs, which is suggestive of independent emergence. None of these 18 clinical isolates produce carbapenemases. The loss of OprD function may be the major determinant of this noncarbapenemase-mediated resistance to carbapenem as shown elsewhere (9). In addition, MexAB-OprM may also play a relevant role in the carbapenem resistance of these isolates, and the Ambler class C β-lactamase enzyme did play a role in a few isolates. It has also been reported that carbapenem-resistant ceftazidime-susceptible and/or cefepime-susceptible P. aeruginosa isolates do not produce carbapenemases, extended-spectrum β-lactamases, or Ambler class C β-lactamase enzymes (4, 5). The relatively low MICs of meropenem observed here are quite different from the high-level resistance to meropenem among IMP6-producing P. aeruginosa isolates, most of which belong to ST235 in the Republic of Korea (9, 10). Although we investigated the carbapenem resistance mechanism to find out if the resistance mechanism may affect susceptibility to β-lactams, such as ceftazidime, cefepime, and piperacillin-tazobactam, we did not find any relationships between them.

We studied the activity of β-lactams against imipenem-resistant ceftazidime-susceptible P. aeruginosa clinical isolates using the time-kill assay. We observed inoculum effects for three β-lactams; although β-lactam antibiotics showed significant killing effects with the standard inocula, they had no effect with the high inocula. Although the isolates were susceptible to β-lactam antibiotics, these bacteria could not be eliminated by β-lactams even at 128 mg/liter when a high bacterial concentration was inoculated. Conversely, inoculum effects for the antibiotics were not evident for the imipenem-susceptible ceftazidime-susceptible isolate. Such an inoculum effect was evident for ceftazidime. It is known that the inoculum effect is associated with the production of β-lactamases (8). Nonetheless, only a few isolates in this study showed AmpC hyperproduction. Therefore, it is assumed that factors other than the amount of AmpC β-lactamase are responsible for the inoculum effect. Other factors, such as quorum sensing, decreasing expression of selected penicillin-binding proteins, and expression of autolysins, have been reported to be related to the inoculum effect (11–13); these factors were not explored in the present study. In addition, Jumbe et al. (14) showed that a substantially higher drug exposure was required to suppress resistance for high-CFU inocula, as the high CFU exceeded the inverse of the mutation frequency. However, we did not evaluate the mutation frequency in time-kill studies. Thus, it is not known if the emergence of resistant mutants caused the inoculum effect in our study.

Over time, colony counts may increase after an initial decrease in the time-kill assay for the lower concentrations of antibiotics. Even for the antibiotic concentrations where bacterial killing was observed over 8 h, bacterial regrowth may occur after that time. Thus, 8 h may be short for the observation of the final effect on bacterial killing. Bacterial regrowth may be due to the selection of resistant mutants, inactivation of the antimicrobial agents, or regrowth of susceptible bacterial cells that have escaped antimicrobial activity by adhering to the wall of the culture vessel.

Although the clinical significance of the inoculum effect is not well established, animal infection models have shown for β-lactams and other antibiotics that a high CFU count or delayed treatment of infection may greatly increase mortality or attenuate anti-infective effects (15, 16). On the basis of our results, we propose that β-lactam monotherapy may not be effective against imipenem-resistant P. aeruginosa infections with large bacterial burdens even though they are susceptible to β-lactams. As indicated recently for carbapenems (17), MIC evaluation alone may not be enough to predict the therapeutic efficacy of β-lactams against ceftazidime-susceptible P. aeruginosa infections. In addition, three β-lactam antibiotics showed a lag of bacterial killing in most cases; that is, killing effects were delayed soon after inoculation even when a significant decrease in bacterial CFU counts was observed after 8 h of treatment. Thus, β-lactams should be used with caution at the early stage of severe infections even at a low bacterial burden.

In this study, one-fourth of imipenem-resistant P. aeruginosa isolates were found to be susceptible to ceftazidime, and they emerged independently mainly due to the loss of OprD function. We also showed that the effects of β-lactam monotherapy against imipenem-resistant ceftazidime-susceptible P. aeruginosa isolates at a high inoculum are attenuated due to the inoculum effect. Thus, we suggest that β-lactam antibiotics be used with caution in patients with imipenem-resistant ceftazidime-susceptible P. aeruginosa isolates, especially in high-inoculum infections such as endocarditis and osteomyelitis.

MATERIALS AND METHODS

Bacterial isolates.

P. aeruginosa isolates were collected from hospitalized patients with sepsis or urinary tract infection in eight hospitals during a prospective surveillance study from November 2012 to August 2013. We included only the first episode of P. aeruginosa infection for each patient in the analysis and excluded polymicrobial infections. Species identification was performed by means of a Vitek 2 system (bioMérieux, Hazelwood, MO, USA).

Antimicrobial susceptibility testing.

Antimicrobial susceptibility was tested by the microdilution method following Clinical and Laboratory Standards Institute (CLSI) guidelines (18). Eight antimicrobial agents were tested: imipenem, meropenem, ceftazidime, cefepime, piperacillin-tazobactam, ciprofloxacin, amikacin, and colistin. Susceptibility was interpreted according to CLSI breakpoints (18). Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 served as quality control strains.

Genotyping.

For genotyping, multilocus sequence typing (MLST) was performed on imipenem-resistant ceftazidime-susceptible isolates as described previously (19). They were screened by PCR for blaIMP, blaVIM, blaSPM, blaGIM, blaSIM, blaNDM, and blaKPC (20–22).

Quantitative reverse-transcription PCR.

mRNA transcription levels of oprD, ampC, mexB, mexD, mexF, and mexY were determined by quantitative reverse transcription PCR (qRT-PCR) in all imipenem-resistant ceftazidime-susceptible P. aeruginosa isolates as described elsewhere (23). Reverse transcription was conducted in accordance with protocol for the use of Omniscript reverse transcriptase (Qiagen GmbH, Hilden, Germany). Quantification of oprD transcripts was carried out using the SYBR green PCR master mix (Applied Biosystems, Foster City, CA). The mRNA levels were normalized to housekeeping gene rpsL and were expressed as a ratio to PAO1 (whose mRNA expression was set to 1.0). Relative quantification was done by the comparative threshold cycle (CT) method (2−ΔΔCT) (24). Reduced oprD expression was considered relevant when it was ≤30% compared with that of reference strain PAO1 (25). Isolates were considered AmpC, MexCD-OprJ, MexEF-OprN, or MexXY-OprM hyperproducers when the expression of gene ampC, mexD, mexF, or mexY, respectively, was ≥10-fold higher than that of the reference strains (the isolates were considered negative for those enzymes if the difference was less than 5-fold). Isolates were considered MexAB-OprM hyperproducers when mexB expression was ≥3-fold higher than that of the reference strain and negative if lower than 2-fold (26).

The in vitro time-kill assay.

Three imipenem-resistant ceftazidime-susceptible isolates were randomly selected among tested isolates for time-kill assays. One imipenem-susceptible ceftazidime-susceptible isolate (K07-089) was also included as a control. Time-kill studies were performed as described previously with some modifications (15). Freshly prepared colonies collected from the surface of an overnight agar culture were suspended in LB broth and were then immediately diluted to a 0.5 McFarland standard (approximately 1.5 × 108 CFU/ml). The bacterial suspensions were washed by phosphate-buffered saline (PBS). An appropriate amount of bacteria was diluted in prewarmed LB broth to achieve the desired CFU count (5 × 105 CFU/ml and 5 × 107 CFU/ml) in a final volume of 10 ml of LB broth. Then, ceftazidime, cefepime, and piperacillin-tazobactam were added to the prepared bacterial suspensions so that the final drug concentration was 0, 1, 4, 16, or 128 μg/ml of ceftazidime, cefepime, or piperacillin-tazobactam, and the cultures were shaken and cultivated at 37°C for 0, 1, 4, or 8 h. Then, 0.1-ml samples from wells were serially diluted and cultured on plates with overnight incubation, and CFU counts were determined for construction of the killing curves of antibiotics. The lower limit of quantitation was 400 CFU/ml (i.e., 2.6 log10 CFU/ml).

ACKNOWLEDGMENTS

All P. aeruginosa isolates used in this study were obtained from the Asian Bacterial Bank (ABB) of the Asia Pacific Foundation for Infectious Diseases (APFID, Seoul, Republic of Korea).

This study was supported by the Samsung Biomedical Research Institute grant and the Korea Healthcare Technology R&D Project, Ministry of Health, Welfare, and Family Affairs, Seoul, Republic of Korea (grant HI15C1733).

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