In vitro Antibacterial Effect of Reduning Combined with Polymyxin on Carbapenem Resistant Klebsiella pneumoniae

Abstract

Objective
This study aimed to investigate the status of carbapenem-resistant strains of Klebsiella pneumoniae isolated from the Department of Microbiology, Zhejiang Tongde Hospital between September 2023 and February 2024, and to examine the in vitro antibacterial effect of Reduning combined with polymyxin on carbapenem-resistant Klebsiella pneumoniae (CRKP), which may provide evidence on the application of Reduning in the clinical anti-infective therapy.

Methods
A total of 50 different isolates of CRKP were collected, and the minimum inhibitory concentrations (MIC) of polymyxin, Reduning and polymyxin plus Reduning were measured with microbroth dilution method. Then, the fractional inhibition concentration index (FICI) was calculated.

Results
A total of 50 strains of CRKP were isolated, sputum and clean urine were the most common source of CRKP, and intensive care unit was the most common source department. More than 90% of CRKP strains were resistant to cefepime, ceftazidime, piperacillin/tazobactam, and cefoperazone/sulbactam. The rate of resistance to levofloxacin was high, but that to tobramycin, tigecycline, and compound sulfamethoxazole was low. In addition, MIC of Reduning plus polymyxin for CRKP was lower than that of Reduning or polymyxin alone. Among 50 strains of CRKP, FICI ≤0.5 was noted in 7 strains, 0.5 < FICI ≤ 1.0 in 43 strains, and none had FICI >1.0. The results showed Reduning combined with polymyxin B exerted additive effect on CRKP and conferred synergistic effect on several strains of CRKP.

Conclusion
Reduning has antibacterial effect on CRKP in vitro, and the addition of Reduning can reduce the dose of polymyxin in the treatment of CRKP.

Full text

ORIGINAL RESEARCH
In vitro Antibacterial Effect of Reduning Combined
with Polymyxin on Carbapenem Resistant Klebsiella
pneumoniae
Zui-Fei Shangguan
1
, Hong-Lei Chen
2
, Yi-Fan Li
3
, Na Shi
4
, Qi-Fen Mao
2
1
Department of Clinical Laboratory, The First Afliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Chinese
Medicine), Hangzhou, Zhejiang, 310006, People’s Republic of China;
2
Department of Clinical Laboratory, Tongde Hospital of Zhejiang Province,
Hangzhou, Zhejiang, 310012, People’s Republic of China;
3
College of Laboratory Medicine and College of Bioengineering, Hangzhou Medical
University, Hangzhou, Zhejiang, 311399, People’s Republic of China;
4
Medical Laboratory, Second Sanatorium of Air Force Healthcare Center for
Special Services, Hangzhou, Zhejiang, 310007, People’s Republic of China
Correspondence: Qi-Fen Mao, Department of Clinical Laboratory, Tongde Hospital of Zhejiang Province, 234#, Gucui Road, Hangzhou, Zhejiang,
310012, People’s Republic of China, Email maoqifen@alu.zcmu.edu.cn
Objective: This study aimed to investigate the status of carbapenem-resistant strains of Klebsiella pneumoniae isolated from the
Department of Microbiology, Zhejiang Tongde Hospital between September 2023 and February 2024, and to examine the in vitro
antibacterial effect of Reduning combined with polymyxin on carbapenem-resistant Klebsiella pneumoniae (CRKP), which may
provide evidence on the application of Reduning in the clinical anti-infective therapy.
Methods: A total of 50 different isolates of CRKP were collected, and the minimum inhibitory concentrations (MIC) of polymyxin,
Reduning and polymyxin plus Reduning were measured with microbroth dilution method. Then, the fractional inhibition concentration
index (FICI) was calculated.
Results: A total of 50 strains of CRKP were isolated, sputum and clean urine were the most common source of CRKP, and intensive
care unit was the most common source department. More than 90% of CRKP strains were resistant to cefepime, ceftazidime,
piperacillin/tazobactam, and cefoperazone/sulbactam. The rate of resistance to levooxacin was high, but that to tobramycin,
tigecycline, and compound sulfamethoxazole was low. In addition, MIC of Reduning plus polymyxin for CRKP was lower than
that of Reduning or polymyxin alone. Among 50 strains of CRKP, FICI ≤0.5 was noted in 7 strains, 0.5 < FICI ≤ 1.0 in 43 strains, and
none had FICI >1.0. The results showed Reduning combined with polymyxin B exerted additive effect on CRKP and conferred
synergistic effect on several strains of CRKP.
Conclusion: Reduning has antibacterial effect on CRKP in vitro, and the addition of Reduning can reduce the dose of polymyxin in
the treatment of CRKP.
Keywords: Reduning, polymyxin, Carbapenem resistant Klebsiella pneumoniae, in vitro antibacterial activity
Introduction
Klebsiella pneumoniae (KP) is a common opportunistic pathogen that causes nosocomial infections, including lung,
blood ow, and urinary tract infections, and in severe cases, it can also lead to critical illnesses such as purulent
meningitis.
1
Carbapenem antibiotics have the characteristics of a wide antibacterial spectrum and high antibacterial
activity and have been widely used in the treatment of KP infection.
2
In recent years, with the increasing use of
carbapenem antibiotics in clinical practice, the detection rate of carbapenem-resistant Klebsiella pneumoniae (CRKP)
and the incidence of CRKP infection are increasing over year.
3
The Chinese Bacterial Resistance Monitoring Network
shows that the detection rates of Klebsiella pneumoniae resistant to meropenem and imipenem are 23.6% and 22.5%,
respectively, between January 2023 and December 2023.
4
Klebsiella pneumoniae produces carbapenemases,
5
such as
KPC, NDM, OXA-48, etc., causing the ineffectiveness of carbapenem antibiotics in the treatment of Klebsiella
pneumoniae infection. CRKP was generally resistant to other antibiotics, and therefore patients with CRKP infection
Infection and Drug Resistance 2025:18 227–237 227
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Infection and Drug Resistance
Open Access Full Text Article
Received: 11 September 2024
Accepted: 19 December 2024
Published: 13 January 2025

have a high risk of death. Patients infected by CRKP usually develop severe and long-lasting symptoms, which poses
a signicant challenge to clinical infection control.
6–8
Polymyxin is a cationic antimicrobial peptide discovered in 1949, mainly including polymyxin B and polymyxin
E (myxin). Polymyxin acts on negatively charged lipids of lipopolysaccharides on cell membrane to destroy the integrity
of cell membrane, exerting bactericidal effect. However, it was once abandoned due to its side effects such as
neurotoxicity and nephrotoxicity.
9
However, due to the emergence of multidrug-resistant bacteria, polymyxin has been
reintroduced to clinical treatment of infections. To date, polymyxin has become a key antibiotic and the last resort for the
treatment of carbapenem-resistant bacteria. With the increasing use of polymyxin in clinical practice, the detection rate of
polymyxin-resistant pathogens has signicantly increased.
10
According to CHINET data in 2023, the detection rate of
CRKP resistant to polymyxin is about 10.1% in China. Therefore, it is particularly crucial to identify a drug that is both
efcient and low toxic and can be used in combination with polymyxin. Traditional Chinese Medicine is highly praised
for its advantages of less drug resistance and minimal side effects.
11
With the deepening of investigation on the efcacy
of Traditional Chinese Medicine, investigators have revealed that the combination of Traditional Chinese Medicine with
antibiotics can not only enhance the therapeutic effect but also help reduce the adverse reactions of drugs. Traditional
Chinese Medicine directly acts on pathogens by intervening with metabolic processes in pathogens and disrupting their
structure and function (such as affecting bacterial cell wall formation, hindering protein synthesis in bacteria, inhibiting
nucleic acid metabolism, or interfering with other pathways important for the metabolism.
12
To date, little is known about
the bacterial resistance to Traditional Chinese Medicine. The combination of Traditional Chinese Medicine and
antibiotics may be a new way to treat CRKP infection. As an iconic heat clearing and detoxifying Traditional Chinese
Medicine, Reduning can effectively suppress inammation and eliminate toxins in the body. In addition, Reduning has
other pharmacological effects, including inhibiting viruses, eliminating bacteria, relieving allergic reactions, suppressing
tumorigenesis, maintaining hepatic health, stimulating bile production, and regulating the immune system.
13
Especially
in the treatment of fungi and bacterial infections, Reduning has particularly outstanding effects. Therefore, we speculate
that Reduning not only has the inhibitory effect on KP infection but also enhances the therapeutic effect of polymyxin
and reduces the toxicity and side effects of polymyxin due to the reduction of dose of polymyxin.
This study aimed to investigate the in vitro inhibitory effect of Reduning on CRKP and explore its synergistic effect
with polymyxin, in order to evaluate the clinical applicability of combined use of Reduning and polymyxin in the
treatment of CRKP, which may provide evidence on the appropriate use of antibiotics in clinical practice.
Materials and Methods
Source of Strains
A total of 50 strains of CRKP were isolated from the Department of Microbiology, Zhejiang Tongde Hospital between
September 2023 and February 2024. The study was approved by the ethic committee of Tongde Hospital of Zhejiang
Province (2022–077), and written informed consent was obtained from each patient. The sources of these strains included
sputum, urine, pleural and peritoneal uid, throat swabs, and catheter drainage. The duplicate strains from the same
patient and the same site were excluded in the collection of bacterial strains. The growth and colony morphology of
different bacterial strains were observed in the culture medium, and the bacterial characteristics were compared under
different culture conditions. All the strains were conrmed as KP by mass spectrometry. After strain identication, the
strains were preserved for future use.
Drugs
Reduning injection (10 mL; Lot number: 230430, Jiangsu Kanion Pharmaceutical Co., Ltd) and Sulfate polymyxin
B injection (500000 U; Lot number: 2308802, SPH No.1 Biochemical & Pharmaceutical Co., Ltd.) were used in the
present study.
Reagents and Instrument
Following instrument and reagents were used in the present study (Table 1).
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Strain Identication and Routine Drug Susceptibility Testing
The specimens were rst seeded on blood agar plates and MacConkey plates, followed by incubation for 18–24 h;
Subsequently, individual colonies were screened on the MacConkey plate. All strains were identied as Klebsiella
pneumoniae using a matrix-assisted laser desorption/ionization time-of-ight mass spectrometry (MALDI-TOF-MS)
identication system. In “Matching antibiotic susceptibility test”, we used the Vitek 2 system for antibiotic susceptibility
testing, using Gram negative bacterial susceptibility card 335 (N335).
Detection of Carbapenemase Type
The enzyme immunochromatography was employed for the detection of carbapenemase type.
14
In the positive speci-
mens, the components of carbapenemase can interact with the monoclonal mouse carbapenem enol antibody coated with
gold particles, forming a specic immune complex. This complex then moves onto the T region of nitrocellulose
membrane and specically binds to the pre-existing monoclonal antibody against carbapenemase, ultimately revealing
a red reaction line. When the remaining gold labeled antibodies pass through the quality control line (C line), the gold
labeled chicken IgY antibodies bind to pre-coated sheep anti-chicken IgY antibodies, exhibiting a red band. In the
negative specimens, color is observable at the quality control line. The whole procedures are show in Figure 1.
After evaluation, when a red line appeared in the rst card, a positive result was considered if a red line was observed
at any one of the KPC, NDM and IMP test lines. For the second card, when the control line was a red line, a positive
result (“+”) was considered if one or more red lines were observed at the VIM and 0XA-48 test lines. If only the control
line was a red line and no test lines turned red, a negative result (“-”) was considered. If there was no line at the site of
C-line, the detection was invalid, and retesting was needed.
Table 1 Instruments and Reagents Used in This Study
Instrument and Reagents Company
Columbia blood agar plate Zhengzhou Antu Biotechnology Co., Ltd
MacConkey Agar Plate Zhengzhou Antu Biotechnology Co., Ltd
Cation Adjusted MH Broth (CAMHB) Wenzhou Kangtai Biotechnology Co., Ltd
VITEK2 Compact automatic microbial identication and drug
sensitivity analysis system
France Biomerieux Co., Ltd
DHP-9272 Digital display electric constant temperature incubator Shanghai Jinghong Experimental Equipment Co., Ltd
Electronic balance Sartorius BSA224S-CW
Carbapenemase detection kit (Immune Colloidal gold technique) Beijing Gold Mountainriver Science and Technology Development Co., Ltd
U-type 96-well plate Hangzhou Lanqian Biotechnology Co., Ltd
MS 5020 automatic biological mass spectrometric system Zhuhai DL Biotech. Co., Ltd
Figure 1 Detection of carbapenemase by gold standard immunochromatography.
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Detection of MIC of a Single Drug
According to the 2022 version of the American Society for Clinical and Laboratory Standards (CLSI) standards,
15
the
minimum inhibitory concentration (MIC) values of 50 experimental strains against Reduning and polymyxin B were
determined using the microdilution method. Firstly, select bacterial colonies cultured for 18 to 24 hours and adjust the
turbidity to 0.5 McPherson turbidity using physiological saline.Then dilute 100 times with cation adjusted MH broth
(CAMHB). Dilute the ratio of Reduning to (1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125μL/mL); Dilute the ratio of
polymyxin B to (32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125μg/mL). Take 100 μL of various concentrations of prepared Reduning
and polymyxin B in sequence and add them to the corresponding wells of a 96 well plate,Then add 100 μL of diluted
bacterial suspension to each well, simultaneously perform negative control and culture at 37°C for 16–18 hours. The
quality control strain is Escherichia coli ATCC25922. Observe the results and record the MIC values of various drugs.
Repeat the experiment three times.
Study of Combination Treatment
In this study, detection was performed strictly following the standard testing procedure provided by CLSI. After dilution
in a multiple ratio series, Reduning and polymyxin B were mixed in different concentration combinations. After
incubation, the MIC values of the single drug and the combination of the two drugs were measured. After the nal
inoculation using the microbroth dilution method, the nal volume of liquid in each well is 100mL, and the nal bacterial
concentration is 5x10
5
cfu/mL. Based on experimental data, the effective concentration range of polymyxin B for the
combined application of Reduning and polymyxin is determined to be 0.25–4μg/mL.
Detection of Fractional Inhibitory Concentration Index (FICI)
The fractional inhibition concentration index (FICI) was used to analyze the mutual inuence between two drugs used for
efcacy analysis.
The synergistic effect of two drugs is considered if the FICI is lower than 0.5. If the FICI ranges from 0.5 to 1.0,
addictive effect is considered. If the FICI ranges from 1.0 to 2.0, no signicant interaction between drugs is considered.
Once the FICI is higher than 2.0, an antagonistic effect can be considered between drugs.
Results
Distribution and Drug Resistance of 50 Strains of CRKP
Sources of 50 CRKP strains: The 50 strains of CRKP were mainly separated from sputum (64%; 32/50) and urine (20%;
10/50), and the other sources of CRKP included pharyngeal swabs and catheters (16%; 8/50) (Figure 2).
Distribution of Patients with CRKP Infection
Patients with CRKP were mainly from intensive care units, including intensive care unit in the Department of Emergency
and Department of Intensive Care Medicine (48%; 24/50). The 50 CRKP strains displayed distinct resistance for different
antibiotics. High resistance rate was noted in cefepime, ceftazidime, piperacillin tazobactam, and cefoperazone/sulbactam
(higher than 90%). The resistance to levooxacin was also evident. Of note, the rate of CRKP strains resistant to
carbapenem antibiotics (such as meropenem and imipenem) was as high as 100%. However, the rate of CRKP strains
resistant to tobramycin, tigecycline, and compound sulfamethoxazole was signicantly lower (Figure 3).
Distribution of Carbapenemase Types in 50 CRKP Strains
The colloidal gold immunochromatography was performed to detect the carbapenemase types. Our results showed that
the 50 CRKP strains in our hospital mainly produced KPC. About 96% (48/50) of CRKP strains produced KPC alone,
and 4% (2/50) CRKP strains could produce KPC and NDM (Figure 4).
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Figure 2 Clinical distribution of 50 CRKP strains.
Figure 3 Drug resistance of 50 CRKP strains in our hospital.
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MIC of Reduning, Polymyxin B and Reduning Plus Polymyxin B for CRKP Strains
In this study, broth microdilution method was used to determine the MIC of Reduning, polymyxin B, and Reduning plus
polymyxin B for 50 CRKP strains. Our results showed that the MIC of Reduning plus polymyxin B for CRKP was lower
than that of Reduning alone. The MIC of polymyxin B plus Reduning was reduced by 2–4 times as compared to the MIC
of polymyxin B alone. (Note: concentration of Reduning: μ l/mL; concentration of Polymyxin B: μ g/mL) (Table 2).
Synergistic Antibacterial Index of Two Drugs
Based on the MIC of Reduning, polymyxin B and Reduning plus polymyxin B for 50 CRKP strains, the FICI of
Reduning, polymyxin B and Reduning plus Polymyxin B were calculated using the algorithm of inhibitory concentration
index. Results reveal 7 strains had FICI lower than 0.5; 43 strains had FICI ranging from 0.5 to 1.0; no strains had FICI
higher than 1.0. Our results showed Reduning and polymyxin B exhibited an additive effect on CRKP, especially several
CRKP strains (Table 3 and Table 4).
Figure 4 Distribution of carbapenemase types of 50 CRKP strains.
Table 2 MIC Values for Drug Monotherapy and Combination Therapy
Drug Single Use MIC Range Combined Use MIC Range
Reduning(μL/mL) 250–500 62.5–250
Polymyxin B(μg / mL) 2–16 0.25–4
Table 3 FICI of Reduning
Plus polymyxinB in 50 CRKP
Strains
No FICI No FICI
1 0.625 26 0.625
2 0.625 27 0.75
3 0.5 28 0.625
4 0.5 29 0.625
5 0.625 30 0.625
6 0.625 31 0.625
7 0.625 32 0.625
(Continued)
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Discussion
Currently, the detection rate of carbapenem-resistant Klebsiella pneumoniae (CRKP) in clinical practice is continuously
increasing worldwide. The Chinese Bacterial Resistance Monitoring Network shows that, from January to
December 2023, the detection rate of KP resistant to meropenem and imipenem was 23.6% and 22.5%, respectively.
Thus, the commonly used carbapenem is currently ineffective for CRKP.
Polymyxin eliminates bacteria by affecting negatively charged phospholipids on cell membrane, thereby disrupting
the structure of cell membrane.
16
However, due to its severe nephrotoxicity, polymyxin has not been commonly used
since 1980s. In recent years, polymyxin has been reintroduced due to the presence of multidrug-resistant bacteria.
Polymyxin can be used for the treatment of ventilator-associated pneumonia,
17
bloodstream infections,
18
abdominal
infections,
19
and urinary tract infections,
20
caused by CRKP regardless of the mechanisms of drug resistance. However,
the heterogeneity of bacterial population usually affects the efcacy of polymyxin alone, and the increasing use of
polymyxin in clinical practice also promotes the presence of resistance to polymyxin.
Traditional Chinese Medicine therapy has a long history in our country, and Traditional Chinese Medicine is rich in
medical wealth and resources. In recent years, clinicians have paid increasing attention to the efcacy of traditional
Chinese medicine and their extracted components in preventing and treating infections. Traditional Chinese medicine can
selectively inhibit pathogenic microorganisms, intervene with their physiological metabolism, and disrupt the biological
structure and function of microorganisms (such as hindering the synthesis of bacterial cell wall, inhibiting protein
production in bacteria, suppressing normal nucleic acids metabolism, and interfering with other biochemical pathways in
microorganisms). In addition, traditional Chinese medicine has some advantages over other pharmacotherapeutics: it is
less likely to cause drug resistance and has few side effects. Few studies have reported the resistance of bacteria to
traditional Chinese medicine, indicating that the combined use of traditional Chinese medicine and antibiotics has broad
application prospects in the treatment of drug-resistant bacteria.
21
Reduning injection is a widely used traditional Chinese
Table 3 (Continued).
No FICI No FICI
8 0.5625 33 0.75
9 0.375 34 0.625
10 0.625 35 0.625
11 0.625 36 0.625
12 0.625 37 0.625
13 0.625 38 0.625
14 0.25 39 0.625
15 0.625 40 0.5625
16 0.5 41 0.5
17 0.625 42 0.625
18 0.625 43 0.625
19 0.625 44 0.625
20 0.625 45 0.625
21 0.625 46 0.625
22 0.625 47 0.375
23 0.75 48 0.625
24 0.75 49 0.625
25 0.625 50 0.625
Table 4 FICI of Reduning Plus polymyxinB in 50 CRKP Strains (%, n)
Combination of Antibacterial Drugs FICI≤0. 5 0. 5<FICI≤1 1<FICI≤2 FICI>2
Reduning plus polymyxin B 14 (7) 86 (43) 0 0
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patent medicine in clinical practice, it shows excellent anti-inammatory, heat clearing and detoxication effects and has
multiple effects such as antiviral, antibacterial, anti-allergic, anti-cancer, hepatoprotective and biliary protective, and
immunoregulative effects, and the main effective ingredients of Reduning include Artemisia annua, honeysuckle, and
Gardenia jasminoides.
22
Modern pharmaceutical study has revealed that Artemisia annua and Lonicera japonica have
signicant inhibitory effect on bacterial growth. Artemisia annua, as a major ingredient, can clear heat from the body,
cool the blood, dissipate surface heat toxin, and had both immunosuppressive and immunoenhancing effects.
23
Honeysuckle, as a ministerial ingredient, helps to clear heat, detoxify, and expel external pathogens.
24
It can disrupt
the peptidoglycan layer in the bacterial cell wall to change the permeability of cell membrane, thereby inhibiting bacterial
growth. Gardenia is an adjunctive ingredient, can clear internal heat in the heart, lung, and stomach, and exert the anti-
infective and temperature-lowering effects.
25
In clinical practice, Reduning is commonly used in antibacterial and
antiviral treatments and has signicant therapeutic efcacy in bacterial or viral infections of upper respiratory tract.
26
A study by Wang et al
27
showed that the combination of Reduning injection and Imipenem Sitastatin Sodium could
signicantly improve the clinical efcacy in severe pneumonia patients, alleviate clinical symptoms and disease severity,
improve lung ventilation, and markedly reduce inammatory reactions. A study by Li et al
28
showed that the combina-
tion of Reduning injection and piperacillin sodium/tazobactam sodium could improve the clinical efcacy in severe
pneumonia patients, improve clinical symptoms and lung ventilation and reduce inammatory reactions with favorable
safety. Xiao et al
29
found that, in the treatment with Reduning plus cefotaxime or cefoperazone/sulbactam, the required
antibacterial concentration for combination therapy was signicantly lower than that of monotherapy. Therefore, this
study aimed to explore the possibility of combined use of Reduning and Polymyxin in the treatment of CRKP infection,
and the therapeutic effect of Reduning plus Polymyxin was further investigated.
In the present study, among 50 CRKP strains, the majority were collected from sputum samples, particularly in
patients in intensive care unit (ICU). This is likely related to the long hospital stay, poor self-resistance capability,
relatively complex disease condition, and multiple invasive procedures (such as indwelling catheterization, tracheotomy,
mechanical ventilation, deep vein catheterization, drainage tube placement, etc.), which increase the risk of concurrent
infections.
30
Studies have reported that inammatory factors such as procalcitonin, interleukin-6, and high-sensitivity
CRP are independent risk factors for bacterial pneumonia.
31
In the present study, our results showed that the levels of
various inammatory indicators increased in 50 CRKP specimens, and thus timely judgments can be made on the
development of disease based on the changes in these indicators.
This experiment conducted in vitro antibacterial experiments on CRKP using both Reduning and polymyxin to determine
their MIC values. The combination of Reduning and polymyxin was then used to determine the MIC values of the two drugs
using the micro-chessboard dilution method, and the fractional inhibitory concentration index (FICI) was calculated. The
experimental results indicate that Reduning can inhibit the growth of CRKP, and when combined with polymyxin, the MIC
values of CRKP are generally lower than those when used separately. Among the 50 clinically isolated CRKP samples, 43
showed additive effects, 7 showed synergistic effects, and no antagonistic phenomena were found. Compared to the MIC
value when using a single drug, combination therapy can reduce the MIC value by 2 to 4 times. As a pure traditional Chinese
medicine preparation, Reduning has shown signicant antibacterial effects when used alone, and its combination therapy can
also produce effective antibacterial effects. The research results of Gao et al
32
show that the combination of Reduning
Injection with polymyxin B and meropenem can signicantly reduce the levels of procalcitonin (PCT), c-reactive protein
(CRP), and serum amyloid protein (SAA) in patients with multidrug-resistant Gram negative pulmonary infections. This
indicates that the combined use of Reduning Injection can indeed signicantly improve patient prognosis and enhance clinical
efcacy. The clinical use of high-dose and high concentration antibiotics may cause toxicity to the liver and kidneys of
patients, and long-term use may also promote bacterial resistance.
33
A study of Gao et al
32
found that the incidence of adverse
reactions in patients after combination therapy did not increase the risk of nephrotoxicity and central neurotoxicity caused by
polymyxin B injection, indicating good drug safety. This indicates that Reduning can enhance the efcacy of polymyxin in
treating CRKP without causing nephrotoxicity. Multiple studies
34–36
have shown that Reduning can effectively shorten
treatment time, reduce treatment costs, and has signicant economic advantages. Therefore, Reduning has practical value in
the treatment of refractory CRKP infections.
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In our study, some results also showed discrepancies with the overall results, which may be ascribed to the
physiological heterogeneity of bacterial colonies of tested strains, resulting in the dispersion of MIC to different extents.
If the test drug is set under multiple ratio conditions, the true MIC of a specic strain may be between two gradient ratios,
and the concomitant subjective observation of turbidity with naked eyes may bias the experimental results to a certain
extent. To standardize the results, a 12 × 8 matrix agar dilution was employed using the principle of agar dilution to
determine the results based on colony formation. At the same time, the manual operation also lacked training and
systematic quality control guarantees, and the aseptic operation was not fully implemented. The freshness of M-H broth
may also cause experimental errors. In our study, the MIC of polymyxin against CRKP obtained by the broth
microdilution method was higher than that detected with the instrument. However, Xie et al
37
found in their study that
the sensitivity of tigecycline to KP measured by the instrument was signicantly lower than that measured with the
manual method, and the evaluated indicators were out of the acceptable range. Their results showed consistency of
results between two methods was very poor, which was similar to the conclusions from other two studies.
38,39
These
ndings suggest that detection with instruments may bias the results to a certain extent. Thus, it is better to compare the
results determined with instrument with those with the manual method, and data detected with two methods were
subjected to further statistical analysis, which may provide better scientic support for the experimental results. The
strains used in this study were separated from the same hospital, and the sample size was relatively small. Therefore, the
accuracy of our results still needs further validation in more multicentered studies with large sample size.
This study preliminarily indicates the antibacterial activity of Reduning in vitro and highlights the necessity for
further investigation into the effects of individual components and combination therapy on their metabolism in vivo. This
study presents the potential of traditional Chinese medicine in preventing and treating infections and provides new
directions and methods for the investigation of treatment targeting drug-resistant bacteria in the future.
Conclusions
Reduning can inhibit the growth of CRKP, and when combined with polymyxin B, the MIC values of CRKP are
generally lower than those when used separately. So, Reduning has antibacterial effect on CRKP in vitro, and the
addition of Reduning can reduce the dose of polymyxin B in the treatment of CRKP.
Acknowledgments
The study was nancially supported by the Project of Zhejiang Medical and Health Science and Technology Plan from
the Health Commission of Zhejiang Province (No: 2020KY197), and the Chinese Medicine Research Program from the
Administration of Traditional Chinese Medicine of Zhejiang Province (No: 2020ZA041, 2023ZL332).
Disclosure
The authors report no conicts of interest in this work.
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