or in combination with other antibiotics against A. baumannii and E. coli

Journal Pre-proof In vitro and in vivo assessment of the antibacterial activity of colistin alone and/or in combination with other antibiotics against A. baumannii and E. coli Yale Wang, He Li, Xiaoqian Xie, XiaoHan Wu, Xinxin Li, Zeyue Zhao, Shasha Luo, Zhijie Wan, Jingjing Liu, Lei Fu, Xiaotian Li

PII:

S2213-7165(19)30246-2

DOI:

https://doi.org/10.1016/j.jgar.2019.09.013

Reference:

JGAR 1050

To appear in:

Journal of Global Antimicrobial Resistance

Received Date:

7 July 2019

Revised Date:

6 September 2019

Accepted Date:

16 September 2019

Please cite this article as: Wang Y, Li H, Xie X, Wu X, Li X, Zhao Z, Luo S, Wan Z, Liu J, Fu L, Li X, In vitro and in vivo assessment of the antibacterial activity of colistin alone and/or in combination with other antibiotics against A. baumannii and E. coli, Journal of Global Antimicrobial Resistance (2019), doi: https://doi.org/10.1016/j.jgar.2019.09.013

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In vitro and in vivo assessment of the antibacterial activity of colistin alone and/or in combination with other antibiotics against A. baumannii and E. coli

Yale Wanga,1, He Lia,b,1, Xiaoqian Xiea, XiaoHan Wua, Xinxin Lia, Zeyue Zhaoa,

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Shasha Luoa, Zhijie Wana, Jingjing Liua, Lei Fua,*, Xiaotian Lia,*

School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue,

Zhengzhou 450001, China

Department of Pharmacy, the First Affiliated Hospital of Zhengzhou University,

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b

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Zhengzhou 450001, China;

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Yale Wang and He Li contribute equally to the manuscript.

*Corresponding authors: Lei Fu; Xiaotian Li

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E-mail: [email protected]; [email protected] Address: School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue

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Avenue, Zhengzhou, Henan 450001, China. Telephone: +86-137836067716

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Highlights 

1 Colistin (COL) monotherapy showed rapid bacterial killing followed by regrowth.

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2 COL monotherapy is not an optimal option especially when MIC is ≥ 1 μg/ml.

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3 COL combined with doripenem showed the highest synergism by

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chequerboard method.

4 All COL-based combination therapies could inhibit or slow bacterial regrowth.

5 Intravenous colistimethate was significantly effective to urinary tract

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infection.

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Abstract

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Objectives

Limited therapeutic options are available for treating severe infections caused by

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multidrug-resistant (MDR) and extensively drug-resistant (XDR) gram-negative pathogens. We investigated the activities of colistin (COL) as a monotherapy and

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combined with antimicrobials against Acinetobacter baumannii in vitro and evaluated the efficacy of intravenous colistimethate sodium (CMS) in a murine model of urinary tract infection (UTI) induced by MDR Escherichia coli. Methods Minimum inhibitory concentration, Monte Carlo simulation, fractional inhibitory

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concentration index, time-kill study and erythrocyte lysis assay were applied to evaluate the effect and cytotoxicity of COL, meropenem (MER), imipenem (IMP), doripenem (DOR) and sulbactam (SUL) alone or in combination. For in vivo experiment, bacterial burden determination and histopathological examination were performed to evaluate the efficacy of CMS against UTI. Results

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98.12% of A. baumannii was susceptible to COL. In a checkboard assay, COL

combined with DOR showed the highest rate of synergism (60%). No antagonism or

cytotoxicity was observed. All COL-based combinations were able to inhibit or slow

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bacterial re-growth in a time-kill assay. In an in vivo activity study, intravenous CMS

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reduced not only the bacterial load but also inflammation and maintained structural integrity of the infected bladders and kidneys.

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Conclusions

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The effectiveness of COL alone in vitro and in vivo suggested that intravenous CMS will be an effective and available therapeutic strategy for UTI due to MDR

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gram-negative pathogens. In-depth in vitro tests demonstrated that COL combined with DOR could be an attractive option, especially when COL MIC of the pathogen is

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≥ 1 μg/ml.

Keywords: colistin; antibacterial activities; urinary tract infection; multidrug resistant; extensively drug-resistant; gram-negative pathogens

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1. Introduction Acinetobacter baumannii (A. baumannii), a gram-negative coccus, has emerged as a critically significant nosocomial pathogen in the 21st century [1]. Quickly acquired and increased resistance of A. baumannii is closely related to the nosocomial infection outbreaks [2]. Furthermore, the striking rate of morbidity and mortality caused by multidrug-resistant (MDR) and carbapenem-resistant A. baumannii

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(CRAB) in the intensive care unit (ICU) has produced an immense challenge to global public health [3]. The World Health Organization listed CRAB as the global “top priority” superbug in 2017 [4]. Furthermore, several studies have suggested that

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A.baumannii can survive on abiotic surfaces in severe conditions for months, and its

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resistance profile can affect its survival duration and biofilm formation [5, 6]. Although carbapenems were once one of the most effective and safe drugs against A.

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baumannii, with the rapid evolution of carbapenem resistance mechanisms, such as

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oxacillinases (OXAs), carbapenems are no longer an optimal treatment option [7]. Colistin (COL), a fairly old antibiotic, is being reappraised and reused alone or in

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combination with a few active antibiotics against CRAB [8, 9]. However, COL monotherapy readily induces the emergence of COL heteroresistance, low plasma

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concentrations, and COL resistance in A. baumannii, which are often associated with treatment failures and rapid growth of resistant strains [10, 11]. Patients who receive high doses of COL are more likely than patients who receive lower doses to develop nephrotoxicity [12]. To date, there is no defined optimal treatment against CRAB. Therefore, COL in

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combination with low-toxicity antibiotics seems to be a more promising option in clinical application. Carbapenems are commonly used as a combination drug for treating infections induced by CRAB [13]. Meanwhile, sulbactam (SUL) is an alternative agent for combination treatment against MDR and XDR A. baumannii infections due to its activity against Acinetobacter spp [14, 15]. Although there are a few studies on combinations including carbapenems, COL and/or SUL [16-18], scant

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data makes it arduous for clinicians to give the most effective combination. As a

consequence, the clinical benefit of systematically evaluating and optimizing the combinations based on these drugs would be considerable.

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There are two available COL forms in the clinic: colistimethate sodium (CMS)

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and COL sulfate [19]. CMS, an inactive prodrug, is hydrolysed to yield the active entity COL. A previous study has already demonstrated that COL sulfate, not CMS,

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should be used for susceptibility studies in vitro [20], while in clinical use, CMS is

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commonly available due to its low toxicity [21]. According to recent reports, the concentration of formed COL in urine and pharmacokinetic (PK) data supports that

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intravenous CMS is used for treating urinary tract infections (UTIs) due to MDR gram-negative pathogens [22, 23]. However, preclinical data on the efficacy of CMS

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are lacking for the treatment of UTI. Therefore, the purpose of this study was to evaluate the activity of carbapenems,

COL and SUL alone or in combination against A. baumannii isolates in vitro and confirm the efficacy of intravenous CMS in a murine model of UTI. 2. Materials and methods

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2.1. Isolates and selection of antibiotics A total of 106 non-repetitive isolates were obtained from patients infected with A. baumannii from May 2017 to November 2018, which were collected from various clinical specimens and identified by a BD Phoenix 100 Automated Microbiology System (Becton, Dickinson and Company, Franklin lakes, NJ, USA) in three tertiary care hospitals in Henan Province, China. Five antibiotics were used, namely, COL,

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SUL, doripenem (DOR), meropenem (MER) and imipenem (IMP), which were

obtained from Meilun Biotechnology Co., Ltd. (Dalian, China). All tests in vitro were performed in duplicate unless stated otherwise [24].

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2.2. Antimicrobial susceptibility testing and identification

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The MICs of COL, SUL, DOR, MER and IMP were determined using the broth microdilution method proposed by the Clinical and Laboratory Standards Institute

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(CLSI, 2018) [25]. Inoculums of 106 colony-forming units per millilitre (CFU/ml),

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freshly cation-adjusted Mueller-Hinton broth and two-fold serially diluted antimicrobial agents were utilized in the susceptibility testing. According to CLSI

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standards, MICs ≤ 2 μg/ml and ≥ 4 μg/ml for COL, MICs ≤ 4 μg/ml and ≥ 16 μg/ml for SUL and MICs ≤ 2 μg/ml and ≥ 8 μg/ml for carbapenems were considered to be

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susceptible and resistant, respectively. ATCC 25922, a quality control strain, was used as a reference for susceptibility testing. Pharmacodynamic (PD) analysis of intravenous CMS was then performed by using Monte Carlo simulation (MCS) based on the distribution of MICs for the A. baumannii isolates and a recent PK study in healthy Chinese subjects [23].

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2.3. Detection of blaOXA-23 genes by polymerase chain reaction (PCR) The presence of the carbapenemase gene blaOXA-23-like was tested by PCR with specific primers to further support resistance to carbapenems [26]. Amplified DNA products were separated by electrophoresis and the DNA bands were then visualized in a gel imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). 2.4. Checkboard assay

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Twenty-five CRAB isolates were randomly selected from 106 clinical strains for the checkboard synergy test. The synergistic effects of 7 combinations of five

antimicrobials were assessed: COL and DOR, COL and MER, COL and IMP, COL

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and SUL, SUL and DOR, SUL and MER, and SUL and IMP. The checkboard

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microdilution method was performed as in a previous study, with some modifications [27]. The concentration of COL ranged from 0.125 to 64 μg/ml while that of the

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carbapenem (MER, IMP and DOR) ranged from 8 to 512 μg/ml. The concentration of

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SUL ranged from 1 to 512 μg/ml (combination with carbapenems) or 8-512 μg/ml (combination with COL). For monotherapy, 100 μl of the serial dilutions of a single

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drug in a combination were added into the wells of row H or column 1 for the determination of the MIC of a single drug. For combined therapy, a total of 100 μl of

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two-fold serial dilutions of two drugs in a combination were added to the wells of columns 2 through row G for the determination of the MICs of drugs in combination. Next, inoculums of 106 CFU/ml were added to each well except column 12 and well H1. 100 μl of serial dilutions of single drug and 100 μl of ATCC25922 bacterial suspensions were added to column 12 except well H12 for the standard quality

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control. Finally, 200 μl of bacterial suspensions and 200 μl of MHB broth were added to wells H1 and H12, respectively. The 96-well plates were then placed in an incubator at 37°C for 18-22 h. The fractional inhibitory concentration index (FICI) for each combination was calculated based on the following formula: FICI = FICA + FICB, where FICA = the MIC of the drug A in combination/the MIC of the drug A alone, and FICB = the MIC

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of the drug B in combination/the MIC of the drug B alone. Interpretation of FICIs was defined as follows: FICI ≤ 0.5, synergy; FICI > 0.5-1.0, addition; FICI > 1.0-2.0, indifference; and > 2.0, antagonism [28].

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2.5. Biofilm formation and measurement

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Twenty-five A. baumannii isolates as well as the above-mentioned isolates were used to determine biofilm formation. The overnight cultures were diluted to a

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bacterial density of 106 CFU/ml in Luria-Bertani (LB) broth. Then, 200 μl of bacterial

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suspensions were added to 96-well flat-bottom polystyrene microtitre plates and incubated at 37℃ for 24 h. The wells were subsequently washed 3 times with 200 μl

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of sterile PBS to gently remove the planktonic cells. Then, 100 μl of 100% methanol was added to fix the bottom biofilm for 15 minutes and was then removed. The wells

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were then naturally dried and stained with 200 μl of 0.1% crystal violet for 30 min. Next, the crystal violet was washed with 200 μl of sterile PBS and the wells were naturally dried. Finally, the remaining crystal violet was solubilized with 200 μl of 95% ethanol for 10 min. The optical density (OD) at 595 nm was measured to quantify the amount of biofilm formed. The method of categorization of biofilm

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producers was performed based on a previous study, and all strains were divided into four classes [29]. 2.6. Time-kill studies Time-kill assays were performed for four representative isolates as in a previous study [30]. Considering synergism as well as achievable plasma concentration, the final drug concentrations used were as follows: COL, 0.5 × MIC for COL-susceptible

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isolates 25992 and 16NE89, 2 mg/l for COL-resistant isolates 198196 and 181006; SUL, 32 mg/l; DOR, 8 mg/l; MER, 8 mg/l; and IMP, 8 mg/l, which was based on

determined resistance of the strains. For monotherapy, 10 ml of bacterial suspensions

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and 10 ml of single antibiotic solution in MH broth were added to the tube, producing

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a bacterial density of 5 × 105 CFU/ml. For combined therapy, 10 ml of bacterial suspensions and a total of 10 ml of antibiotics solution of two drugs in a combination

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were added to the other tube. Simultaneously, the control groups were set, containing

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20 ml of MH broth and 20 ml of bacterial suspensions of 5 × 105 CFU/ml, respectively. These tubes were then incubated in an incubator at 37℃ for 24 h with

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constant shaking. Then, 100 μl of suspensions were acquired to determine the viable counts at the predetermined time points (0, 2, 4, 8, and 24 h), and serial 10-fold

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dilutions were spread onto MH agar plates to test the number of CFUs at 37℃ after 24 h. Subsequently, time-kill curves were plotted. According to a previous report [24], bactericidal activity was defined as a ≥ 3-log10 CFU/ml reduction compared with the concentration of the initial inoculums, and synergistic effects were defined as a decrease of ≥ 2-log10 CFU/ml between the combination and the most active drug

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alone at 24 h. 2.7. Erythrocyte lysis assay An erythrocyte lysis assay was carried out according to a previous study, with some modifications [31]. The most effective combination was used to test the cytotoxicity according to the results above. Five millilitres of fresh blood was drawn from a healthy donor and centrifuged for 5 min at 1000 × g. Red blood cells (RBCs)

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were washed three times with 0.9% NaCl and resuspended to 10% in 0.9% NaCl. For

monotherapy, the final concentration of a single drug ranged from 0.125 to 256 μg/ml. For combined therapy, the final concentration of two drugs in a combination ranged

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from 0.125 ×0.125 to 256 × 256 μg/ml. Then, 100 μl of RBC suspensions were

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cultured with 100 μl of two-fold serial dilutions at 37°C for 1 h. Afterwards, the test samples were centrifuged for 5 min at 1000 × g. The absorbance of the supernatant

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was then measured at 540 nm to determine the amount of hemoglobin released from

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the RBCs. 0.1% Triton X-100 and 0.9% NaCl were used as a positive control and a negative control, respectively. The percentage of hemolysis was determined by

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comparing the absorbance of the sample and that of the positive [32]. The experiment was performed in triplicate.

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2.8. In vivo experiments 2.8.1. Bacterial isolates

Twenty-two clinical E. coli isolates from patients with UTIs were collected to determine the virulence genes and resistant genes by PCR. Subsequently, a strain with a high degree of urinary tract pathogenicity and resistance was used for subsequent

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animal experiments. 2.8.2. Antibiotics, dosage, and sampling Animal studies were approved by the Experimental Animal Administration and Ethics Committee in Experimental Animal Center of Zhengzhou University. The ascending UTI model was established as previously reported [33]. Immunocompetent KM female mice (weight, 20 ± 2 g) were used, which were obtained from Henan

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Province Laboratory Animal Center, China. After anesthetizing animals with 10%

chloral hydrate and gently emptying the bladder [34], the bladders were injected with

50 μl of E. coli suspensions in PBS containing approximately 109 CFU/ml by a plastic

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catheter. The next day after infection, mice were treated intravenously with CMS

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(Forest Laboratories, Kent, UK). The dosage regimens (total daily-doses of 23.4 to 93.6 mg/kg CMS) were as follows: once-daily (q 24 h) 93.6, 46.8 and 23.4 mg/kg

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CMS and twice-daily (q 12 h) 46.8, 23.4 and 11.7 mg/kg CMS. Six mice were treated

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for each therapeutic regimen. At 24 h after treatment, urine from the mice was collected by gentle compression on the abdomen. The bladders and kidneys were then

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removed aseptically. One half of the organ was placed in a 10% formaldehyde solution and histological examination was performed. The other half was

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homogenized with 1 ml of 0.9% saline to determine the viable counts. 2.8.3 Histopathological examination The bladder and kidney tissues were fixed in 10% formaldehyde solution overnight, dehydrated in ethanol and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (HE) and images were observed using K-Viewer

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software (KFBIO Technology for Health Co., Ltd., China). Animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.9. Statistical analysis In the in vivo activity study, the results are presented as median counts. The

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Mann-Whitney U test was performed to compare the number of CFU in the urine,

bladders and kidneys of different groups (SPSS Software, SPSS Inc., Chicago, USA) [version 22]. The significance level was defined as a P value less than 0.05.

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3. Results

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3.1. Determination of minimum inhibitory concentrations (MICs)

The MIC50 values, MIC90 values, MIC range and resistance ratios for five

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antimicrobial agents against 106 clinical A. baumannii isolates are presented in Table

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1. The susceptibility testing showed that the majority of strains were resistant to SUL, DOR, MER and IMP. Only 1.88% (2/106) of the isolates were resistant to COL, and

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their MICs were 8 and 32 μg/ml. The MCS results showed that the probability of target attainment (PTA) of 300 mg of COL base activity (CBA) per day was less than

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90% when the MIC was ≥ 1 μg/ml. 3.2. The fractional inhibitory concentration index and resistance gene (blaOXA-23) detection Table 2 shows the distribution of blaOXA-23 genes and synergy testing results of 7 antibiotic combinations against 25 CRAB strains. The COL/DOR combination was

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the most effective combination, exhibiting a synergistic effect for 60% (15/25) of the isolates and exhibiting an additive effect for the remaining 10 strains (40%). Next, the IMP/SUL combination showed synergy against 56% (14/25) of the strains, followed by both the COL/IMP and COL/MER combinations [synergy against 13/25 (52%) strains], and the SUL/MER combination [synergy against 12/25 (48%) strains]. The COL/SUL and SUL/DOR combinations showed similar additive effects but synergism

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against 32% and 28% of isolates, respectively.

The MICs of four antibiotics combined with COL decreased 2-128-fold in the

presence of sub-inhibitory concentrations of COL (0.5 × and 0.25 × MIC). The CIR

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curves for COL alone and/or in combinations are shown in Fig. 1. The CIR curves for

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COL in combination with different antimicrobials moved to the left by different levels, indicating that COL in combination therapy is beneficial.

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3.3. Biofilm formation and measurement

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The biofilm production capability of 25 isolates is shown in Table 3. Most of the isolates (80%) were able to produce biofilm and more than 50% of the isolates were

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the medium biofilm producers. 3.4. Time-kill assay

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Time-kill curves of seven combinations of five antibiotics are displayed in Fig. 2.

Regarding COL-based combinations, COL/IMP, COL/MER, COL/DOR and COL/SUL exhibited bactericidal and synergistic activity against the COL-sensitive isolate 255005 at 24 h, and all four combinations were able to achieve a ≥ 3-log10 CFU/ml reduction compared to that of the most active single antibiotic. There was no

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significant difference among the synergisms of the four combinations. For the strain 16NE89, both COL as a monotherapy and in combinations were able to achieve durable killing. For the isolates 198196 and 181006, COL alone and combined with antimicrobials were able to inhibit growth at only the initial 2 h, followed by regrowth. SUL–based combinations showed the maximum reductions in bacterial counts against the isolate 255005 at 8 h, with subsequent re-growth and no

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bactericidal and synergistic effect was observed. 3.5. Erythrocyte toxicity

The effects of COL alone, DOR alone and COL in combination with DOR on

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RBC hemolysis are shown in Fig. 3. The cell lysis of 0.1% Triton X-100 was 100%.

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COL alone, DOR alone and COL in combination with DOR did not present increased

concentration range tested.

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3.6. In vivo studies

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hemolytic activity compared with 1.41% of cell lysis for the 0.9% NaCl control at the

3.6.1. In vivo activity studies

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The uropathogenic Escherichia coli (E. coli) (UPEC) isolate 198776 isolated from UTI patients was able to successfully induce the ascending urinary tract

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infection model. The MIC of COL for the isolate 198776 was 0.25 μg/ml. The bacterial counts in the urine and bladder are shown in Fig. 4. At 24 h of treatment with CMS, there was a significant decrease in bacterial counts for the 93.6 and 46.8 mg/kg daily-doses (P < 0.05), and the medians were below the detection line in urine samples. No significant reduction was observed for the daily-dose of 23.4 mg/kg. For

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the bacterial counts from bladder samples, there was a significant reduction for the 93.6 and 46.8 mg/kg daily-doses (P < 0.05), while the reduction was not significant for the 23.4 mg/kg dose. For the kidney samples, the bacterial counts in all six CMStreated groups showed a significant reduction compared with those of the control group (P < 0.05). 3.6.2. Histopathological examination

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Histopathological changes in bladders and kidneys are shown in Fig. 5.

Compared with that of the normal group (Fig. 5A), the bladder section of the model

group (Fig. 5B) showed severe neutrophil infiltration and desquamation of epithelial

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cells of the bladder mucosa. The treatment group showed decreased the inflammatory

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cell infiltration and exfoliation of epithelial cells (Fig. 5C). Compared with that of the normal group, the kidney section of the model group (Fig. 5E) showed severe

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neutrophil infiltration, necrosis and a reduction in the glomerular endothelium cells

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and loss of the brush border and vacuolization in the renal tubule (Fig. 5D). No obvious histopathological changes were observed in the treatment group (Fig. 5F).

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4. Discussion

The increasing resistance of gram-negative bacteria (GNB) has been a hot topic

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in the healthcare setting. In this study, data showed that more than 75% of the A. baumannii isolates to three common carbapenems were resistant, and the MIC50 and MIC90 values were 64 μg/mL and 128 μg/ml for SUL, which were inconsistent with previous reports [16, 35]. The high rate of susceptibility (98.12%) of A. baumannii to COL and effectiveness of intravenous CMS against UTI suggested that COL remains

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an available treatment option for MDR GNB. However, the emergence of heteroresistance with COL in monotherapy was found in our study [24], and PD analysis by MCS suggested that intravenous CMS as a monotherapy may not be an optimal option when the MIC of the pathogen is  ≥ 1  μg/ml [23, 36]. Therefore, we systematically investigated the antibacterial activity of COL in combination with other antibiotics against A. baumannii.

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Biofilm production, a virulence trait, makes it difficult to eliminate A.baumannii with multiple resistance mechanisms, which often induce repeated infections. This research, in accordance with the recent study [37], showed that 80% of clinical

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A.baumannii isolates could produce the biofilm. However, the association of the

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resistance spectrum and biofilm production is still doubtful and disputed. According to previous observations, MDR A.baumannii strains are more likely than susceptible

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strains to produce biofilms [38, 39]. Future studies should be focused on exploring the

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mechanism of development and eradication of biofilms in more clinical samples. Because antimicrobials in combination therapy may produce better

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pharmacokinetic effects and synergism than monotherapies, combination therapy has been recommended as the standard for infections by CRAB [20]. Similar to an earlier

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study, COL combined with DOR showed the highest synergism rate by the checkboard method, although different antibiotic combinations were previously evaluated [40]. In addition, six other combinations showed different synergy and no antagonism was observed. Encouragingly, the MIC values for COL in combination exhibited a 2- to 16-fold reduction, suggesting that combination therapies are

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necessary and beneficial for the treatment of CRAB and reduction in COL toxicity. The time-kill assay can not only evaluate the synergistic effect but also determine the bactericidal activity and bacterial re-growth associated with an antibiotic [41], it was thus employed to test the synergy of seven combinations against four A. baumannii isolates. In this research, similar antibacterial activities of MER, IMP and DOR were observed, and COL-based combinations consistently showed the

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maximum antibacterial effect, including inhibiting or slowing bacterial re-growth. This effect was observed in the first few hours, suggesting that it is important to

increase antibiotic exposure in the early stages of treatment to eliminate resistant

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subpopulations. Surprisingly, no synergism was found for SUL-based combinations in

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the time-kill assay. We hypothesize that this result may be related to the severe resistance of local bacteria to SUL, for which MICs were ≥ 64 μg/ml. SUL-based

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combinations are suggested for use as the treatment for infections induced by XDR A.

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baumannii in a Chinese consensus statement [15], which should therefore be carefully considered. However, the addition of SUL restored the bactericidal activity of COL

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and DOR combination therapy against recurrent and COL-resistant isolates, which seems to be a promising treatment option for SUL against A. baumannii [42].

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Interestingly, even 0.5 × MIC of COL in monotherapy was bactericidal against

the isolate 16NE89, but a similar effect was not observed for SUL alone. Furthermore, we noted that the therapeutic effect varied with the MIC of the isolate. Thus, the individualized treatment should to be given when choosing antimicrobials for treating A. baumannii infections.

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Hemoglobin release from damaged RBCs was used to evaluate drug cytotoxicity, which is a widely accepted model [32]. As far as we know, once the RBC membrane integrity is destroyed, RBCs will die rapidly. Therefore, it is necessary to determine drug cytotoxicity. In the erythrocyte lysis assay, we found that both single drug and combination therapy (COL with DOR) had no effect on RBC membranes, suggesting that this combination is safe and noncytotoxic for human RBCs.

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To date, a few studies have investigated the effects of the combination of COL and DOR on gram-negative pathogens [40, 43-46]. These studies support the

combination of COL and DOR against MDR and XDR gram-negative pathogens in

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vitro due to its high proportion of synergism, bactericidal activity and lack of

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antagonism. A recent clinical study reported that combination therapy with active COL and prolonged infusion high-dose DOR was related to longer survival time,

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lower mortality and better microbiological eradication than active single drug therapy

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[45]. In addition, subpopulation synergy and mechanistic synergy were considered to be the mechanism of synergy of COL combined with DOR [46]. For subpopulation

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synergy, COL and DOR show the complementary effects by acting on different bacterial subpopulations with different susceptibilities to antibiotics [46]. For

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mechanistic synergy, it has been suggested that COL can damage the bacterial outer membrane, increasing its permeability, which in turn allows elevated amounts of DOR to enter the bacteria, increasing access to the acetylate penicillin-binding proteins and interferes with the production of peptidoglycan [46, 47]. Recent studies have supplemented the mechanism of synergy of COL and DOR combination therapy via

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metabolomics and genomics on the basis of the damage of COL to the outer membrane [48, 49]. It was reported that 75 to 90% of community-acquired UTIs are caused by Escherichia coli [50], so a clinical MDR E. coli isolate obtained from a UTI patient was used in vivo. To our knowledge, this report is the first study demonstrating the efficacy of intravenous CMS in a murine model of UTI model due to MDR E. coli.

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According to previous studies on CMS PK, high concentrations of formed COL in urine by intravenous CMS have been proven [23, 51-53]. Therefore, we used

humanized CMS dosing regimens in this study. After 24 h of treatment, bacterial

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counts in the urine and bladders showed a significant reduction for the total daily-

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doses of 93.6 and 46.8 mg/kg of intravenous CMS, and there was no difference in efficacy between the once and twice a day dosing regimens. Furthermore, we found

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that the number of CFU in kidney homogenates in the treatment group was slightly

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affected by the dosage of CMS, which remains to be further researched. Histopathology examination also identified the efficacy of CMS for UTI. Therefore,

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this study confirmed that intravenous CMS decreased not only bacterial counts but also the severity of inflammation of bladders and kidneys. At last, we note that the

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efficacy against only one MDR E. coli was evaluated. However, three E. coli isolates with different resistance profiles successfully induced the ascending murine UTI model in our pre-experiment. In addition, a total daily dose of 46.8 mg/kg CMS (equivalent to 4.5 million IU in human) showed good efficacy, and the MDR E. coli we used was obtained from a patient suffering from UTI, so other E. coli strains may

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exhibit similar effects. 5. Conclusions In conclusion, COL remains a last-line treatment for MDR GNB. The high rate of susceptibility of A. baumannii to COL alone in vitro and in-depth study in vivo study demonstrate that intravenous CMS will be an effective and available therapeutic option for UTI induced by MDR GNB. In addition, our study shows the highest rate

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of synergism for COL and DOR in combination against A. baumannii and no

antagonism or cytotoxicity was found. In vitro data support the combination of COL and DOR, and further study is needed to explore the efficacy of COL combination

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therapy in vivo, especially when the COL MIC of the strain is ≥ 1 μg/ml.

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Declarations

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Funding: This work was supported by Science and Technology Research Project of

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Henan Province, China (No. 162102310540).

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Competing Interests: None declared.

Ethical Approval: Not required.

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49. Henry R, Crane B, Powell D, Deveson Lucas D, Li Z, Aranda J et al. Aranda J. The transcriptomic response of Acinetobacter baumannii to colistin and doripenem

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50. Zykov IN, Samuelsen O, Jakobsen L, Smabrekke L, Andersson DI, Sundsfjord A

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et al. Pharmacokinetics and Pharmacodynamics of Fosfomycin and Its Activity against Extended-Spectrum-beta-Lactamase-, Plasmid-Mediated AmpC-, and

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Carbapenemase-Producing Escherichia coli in a Murine Urinary Tract Infection Model. Antimicrob Agents Chemother 2018; 62: e02560-17. 51. Couet W, Gregoire N, Gobin P, Saulnier PJ, Frasca D, Marchand S et al. Pharmacokinetics of colistin and colistimethate sodium after a single 80-mg intravenous dose of CMS in young healthy volunteers. Clin Pharmacol Ther 2011; 89:

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875-879. 52. Yapa SWS, Li J, Patel K, Wilson JW, Dooley MJ, George J et al. Pulmonary and systemic pharmacokinetics of inhaled and intravenous colistin methanesulfonate in cystic fibrosis patients: targeting advantage of inhalational administration. Antimicrob Agents Chemother 2014; 58: 2570-2579. 53. Li J, Milne RW, Nation RL, Turnidge JD, Smeaton TC, Coulthard K.

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Pharmacokinetics of colistin methanesulphonate and colistin in rats following an

intravenous dose of colistin methanesulphonate. J Antimicrob Chemoth 2004; 53:

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837-840.

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Figure:

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Fig. 1. Cumulative inhibition ratio (CIR) of COL alone or in combination against CR A. baumannii isolates (n = 25)

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Fig. 2. Time-kill curve of seven combinations of five antibiotics for four A. baumannii isolates

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Fig. 3. Cytotoxicity of COL alone, DOR alone and COL in combination with DOR against Red blood cells (RBCs)

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Fig. 4. Effect of CMS with six dosing regimens on UTI caused by Uropathogenic Escherichia coli (UPEC)

33

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Fig. 5. Histopathological examination of bladder and kidney sections

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Legends to figures:

Fig. 1. Cumulative inhibition ratio (CIR) of colistin alone or in combination against CR A. baumannii isolates (n = 25) CIR is defined as the percentage of strains that were inhibited at a certain concentration of colistin. COL+SUL, colistin combination with sulbatam; COL+DOR, colistin combination with doripenem; COL+IMP, colistin combination

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with imipenem; COL+MER, colistin combination with meropenem. The CIR curves of COL+DOR and COL+IMP are coincident.

Fig. 2. Time-kill curve of seven combinations of five antibiotics for four A. baumannii

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isolates

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Two COL-susceptible strains: 255005 and 16NE89. Two COL-resistant strains:

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198196 and 181006. Antimicrobial concentrations: COL, 0.5 MIC for 255005 and 16NE89; 2 µg/ml for 198196 and 181006; SUL, 32 µg/ml; IMP, MER and DOR, 8

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µg/ml for four isolates.

Fig. 3. Cytotoxicity of COL alone, DOR alone and COL in combination with DOR

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against Red blood cells (RBCs)

Fig. 4. Effect of CMS with six dosing regimens on UTI caused by Uropathogenic

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Escherichia coli (UPEC)

Each point represents the bacterial counts per milliliter of urine, each bladder or kidney. Solid horizontal line represents the median bacterial count. Dashed line represents the limit of detection (LOD). The number of urine samples in 11.7mg/kg q12h dose group was 5 due to failure urine sample collection. 35

Fig. 5. Histopathological examination of bladder and kidney sections A-C represented the bladder section of normal group, model group and treatment group, respectively; D-F represented the kidney section of normal group, model group

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and treatment group, respectively. a: bladder mucosal; b: glomerular endothelial cells.

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Table: Table 1 MIC50, MIC90, MIC range and percentage of resistance of five antibiotics against 106 clinical A. baumannii isolates MIC（µg/ml） MIC50

MIC90

MIC range

Resistance (%)

COL

0.5

1

0.125-32

1.88

SUL

64

128

2-1024

81.2

MER

64

128

0.5-512

79.3

IMP

32

64

0.25-256

77.4

DOR

64

128

0.25-256

79.3

Antibiotics

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COL, colistin; SUL, sulbactam; MER, meropenem; IMP, imipenem; DOR, doripenem.

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Table 2 Carbapenemase gene distribution and the effect of antimicrobial combination for 25 CR A. baumannii isolates

Isolate

bla

FICI COL/ MER

COL/ IMP

COL/ DOR

COL/ SUL

SUL/ MER

SUL/ IMP

SUL/ DOR

1

√

0.375

0.75

0.5

0.53

1

0.5

0.625

2

√

0.506

0.75

0.75

0.5

0.5

1.03

1

3

√

0.625

0.281

0.5

0.503

0.625

0.51

0.75

4

√

0.5

0.375

0.5

1

0.375

0.18

0.25

5

√

1

0.5

0.625

0.625

0.375

0.375

0.625

6

√

0.75

1

0.375

0.75

0.513

0.5

0.75

7

√

0.52

0.31

0.56

0.5

0.52

0.375

0.75

8

√

0.5

0.75

0.53

0.51

0.375

0.188

0.53

9

√

0.5

0.75

0.375

0.375

0.75

0.28

0.75

10

√

0.5

0.5

0.51

0.375

0.5

0.375

0.31

11

√

0.5

0.5

0.5

0.75

0.375

0.375

0.375

12

√

0.375

0.5

0.75

0.56

0.28

0.31

1

13

√

0.5

0.75

0.5

1.004

1

0.75

0.75

14

√

0.75

0.375

0.5

0.31

0.75

0.625

0.375

15

√

0.75

0.56

0.625

1.02

0.506

0.25

0.75

16

√

0.56

0.5

0.56

0.53

0.5

0.75

0.75

17

√

0.5

0.5

0.375

0.75

0.5

0.75

0.53

18

√

0.31

0.375

0.5

0.375

0.5

0.51

0.375

19

√

0.5

0.75

0.75

1

0.75

0.75

0.75

-p

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lP

na

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OXA-23

20

√

0.5

0.75

0.52

0.75

0.506

0.5

0.625

21

√

0.75

1

0.5

0.5

0.56

0.75

0.52

22

√

0.31

0.625

0.375

1

0.75

0.75

0.14

23

√

0.75

0.5

0.375

0.75

0.5

0.51

0.51

24

√

0.515

0.75

0.5

0.5

0.5

0.25

0.75

38

25

√

0.75

0.5

0.5

0.53

0.53

0.5

0.5

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FICI, fractional inhibitory concentration index. √, blaOXA-23 gene present.

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Table 3 Categorization of capability of biofilm formation for 25 isolates Capability of biofilm production No. of strains

No. of strains (%)

Median

OD range

Non-biofilm producers

4

16

0.066

0.065-0.072

Low biofilm producers

7

28

0.096

0.086-0.12

Moderate biofilm producers

12

48

0.15

0.12-0.37

High biofilm producers

2

8

0.61

0.48-0.75

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OD, optical density.

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