Applied Biochemistry and Biotechnology (2024) 196:690–700

https://doi.org/10.1007/s12010-023-04516-8

ORIGINAL ARTICLE

Study on the Mechanism of Levofloxacin Combined

with Imipenem Against Pseudomonas aeruginosa

Qiang Lu1 · Qingqing Yang1

Accepted: 11 April 2023 / Published online: 13 May 2023

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023

Abstract
Pseudomonas aeruginosa can develop resistance. Therefore, it is necessary to design proper
treatment for it. Pseudomonas aeruginosa can develop resistance against levofloxacin due to the
development of efflux pumps. However, the development of these efflux pumps cannot develop
resistance against imipenem. Additionally, the MexCDOprJ efflux system which is responsible for
the resistance of Pseudomonas aeruginosa to levofloxacin is highly susceptible to imipenem. The
objective of the study was to evaluate the emergence of resistance of Pseudomonas aeruginosa
against 750 mg levofloxacin, 250 mg imipenem, and a combination of 750 mg levofloxacin and
250 mg imipenem. An in vitro pharmacodynamic model was selected for the evaluation of the
emergence of resistance. Pseudomonas aeruginosa strain 236, Pseudomonas aeruginosa strain
GB2, and Pseudomonas aeruginosa strain GB65 were selected. Susceptibility testing of both
antibiotics was done by agar dilution methodology. A disk diffusion bioassay was performed for
antibiotics. RT-PCR measurement was done for the evaluation of expressions of Pseudomonas
aeruginosa genes. Samples were tested at 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, 24 h, and 30 h. Levofloxa-
cin and imipenem both individually reported a decrease in colony-forming unit per milliliter of
strength in the initial stage but in the later stage both develop resistance individually. Levofloxacin
with imipenem had no resistance to Pseudomonas aeruginosa during 30 h. Time after the start of
development of resistance or decrease in clinical efficacy was higher for levofloxacin and imipe-
nem combination in all strains. The concentration of Pseudomonas aeruginosa at the time after
the start of development of resistance or decrease in clinical efficacy was fewer for levofloxacin
and imipenem combination. Levofloxacin with imipenem is recommended for the treatment of
infection due to Pseudomonas aeruginosa.

Keywords Antibiotics · Drug resistance · Imipenem · Levofloxacin · Pharmacodynamic

model · Pseudomonas aeruginosa

* Qiang Lu
qianglu0721@163.com
1
Northern Jiangsu People’s Hospital, Nantong West Road No. 98, Yangzhou 225001, Jiangsu,
China

1 Vol:.(1234567890)
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Introduction

Pseudomonas aeruginosa is deadly for immunocompromised patients [1]. These pathogens

can develop resistance. Therefore, it is necessary to design proper treatment for infections
due to Pseudomonas aeruginosa [2]. Usually, aminoglycosides with β-lactam are used
for the treatment of infections due to Pseudomonas aeruginosa [1]. However, this is not
always an effective treatment [3]. That is why a more effective combination of antibiotics
is required.
Pseudomonas aeruginosa can develop resistance against levofloxacin due to the devel-
opment of efflux pumps. However, the development of these efflux pumps cannot develop
resistance against imipenem [4]. Additionally, the MexCDOprJ efflux system which is
responsible for the resistance of Pseudomonas aeruginosa to levofloxacin is highly suscep-
tible to imipenem[5]. Also, the OprD porin expressions are decreased in the outer mem-
brane leading to develop resistance of Pseudomonas aeruginosa to levofloxacin which is
highly susceptible to imipenem [6]. However, overexpression of the MexEF-OprN efflux
pump leads to the development of resistance against levofloxacin and imipenem [7]. Theo-
retically, it is irrational.
The objective of the study was to evaluate the emergence of resistance of Pseudomonas
aeruginosa against 750 mg levofloxacin, 250 mg imipenem, and a combination of 750 mg
levofloxacin and 250 mg imipenem.

Materials and Methods

Bacterial Strains and Culture Conditions

Pseudomonas aeruginosa strain 236, Pseudomonas aeruginosa strain GB2, and Pseu-
domonas aeruginosa strain GB65 were selected and stored at – 70 °C in 50% brain–heart
infusion broth and 50% sterile horse serum.

Fig. 1  Pharmacodynamics of Pseudomonas aeruginosa strain 236

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692 Applied Biochemistry and Biotechnology (2024) 196:690–700

Table 1  Pharmacodynamics of Time (h) log10 CFU/mL

Pseudomonas aeruginosa strain
236 Levofloxacin Imipenem Levofloxa-
cin + imipe-
nem

0 8 8 8
2 7.6 7.7 7
4 5.5 6.2 5
6 5.1 5.4 4.9
8 5 4.2 4.8
10 5.4 4 4.7
12 5.8 3.8 4.5
14 6 3.9 4
16 6.5 4.1 3.5
18 7.1 4.2 2.9
20 7.3 4.5 2.8
22 7.5 4.7 2.7
24 7.6 4.8 2.65

Antibiotics and Susceptibility Testing

Levofloxacin was purchased from Sanofi Aventis Pharma, Bridgewater Township, NJ,
USA. Imipenem was purchased from Novartis Pharmaceuticals Corporation, East Hanover,
NJ, USA. Antibiotics were solubilized in 0.2 mL 0.1 N NaOH and made 1000 mL with dis-
tilled water. Then, it was filtered through a membrane filter. Susceptibility testing of both
antibiotics was done by agar dilution methodology [8].

Fig. 2  Pharmacodynamics of Pseudomonas aeruginosa strain GB2

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Table 2  Pharmacodynamics of Time (h) log10 CFU/mL

Pseudomonas aeruginosa strain
GB2 Levofloxacin Imipenem Levofloxa-
cin + imipe-
nem

0 8 8 8
2 7.5 7.8 7.1
4 6 7.1 7.3
6 5.9 6.5 7
8 5.2 6 6.5
10 5 5.5 6
12 4.7 5 5.5
14 4.9 5.4 5
16 5.7 5.7 4.8
18 6.2 6.2 4.2
20 6.8 6.8 3.8
22 6.9 6.9 3.4
24 7 7.1 3

In Vivo Pharmacokinetic Model

Two compartments kinetic model was performed for the experiment [9]. The volume of
levofloxacin-containing media in the central reservoir was 500 mL and that for imipenem-
containing media was 200 mL for experiments and that for levofloxacin and imipenem-
containing media in the central reservoir was 500 mL.

Pharmacodynamics Against Pseudomonas aeruginosa

1 × ­108 CFU/mL of Pseudomonas aeruginosa was cultured in each compartment. In control,

1 × ­106 CFU/mL of Pseudomonas aeruginosa was cultured. Dosing of levofloxacin was done

Fig. 3  Pharmacodynamics of Pseudomonas aeruginosa strain GB65

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694 Applied Biochemistry and Biotechnology (2024) 196:690–700

Table 3  Pharmacodynamics of Time (h) log10 CFU/mL

Pseudomonas aeruginosa strain
GB65 Levofloxacin Imipenem Levofloxa-
cin + imipe-
nem

0 8 8 8
2 7.4 7.9 7.3
4 7 7.2 7.1
6 6.6 7 7
8 6 6.6 6.4
10 5.2 6 6
12 4.7 5.5 5.1
14 4.2 5 5
16 5 6 4.3
18 6 7 4
20 7 7.5 3.5
22 7.5 7.8 3
24 7.7 7.91 2

at 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, 24 h, and 30 h dosing of imipenem was done at 2 h, 4 h, 6 h,

8 h, 12 h, 16 h, 24 h, and 30 h, and that of the combination was done at 2 h, 4 h, 6 h, 8 h, 12 h,
16 h, 24 h, and 30 h [10]. A disk diffusion bioassay was performed for antibiotics [11].

RT‑PCR Measurement

RT-PCR measurement was done for the evaluation of expressions of Pseudomonas aerugi-
nosa genes.

Results

Levofloxacin and imipenem both individually reported a decrease in colony-forming unit

per milliliter of strength in the initial stage but in the later stage both develop resistance
individually. Levofloxacin with imipenem had no resistance to Pseudomonas aeruginosa
during 30 h. Pharmacodynamics of Pseudomonas aeruginosa strain 236, Pseudomonas

Table 4  Time after the start of Pseudomonas aer- Time (h)

development of resistance or uginosa strains
decrease in clinical efficacy Levofloxacin Imipenem Levofloxa-
cin + imipe-
nem

236 8 14 24
GB2 14 12 24
GB65 16 14 24

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Table 5  The concentration Pseudomonas aer- CFU/mL

of Pseudomonas aeruginosa uginosa strains
at a time after the start of Levofloxacin Imipenem Levofloxa-
development of resistance or cin + imipe-
decrease in clinical efficacy nem

236 105 104 317

5
GB2 10 3.16 × ­105 300
GB65 105 105 310

aeruginosa strain GB2, and Pseudomonas aeruginosa strain GB65 were reported in Fig. 1
and Table 1, Fig. 2 and Table 2, and Fig. 3 and Table 3, respectively.

Pharmacodynamics

Time after the start of development of resistance or decrease in clinical efficacy was higher
for levofloxacin and imipenem combination in all strains (Table 4). Also, colony-forming
unit per milliliter of Pseudomonas aeruginosa at the time of start development of resist-
ance or decrease in clinical efficacy is reported in Table 5.

RT‑PCR Measurement

Expressions of a mutant gene were fewer for the combination of levofloxacin and imi-
penem than those of levofloxacin alone and imipenem alone. Expressions of a mutant
gene of Pseudomonas aeruginosa strain 236, Pseudomonas aeruginosa strain GB2, and
Pseudomonas aeruginosa strain GB65 for levofloxacin, imipenem, and a combination
of levofloxacin with imipenem were reported in Fig. 4, Fig. 5, and Fig. 6 and Table 5,
Table 6, and Table 7, respectively.

Fig. 4  Expressions of a mutant gene of Pseudomonas aeruginosa strain 236

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Fig. 5  Expressions of a mutant gene of Pseudomonas aeruginosa strain GB2

All strains of Pseudomonas aeruginosa at the time of development of resistance had

higher Pseudomonas aeruginosa concentration if levofloxacin alone or imipenem alone
were treatments. However, if the combination of levofloxacin and imipenem had fewer
Pseudomonas aeruginosa concentration at the time of development of resistance. Rep-
resentative microscopic image for Pseudomonas aeruginosa strain 236 at the time of
development of resistance is presented in Fig. 7 (Table 8).

Discussion

Treatment infections caused by Pseudomonas aeruginosa are difficult to treat because

they may acquire resistance [1]. Mutation in the gene of Pseudomonas aeruginosa leads
to the development of multi-drug resistance of it during treatment. In the current study,
levofloxacin and imipenem both individually reported a decrease in colony-forming unit

Fig. 6  Expressions of a mutant gene of Pseudomonas aeruginosa strain GB65

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Table 6  Expressions of mutant Parameters Levofloxacin Imipenem Levofloxa-

gene Pseudomonas aeruginosa cin + imipe-
strain 236 nem

Average 25 15 5
SD 5 3 1
Minimum 15 10 1
Maximum 30 18 7
Mode 20 12 5

per milliliter of strength in the initial stage but in a later stage both develop resistance indi-
vidually. Pseudomonas aeruginosa always treated with antibiotics in the initial stage shows
activity but in the later stage develops resistance [1]. This phenomenon is reported for
ticarcillin, carbenicillin, ticarcillin-clavulanate, aztreonam, piperacillin, and the extended-
spectrum cephalosporins [12]. This leads to clinical failure in about 50% of patients [1].
When Pseudomonas aeruginosa infection was outside the urinary tract, particularly in
cases of cystic fibrosis or neutropenia, the clinical failure rate is much higher [13]. The
combination of antipseudomonal β-lactam and an aminoglycoside is preferred in the treat-
ment of Pseudomonas aeruginosa because there is less emergence of resistance [3].
The study found that levofloxacin with imipenem had no resistance to Pseudomonas
aeruginosa during 30 h. The theory behind the development of resistance for Pseudomonas
aeruginosa against levofloxacin and that for imipenem do not overlap [1]. Therefore, levo-
floxacin with imipenem combination remains effective against Pseudomonas aeruginosa.
The study reported a few mutant resistances for Pseudomonas aeruginosa against
levofloxacin with imipenem combination. This is because of the development of the
MexEF-OprN efflux pump in the Pseudomonas aeruginosa strains [14]. The combina-
tion of levofloxacin with imipenem also has the chance of development of resistance
due to mutation in Pseudomonas aeruginosa. Also, the resistance development potential
of Pseudomonas aeruginosa is not fully recognized.
The study used three strains (236, GB2, and GB65) of Pseudomonas aeruginosa.
Minimum inhibitory concentration (MIC) values for these strains of Pseudomonas aer-
uginosa for levofloxacin were 2–4 μG/mL and that for imipenem was 1–2 μG/mL. These
all strains have fast development of resistance against several antibiotics [1].
The study used 750 mg twice the dose of levofloxacin and 250 mg twice the dose of
imipenem for the model. These were the lowest possible doses of levofloxacin and imipe-
nem for drug action in humans [1]. Therefore, to develop an environment like human tis-
sue, 750 mg levofloxacin and 250 mg imipenem were designed for study.

Table 7  Expressions of mutant Parameters Levofloxacin Imipenem Levofloxa-

gene Pseudomonas aeruginosa cin + imipe-
strain GB2 nem

Average 27 17 6
SD 7 3 1
Minimum 14 12 2
Maximum 32 20 10
Mode 21 14 5

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698 Applied Biochemistry and Biotechnology (2024) 196:690–700

Fig. 7  Microscopic view of Pseudomonas aeruginosa strain 236 at the time of development of resistance. A
Pseudomonas aeruginosa treated with levofloxacin alone. B Pseudomonas aeruginosa treated with imipe-
nem alone. C Pseudomonas aeruginosa treated with combination of levofloxacin and imipenem

Time after the start of development of resistance or decrease in clinical efficacy was higher for
levofloxacin and imipenem combination in all strains. Resistance of levofloxacin and imipenem
for Pseudomonas aeruginosa are not overlapped with each other [1]. The treatment period of
levofloxacin and imipenem combination could be higher comparatively other antibiotics.
The concentration of Pseudomonas aeruginosa at the time after the start of develop-
ment of resistance or decrease in clinical efficacy was fewer for levofloxacin and imipenem
combination [15]. The resistance of levofloxacin is susceptible to imipenem [5, 6]. Also,
the least resistance of Pseudomonas aeruginosa is for imipenem. Levofloxacin and imipe-
nem combination last longer for treatment [16].
The concentration of Pseudomonas aeruginosa was not zero for levofloxacin and imi-
penem combination at the time after the start of development of resistance or decrease
in clinical efficacy [17]. The development of the MexEF-OprN efflux pump in the Pseu-
domonas aeruginosa strains is responsible for the decline of clinical efficacy for levofloxa-
cin and imipenem combination [14–18]. The dose oscillation study might be effective to
overcome these issues [19].
In the limitation of the study, for example, ciprofloxacin also has efficacy against Pseu-
domonas aeruginosa [18] but it was not tested.

Conclusions

Levofloxacin with imipenem has the least resistance against Pseudomonas aerugi-
nosa. The treatment period of levofloxacin and imipenem combination could be higher
comparatively other antibiotics in Pseudomonas aeruginosa infections. Levofloxacin

Table 8  Expressions of mutant Parameters Levofloxacin Imipenem Levofloxa-

gene Pseudomonas aeruginosa cin + imipe-
strain GB65 nem

Average 28 20 6
SD 5 5 2
Minimum 12 12 2
Maximum 33 21 11
Mode 20 15 9

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with imipenem is recommended for the treatment of infection due to Pseudomonas

aeruginosa.

Author Contribution All authors have contributed equally to this manuscript.

Data Availability The data of the study has been included in this study.

Declarations
Ethical Approval The Institutional Ethical Committees of Chengdu Medical College has approved the study.

Consent to Participate Not applicable.

Consent to Publish Not applicable.

Conflict of Interest The authors declare no competing interests.

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