SUPPLEMENTARY APPENDIX Efficacy and Safety of PL-5 (Peceleganan) Spray for Wound Infections: A Phase IIb Randomized Clinical Trial

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SUPPLEMENTARY APPENDIX

Efficacy and Safety of PL-5 (Peceleganan) Spray for Wound Infections:

A Phase IIb Randomized Clinical Trial

Yating Wei, PhD¹, Jun Wu, MD^{1, 2}*, Yuxin Chen, PhD³*, Kunwu Fan, MD¹, Xuming Yu, MD¹, Xiaojian Li, MD⁴, Yaohua Zhao, MD⁵, Yi Li, MD⁶, Guozhong Lv, MD⁷, Guodong Song, MD⁸, Xinzhou Rong, MD⁹, Cai Lin, MD¹⁰, Haitao Wang, MD¹¹, Xiaodong Chen, MD¹², Pihong Zhang, MD¹³, Chunmao Han, MD¹⁴, Hongxu Zu, MD¹⁵, Wenjun Liu, MD¹⁶, Yi Zhang, MD¹⁷, Chang Liu, MD¹⁸, Yongtao Su, MD¹⁹, Baolin Zhang, MD²⁰, Bingwei Sun, MD²¹, Lei Wang, MD²², Wen Lai, MD²³, Jinhui Liu, MD²⁴, Chengde Xia, MD²⁵, Geng Ji, MD²⁶, Feng Zhu, MD²⁷, Jia’ao Yu, MD²⁸, Akebaer.Ahemaiti MD²⁹, Hu Dong, Msc³, Mingxia Chen, Msc³, on behalf of the PL-5 Investigators

TABLE OF CONTENTS

List of Investigators and Study Sites……….1

In vitro tests of antibacterial activity……….3

Overview of Preclinical Pharmacokinetic Study……….15

Toxicity tests………...16

Allergy test……….19

Irritation test………...20

Phase I clinical trial……….21

Phase IIa clinical trial………..23

Table S1. The Skin Infection Rating Scales (SIRS)……… ………..25

Table S2. Inclusion and exclusion criteria of the phase IIb clinical trial………26

Table S3. Demographic and clinical characteristics of the patients at baseline ………...……….27

Table S4. The SIRS evaluations before and after interventions……….28

Table S5. The results of bacteria cultures at baseline ………..31

Table S6. The results of adverse event ………32

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List of Investigators and Study Sites

Dr. Yating Wei, Shenzhen Second People’s Hospital, No. 3002, Sungang West Road, Futian District, Shenzhen, Guangdong Province, China 518035

Dr. Dr. Jun Wu, Shenzhen Second People’s Hospital, No. 3002, Sungang West Road, Futian District, Shenzhen, Guangdong Province, China 518035

Dr. Kunwu Fan, Shenzhen Second People’s Hospital, No. 3002, Sungang West Road, Futian District, Shenzhen, Guangdong Province, China 518035

Dr. Xuming Yu, Shenzhen Second People’s Hospital, No. 3002, Sungang West Road, Futian District, Shenzhen, Guangdong Province, China 518035

Dr. Xiaojian Li, Guangzhou Red Cross Hospital, No.396 ,Tongfu Middle Road, Guangzhou, Guangdong Province, China 510240

Dr. Yaohua Zhao, Jiangyin People’s Hospital, No.163 Shoushan Road, Jiangyin, Wuxi, Jiangsu Province, China 214499

Dr. Yi Li, Qinghai University Affiliated Hospital, No. 29 Tongren Road, Xining, Qinghai Province, China 810012 Dr. Guozhong Lv, Affiliated Hospital of Jiangnan University, No. 1000 He Feng Road, Wuxi, Jiangsu Province, China 214043

Dr. Guodong Song, Jinan Central Hospital, NO.105, Jiefang Road, Lixia District, Jinan, Shandong Province, China 250013

Dr. Xinzhou Rong, Guangzhou First people's Hospital, No.1, Panfu Road, Yuexiu District, Guangzhou, Guangdong Province, China 510180

Dr. Cai Lin, The First Affiliated Hospital of WMU, New District of the First Hospital of Wenyi Hospital, Nanbaixiang, Ouhai District, Wenzhou, Zhejiang Province, China 325015

Dr. Haitao Wang, Weihai Municipal Hospital, NO.70, Heping Road, Huancui District, Weihai, Shandong Province, China 264299

Dr. Xiaodong Chen, The First People's Hospital of Foshan, No. 81, North Lingnan Avenue, Chancheng District, Foshan, Guangdong Province, China 528010

Dr. Pihong Zhang, Xiangya Hospital of Central South University, No.87, Xiangya Road, Changsha, Hunan Province, China 410008

Dr. Chunmao Han, The Second Affiliated Hospital Zhejiang University School of Medicine, NO.88, Jiefang Road, Shangcheng District, Hangzhou, China 310003

Dr. Hongxu Zu, The First Affiliated Hospital of Henan University of Science and Technology, NO.24,Jinghua Road, Jianxi District, Luoyang, Henan Province, China 450052

Dr. Wenjun Liu, The Second Affiliated Hospital of Kunming Medical University, No. 374, Dian Mian Road, Wuhua District, Kunming, Yunnan Province, China 650106

Dr. Yi Zhang, Affiliated Hospital of Nantong University, NO.20, Xisi Road, Nantong, Jiangsu Province, China 226006

Dr. Chang Liu, Affiliated Hospital of Jiangsu University, No.438 Jiefang Road, Zhenjiang, Jiangsu Province, China 212001

Dr. Yongtao Su, PKU care Luzhong Hospital, NO.65,Taigong Road, Linzi District, Zibo, Shandong Province, China 045099

Dr. Baolin Zhang, First Hospital of Shanxi Medical University, NO.85, Jiefang South Road, Yingze District, Taiyuan, Shanxi Province, China 030001

Dr. Bingwei Sun, Suzhou Municipal Hospital, NO.242, Guangji Road, Gusu District, Suzhou, Jiangsu Province, China 215008

Dr. Lei Wang, Zhongda Hospital Southeast University, No.87 Dingjiaqiao, Gulou District, Nanjing, Jiangsu Province, China 210009

Dr. Wen Lai, Guangdong Provincial People's Hospital, No. 106, Zhongshan 2nd Road, Yuexiu District, Guangzhou, Guangdong Province, China 510080

Dr. Jinhui Liu, Jilin Province People's Hospital, No. 1183 Gongnong Road, Chaoyang District, Changchun, Jilin Province, China 130021

Dr. Chengde Xia, Zhengzhou First People’s Hospital, NO.56, East Street, Huizu District, Guancheng, Zhengzhou, Henan Province, China 451475

Dr. Geng Ji, Taizhou People's Hospital, No.366 Taihu Road, Taizhou Medical High-tech Zone, Taizhou, Jiangsu Province, China 225399

Dr. Feng Zhu, Sir Run Run Hospital Nanjing Medical University, No. 109 Longmian Dadao, Jiangning District, Nanjing, Jiangsu Province, China 211112

Dr. Jia’ao Yu, The First Hospital of Jilin University, No.1 Xinmin Dajie, Chaoyang District, Changchun City, Jilin Province, China 130031

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Dr. Akebaer.Ahemaiti, The First Affiliated Hospital of Xinjiang Medical University, No. 137 Li Yu Shan Nan Lu, Urumqi, Xinjiang Uygur Autonomous Region, China 830011

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In vitro tests of antibacterial activity

Antibacterial and bactericidal effects of PL-5 against 13 strains in vitro (MIC and MBC determination) 1. Antibacterial and bactericidal effects of antimicrobial peptide PL-5 on Staphylococcus in vitro

The antibacterial and bactericidal effects of antimicrobial peptide PL-5 on 130 strains of methicillin resistant Staphylococcus aureus (MRSA), 67 strains of methicillin sensitive Staphylococcus aureus (MSSA), 121 strains of methicillin resistant coagulase negative Staphylococcus (MRSCNS) and 61 strains of methicillin sensitive coagulase negative Staphylococcus (MSSCNS) were determined using microdilution method and nutrient agar plate culture method, and vancomycin was used as the control drug.

The results showed that the MIC range of PL-5 to four kinds of Staphylococcus were 2~16 μg/ml, 2~16 μg/ml, 2~16 μg/ml and 2~16μg/ml; the geometric mean of MIC was 5.54μg/ml, 2.43μg/ml, 4.49 μg/ml and 4.48 μg/ml respectively; MIC50 were 4 μg/ml, 4 μg/ml, 4 μg/ml and 4 μg/ml, respectively; MIC90

were 8 μg/ml, 8 μg/ml, 8 μg/ml and 8 μg/ml, respectively. The cumulative antibacterial percentage showed that at the concentration of 16 μg/ml, PL-5 could 100% inhibit the growth of four kinds of Staphylococcus. The MBC ranges of four staphylococci were 4 ~ > 64 μg/ml, 2 ~ > 64 μg/ml, 2 ~ > 64 μg/ml and 2 ~ > 64 μg/ml respectively; MBC50 were 8 μg/ml, 4 μg/ml, 8 μg/ml, and 8 μg/ml respectively;

MBC90 is 32 μg/ml, 16 μg/ml, 64 μg/ml and 64 μg/ml. The cumulative bactericidal percentage of MRSA and MSSA at the concentration of 32 μg/ml exceeded 90%; while at the concentration of 64g/ml, the cumulative bactericidal percentage was more than 90%.

The above results showed that antimicrobial peptide PL-5 had strong antibacterial and bactericidal effects on the growth of four kinds of Staphylococcus. Considering the content of pure peptide to be 84.34%, the MIC range and MBC range of MRSA were 1.6868 ~ 13.4944 μg/ml and 3.3736 ~ > 53.9776 μg/ml respectively; MIC90 and MBC90 were 6.7472 μg/ml and 26.9888 μ g/ml respectively; The MIC range and MBC range of MSSA were 1.6868 ~ 13.4944 μg/ml and 1.6868 ~ > 53.9776 μg/ml respectively, and MIC90 and MBC90 were 6.7472 μg/ml and 13.4944 μ g/ml respectively; The MIC range and MBC range of MRSCNS were 1.6868 ~ 13.4944 μg/ml and 1.6868 ~ > 53.9776 μg/ml respectively; and MIC90 and MBC90 were 6.7472, respectively μ G / ml and 53.9776 μ g/ml； The MIC range and MBC range of MSSCNS were 1.6868 ~ 13.4944 μg/ml and 1.6868 ~ > 53.9776 μg/ml respectively , MIC90 and MBC90

were 6.7472 μg/ml and 53.9776 μ g/ml respectively.

Antibacterial effect of antimicrobial peptide PL-5 and vancomycin on Staphylococcus aureus and coagulase negative Staphylococcus in vitro

Sample Bacteria Strains Number of Strains

Strains of bacteria not growing under different drug concentrations (μg/ml) MIC range (μg/ml)

MIC50

(μg/ml) MIC90

(μg/ml) MIC Geometric mean (μg/ml)

64 32 16 8 4 2 1 1/2 1/4 solvent Culture

medium

PL-5

Staphylococcus aureus

(ATCC 29213） 1 1 1 1 1 0 0 0 0 - 0 1 8 8 8 8

（MRSA） 130 130 130 130 124 70 5 0 0 - 0 130 2～16 4 8 5.54 Staphylococcus

aureus

（MSSA） 67 67 67 67 65 47 3 0 0 - 0 67 2～16 4 8 4.87 Coagulase

negative Staphylococcus (MRSCNS)

121 121 121 121 117 90 15 0 0 - 0 121 2～16 4 8 4.49

Coagulase negative Staphylococcus (MSSCNS)

61 61 61 61 58 45 9 0 0 - 0 61 2～16 4 8 4.48

Vanco- mycin

(ATCC 29213） 1 - 1 1 1 1 1 1 0 0 0 1 1 1 1 1

（MRSA） 130 - 130 130 130 130 127 118 43 0 0 130 0.5～4 1 1 0.86 Staphylococcus

aureus

（MSSA） 67 - 67 67 67 67 63 59 26 - 0 67 0.5～4 1 2 0.87 Coagulase

121 - 121 121 121 121 120 105 25 0 0 121 0.5～4 1 2 0.96

61 - 61 61 61 61 58 45 15 0 0 61 0.5～4 1 2 1.05

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Bactericidal effect of antimicrobial peptide PL-5 and vancomycin on Staphylococcus aureus and coagulase negative Staphylococcus in vitro

Strains of bacteria not growing under different drug concentrations (μg/ml) MBC range (μg/ml)

MBC50

(μg/ml) MBC90

(μg/ml)

64 32 16 8 4 2 1 1/2 1/4 solvent Culture

medium

PL-5

(ATCC 29213） 1 1 1 1 1 0 0 0 0 - 0 1 8 8 8

（MRSA） 130 122 119 112 92 18 0 0 0 - 0 130 4~＞64 8 32 Staphylococcus

aureus

（MSSA） 67 66 62 55 38 17 1 0 0 - 0 67 2~＞64 4 16 Coagulase

121 120 108 95 67 33 5 0 0 - 0 121 2~＞64 8 64

61 60 51 42 32 18 3 0 0 - 0 61 2~＞64 8 64

Vanco- mycin

(ATCC 29213） 1 - - 1 1 1 0 0 0 0 1 4 4 4 1

（MRSA） 130 - - 94 81 69 52 25 1 0 0 130 0.5~

＞16 4 ＞16 Staphylococcus

aureus

（MSSA） 130 - - 94 81 69 52 25 1 0 0 130 0.5~

＞16 4 ＞16 Coagulase

121 - - 89 77 63 33 13 3 0 0 121 0.5~

＞16 4 ＞16 Coagulase

negative Staphylococcus (MSSCNS)

61 - - 47 42 37 28 13 4 0 0 61 0.5~

＞16 4 ＞16

Cumulative antibacterial percentage of antimicrobial peptide PL-5 and vancomycin on Staphylococcus aureus and coagulase negative Staphylococcus in vitro

Sample Bacteria Strains Number of Strains Cumulative antibacterial percentage of different concentrations (μg/ml) of tested drugs (%) MBC50/MIC50 MBC90/MIC90

64 32 16 8 4 2 1 0.5 0.25

PL-5

（MRSA） 130 100 100 100 95 54 4 0 0 - 2 4 Staphylococcus aureus

（MSSA） 67 100 100 100 97 70 4 0 0 - 1 2 Coagulase negative

Staphylococcus

(MRSCNS) 121 120 108 95 67 33 5 0 0 - 2 8

61 100 100 100 95 74 15 0 0 - 2 8

Vanco- mycin

（MRSA） 130 - 100 100 100 100 98 91 33 0 4 ＞16 Staphylococcus aureus

（MSSA） 67 - 100 100 100 100 94 88 39 0 4 ＞8 Coagulase negative

Staphylococcus (MRSCNS)

121 - 100 100 100 100 99 87 21 0 4 ＞8

61 - 100 100 100 100 95 74 25 0 4 ＞8

Cumulative bactericidal percentage of antimicrobial peptide PL-5 and vancomycin on Staphylococcus aureus and coagulase negative Staphylococcus in vitro

Sample Bacteria Strains Number of

Strains Cumulative bactericidal percentage of different concentrations (μg/ml) of tested drugs (%)

64 32 16 8 4 2 1 0.5 0.25

PL-5

（MRSA） 130 94 92 86 71 14 0 0 0 - Staphylococcus aureus

（MSSA） 67 99 93 82 57 25 1 0 0 - Coagulase negative

Staphylococcus

(MRSCNS) 121 99 89 79 55 27 4 0 0 -

Coagulase negative Staphylococcus

(MSSCNS) 61 98 84 69 52 30 5 0 0 -

Vanco- mycin

（MRSA） 130 - - 100 100 100 98 91 33 0 Staphylococcus aureus

（MSSA） 67 - - 70 63 55 42 28 3 0 Coagulase negative

Staphylococcus

(MRSCNS) 121 - - 74 64 52 27 11 2 0

Coagulase negative Staphylococcus

(MSSCNS) 61 - - 77 69 61 46 21 7 0

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2. Antibacterial and bactericidal effects of antimicrobial peptide PL-5 on Streptococcus pyogenes in vitro The antibacterial effect and bactericidal efficacy of antimicrobial peptide PL-5 on 19 erythromycin resistant strains and 21 erythromycin sensitive strains of Streptococcus pyogenes were tested in vitro using microdilution method and nutrient agar plate culture method, and vancomycin was used as the control drug.

The results showed that the MIC range of PL-5 to erythromycin resistant and erythromycin sensitive strains of Streptococcus pyogenes were 0.5~64 μg/ml and <0.03125~64μg/ml; the geometric mean of MIC were 23.0448μg/ml and 15.48μg/ml, respectively; MIC50 and MIC90 were both 32μg/ml. At the concentration of 64 μg/ml, PL-5 could 100% inhibit the growth of two kinds of Streptococcus pyogenes.

The MBC ranges of two kinds of Streptococcus pyogenes were 1~>64 and <0.03125~64μg/ml respectively; MBC50 were 32μg/ml, and MBC90 is 64 μg/ml. At the concentration of 64 μg/ml, the cumulative bactericidal percentage of PL-5 on erythromycin resistant strains reached 95%; while that was 100% on erythromycin sensitive strains. MBC50/MIC50 was 1 and MBC90/MIC90 was 2.

The results showed that antimicrobial peptide pl-5 had certain antibacterial and bactericidal effects on erythromycin resistant and erythromycin sensitive strains of Streptococcus pyogenes. Considering the content of pure peptide to be 84.34%, the MIC range and MBC range of erythromycin resistant strains of Streptococcus pyogenes were 0.4217~53.9776μg/ml and 0.8434~>53.9776μg/ml respectively; MIC90

and MBC90 were 26.9888μg/ml and 53.9776μg/ml respectively; The MIC range and MBC range of erythromycin sensitive strains of Streptococcus pyogenes were <0.0264~53.9776μg/ml and

<0.0264~53.9776μg/ml respectively, and MIC90 and MBC90 were 26.9888μg/ml and 53.9776μg/ml respectively.

Antibacterial effect of antimicrobial peptide PL-5 and vancomycin on Streptococcus pyogenes in vitro

Sampl

e Bacteria

Strains Num- ber of Strain s

Strains of bacteria not growing under different drug concentrations (μg/ml) MIC

range (μg/ml)

MIC50

(μg/ml) MIC90

64 32 16 8 4 2 1 1/2 1/4 1/8 1/16 1/32 1/64 solvent Culture

medium

PL-5

ATCC19615 1 1 1 1 1 0 0 0 0 0 0 0 0 - 0 1 8 8 8 8

ATCC13813 1 1 1 1 0 0 0 0 0 0 0 0 0 - 0 1 16 16 16 16

Streptococcus pyogenes Erythromycin resistant strain

19 19 18 5 1 1 1 1 1 0 0 0 0 - 0 19 0.5~64 32 32 23.04

Streptococcus pyogenes Erythromycin sensitive strain

21 21 19 9 5 2 2 2 1 1 1 1 1 - 0 21 <0.031

25~64 32 32 15.48

Vanc o- mycin

ATCC19615 1 - 1 1 1 1 1 1 1 1 0 0 0 0 0 1 0.25 0.25 0.25 0.25

ATCC13813 1 - 1 1 1 1 1 1 1 0 0 0 0 0 0 1 0.5 0.5 0.5 0.5

19 - 19 19 19 19 19 19 19 16 1 0 0 0 0 19 0.125~

0.5 0.25 0.5 0.27

21 - 19 19 19 19 19 19 19 15 2 1 0 0 0 21 0.0625

~>32 0.25 0.5 0.41

Bactericidal effect of antimicrobial peptide PL-5 and vancomycin on Streptococcus pyogenes in vitro

Sample Bacteria Strains Num-

ber of Strains

Strains of bacteria not growing under different drug concentrations (μg/ml)

MBC range

(μg/ml) MBC50

(μg/ml) MBC90

(μg/ml)

64 32 16 8 4 2 1 1/2 1/4 1/8 1/16 1/32 1/64 solvent Culture

medium

PL-5

ATCC19615 1 1 1 1 0 0 0 0 0 0 0 0 0 - 0 1 8 16 16

ATCC13813 1 1 1 1 0 0 0 0 0 0 0 0 0 - 0 1 16 16 16

19 18 17 5 1 1 1 1 0 0 0 0 0 - 0 19 1~>64 32 64

21 21 17 7 1 1 2 1 1 1 1 1 1 - 0 21 <0.03125~64 32 64

Vanco- mycin

ATCC19615 1 - 1 1 1 1 1 1 0 0 0 0 0 0 0 1 1 1 1

ATCC13813 1 - 0 0 0 0 0 0 0 0 0 0 0 0 0 1 >32 >32 >32

19 - 16 13 11 7 4 4 3 2 0 0 0 0 0 19 0.25~>32 8 >32

21 - 15 11 11 9 8 8 7 5 1 1 0 0 0 21 0.0625~>32 8 >32

Cumulative antibacterial percentage of antimicrobial peptide PL-5 and vancomycin on Streptococcus pyogenes in vitro

Sample Bacteria Strains

Number of Strains

Cumulative antibacterial percentage of different concentrations (μg/ml) of tested drugs (%) MBC50/MIC50 MBC90/MIC90

64 32 16 8 4 2 1 1/2 1/4 1/8 1/16 1/32 1/64

PL-5

19 100 95 26 5 5 5 5 5 0 0 0 0 - 1 2

21 100 90 43 24 10 10 10 5 5 5 5 5 - 1 2

Vanco- mycin

19 - 100 100 100 100 100 100 100 84 5 0 0 0 32 ＞64

21 - 90 90 90 90 90 90 90 71 10 5 0 0 32 ＞64

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Cumulative bactericidal percentage of antimicrobial peptide PL-5 and vancomycin on Streptococcus pyogenes in vitro

Cumulative antibacterial percentage of different concentrations (μg/ml) of tested drugs (%)

64 32 16 8 4 2 1 1/2 1/4 1/8 1/16 1/32 1/64

PL-5

19 95 89 26 5 5 5 5 0 0 0 0 0 -

21 100 81 33 5 5 5 5 5 5 5 5 5 -

Vanco- mycin

19 - 84 68 58 37 21 21 16 11 0 0 0 0

21 - 71 52 52 43 38 38 33 24 5 5 0 0

3. Antibacterial and bactericidal effects of antimicrobial peptide PL-5 on Enterococcus in vitro

The antibacterial effect and bactericidal efficacy of antimicrobial peptide PL-5 on 40 strains of Enterococcus faecalis and 40 clinical isolates of Enterococcus faecium were tested in vitro using microdilution method and nutrient agar plate culture method, and vancomycin was used as the control drug.

The results showed that the MIC range of PL-5 to Enterococcus faecalis and Enterococcus faecium were both 2~>64μg/ml; the geometric mean of MIC was 14.17 and 11.31μg/ml, respectively; MIC50 were 16 and 4μg/ml respectively, and MIC90 were >64 and 64μg/ml respectively. At the concentration of 64 μg/ml, PL-5 could inhibit the growth of Enterococcus faecalis and Enterococcus faecium by 80% and 93%

respectively. The MBC ranges of two kinds of bacteria were both 2~>64μg/ml; MBC50 were 16μg/ml, and MBC90 is >64 μg/ml. At the concentration of 64 μg/ml, the cumulative bactericidal percentage of PL-5 on both bacteria reached 78% and 85% respectively.

The above results showed that antimicrobial peptide PL-5 had certain antibacterial and bactericidal effects on Enterococcus faecalis and Enterococcus faecium only at high concentrations. Considering the content of pure peptide to be 84.34%, the MIC range and MBC range of Enterococcus faecalis and Enterococcus faecium were 1.6868~>53.9776μg/ml and 1.6868~>53.9776μg/ml respectively; MIC90

and MBC90 were > 53.9776μg/ml and >53.9776μg/ml respectively; The MIC range and MBC range of Enterococcus faecalis and Enterococcus faecium were 1.6868~>53.9776μg/ml and 1.6868~>53.9776μg/ml respectively; the MIC90 and MBC90 were 53.9776μg/ml and > 53.9776μg/ml respectively.

Antibacterial effect of antimicrobial peptide PL-5 and vancomycin on Enterococcus faecalis and Enterococcus faecium in vitro

Strains of bacteria not growing under different drug concentrations (μg/ml) MIC range (μg/ml)

MIC50

(μg/ml) MIC90

64 32 16 8 4 2 1 1/2 1/4 solvent Culture

medium

PL-5

Enterococcus faecalis

(ATCC 29212） 1 1 1 0 0 0 0 0 0 - 0 1 32 32 32 32

Enterococcus

faecalis 40 32 26 22 19 17 3 0 0 - 0 40 2~>64 16 >64 14.17

Enterococcus

faecium 40 37 29 25 22 21 3 0 0 - 0 40 2~>64 4 64 11.31

Vanco- mycin

(ATCC 29212） 1 - 1 1 1 1 1 1 0 0 0 1 1 1 1 1

Enterococcus

faecalis 40 - 40 40 40 40 40 39 23 0 0 40 0.5～2 0.5 1 0.68

Enterococcus

faecium 40 - 32 32 32 32 32 26 15 0 0 40 0.5 ～

32 1 >32 1.71

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Bactericidal effect of antimicrobial peptide PL-5 and vancomycin on Enterococcus faecalis and Enterococcus faecium in vitro

MBC50

(μg/ml) MBC90

(μg/ml)

64 32 16 8 4 2 1 1/2 1/4 solvent Culture

medium

PL-5

(ATCC 29212） 1 1 1 0 0 0 0 0 0 - 0 1 32 32 32

Enterococcus

faecalis 40 31 25 22 16 4 1 0 0 - 0 40 2~＞64 16 >64

Enterococcus

faecium 40 34 27 22 19 10 1 0 0 - 0 40 2~＞64 16 >64

Vanco- mycin

(ATCC 29212） 1 - 0 0 0 0 0 0 0 0 0 1 >32 >32 >32

Enterococcus

faecalis 40 - 33 10 3 0 0 0 0 0 0 40 8~>32 32 >32

Enterococcus

faecium 40 - 20 4 0 0 0 0 0 0 0 40 16~>32 32 >32

Cumulative antibacterial percentage of antimicrobial peptide PL-5 and vancomycin on Enterococcus faecalis and Enterococcus faecium in vitro

Sample Bacteria Strains Number of Strains Cumulative antibacterial percentage of different concentrations (μg/ml) of tested drugs (%) MBC50/MIC50 MBC90/MIC90

64 32 16 8 4 2 1 0.5 0.25

PL-5 Enterococcus faecalis 40 80 65 55 48 43 8 0 0 - 1 -

Enterococcus faecium 40 93 73 63 55 53 8 0 0 - 4 >1

Vanco- mycin

Enterococcus faecalis 40 - 100 100 100 100 100 98 58 0 64 ＞32

Enterococcus faecium 40 - 80 80 80 80 80 65 38 0 32 -

Cumulative bactericidal percentage of antimicrobial peptide PL-5 and vancomycin on Enterococcus faecalis and Enterococcus faecium in vitro

Cumulative bactericidal percentage of different concentrations (μg/ml) of tested drugs (%)

64 32 16 8 4 2 1 0.5 0.25

PL-5 Enterococcus faecalis 40 78 63 55 40 14 10 3 0 -

Enterococcus faecium 40 85 68 55 48 25 25 3 0 -

Vanco- mycin

Enterococcus faecalis 40 - 83 25 8 100 0 0 0 0

Enterococcus faecium 40 - 50 10 0 55 0 0 0 0

4. Antibacterial and bactericidal effects of antimicrobial peptide PL-5 on Escherichia coli and Klebsiella pneumoniae in vitro

The antibacterial effect and bactericidal efficacy of antimicrobial peptide PL-5 on

119 clinical isolates of Escherichia coli (79 strains of producing extended spectrum beta lactamases ESBLs and 40 non-producing ESBLs) and 120 Klebsiella pneumoniae (80 strains of producing ESBLs and 40 non-producing ESBLs) were tested in vitro using microdilution method and nutrient agar plate culture method, and Imipenem Cilastatin sodium was used as the control drug.

The results showed that the MIC range of PL-5 to ESBLs producing Escherichia coli and ESBLs non- producing Escherichia coli were both 2~>64μg/ml; the geometric mean of MIC was 4.68 and 8.14μg/ml, respectively; MIC50 were 4 and 8μg/ml respectively, and MIC90 were 32 and 64μg/ml respectively.

According to the cumulative antibacterial percentage, at the concentration of 64 μg/ml, PL-5 could inhibit the growth of ESBLs producing Escherichia coli and ESBLs non-producing Escherichia coli by 94%

and 93% respectively. The MBC ranges of two kinds of bacteria were both 2~>64μg/ml; MBC50 were 4μg/ml and 8μg/ml, and MBC90 were 64 and >64 μg/ml. At the concentration of 64 μg/ml, the cumulative bactericidal percentage of PL-5 on both Escherichia coli reached 94% and 88% respectively; MIC range of PL-5 to ESBLs producing Klebsiella pneumoniae and ESBLs non-producing Klebsiella pneumoniae were 1~>64μg/ml and 1~>64μg/ml respectively; the geometric mean of MIC were 6.22 and 5.96μg/ml, respectively; MIC50 were 4 and 8μg/ml respectively, and MIC90 were both 8μg/ml. According to the cumulative antibacterial percentage, at the concentration of 64 μg/ml, PL-5 could inhibit both the growth of ESBLs producing Klebsiella pneumoniae and ESBLs non-producing Klebsiella pneumoniae by 100%.

The MBC ranges of two kinds of bacteria were 2~>64μg/ml and 2~64μg/ml respectively; MBC50 were both 8μg/ml, and MBC90 were 64 and >64 μg/ml. At the concentration of 64 μg/ml, the cumulative bactericidal percentage of PL-5 on ESBLs producing Klebsiella pneumoniae and ESBLs non-producing Klebsiella pneumoniae were 96% and 100% respectively.

The above results showed that antimicrobial peptide PL-5 had antibacterial and bactericidal effects on clinical isolates of ESBLs producing Escherichia coli, ESBLs non-producing Escherichia coli, ESBLs producing Klebsiella pneumoniae and ESBLs non-producing Klebsiella pneumoniae. Considering the content of pure peptide to be 84.34%, the MIC range and MBC range of ESBLs producing Escherichia coli were 1.6868~>53.9776μg/ml and 1.6868~>53.9776μg/ml respectively; MIC90 and MBC90 were >

26.9888μg/ml and >53.9776μg/ml respectively; The MIC range and MBC range of ESBLs non-

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producing Escherichia coli were 1.6868~> 53.9776μg/ml and 1.6868~>53.9776μg/ml respectively; the MIC90 and MBC90 were 53.9776μg/ml and > 53.9776μg/ml respectively. The MIC range and MBC range of ESBLs producing Klebsiella pneumoniae were 0.8434~53.9776μg/ml and 1.6868~>53.9776μg/ml respectively; the MIC90 and MBC90 were 6.7472μg/ml and > 53.9776μg/ml respectively. The MIC range and MBC range of ESBLs non-producing Klebsiella pneumoniae were 1.6868~53.9776μg/ml and 1.6868~>53.9776μg/ml respectively; the MIC90 and MBC90 were 6.7472μg/ml and 26.9888μg/ml respectively.

Antibacterial effect of antimicrobial peptide PL-5 and imipenem cilastatin sodium on Escherichia coli in vitro

Sample Bacteria

Strains NO. of

Strains

Strains of bacteria not growing under different drug concentrations (μg/ml) MIC range

(μg/ml) MIC50

(μg/ml) MIC90

64 32 16 8 4 2 1 1/2 1/4 1/8 solvent Culture

medium

PL-5

ESBLs E. Coli (ATCC

35218） 1 1 1 1 1 1 0 0 0 - - 0 1 4 4 4 4

Non-ESBLs E.

Coli（ATCC

25922） 1 1 1 1 1 1 0 0 0 - - 0 1 4 4 4 4

ESBLs E. Coli 79 74 73 71 70 61 23 0 0 - - 0 79 2～>64 4 32 4.68

Non-ESBLs E.

Coli 40 37 33 33 33 18 2 0 0 - - 0 40 2～>64 8 64 8.14

imipenem cilastatin sodium

ESBLs E. Coli (ATCC

35218） 1 - - 1 1 1 1 1 1 1 0 0 1 0.25 0.25 0.25 0.25

Non-ESBLs E.

Coli (ATCC

25922） 1 - 1 1 1 1 1 1 1 1 0 0 1 0.25 0.25 0.25 0.25

ESBLs E. Coli 79 - 77 77 73 73 61 54 47 30 2 0 79 <0.125~>32 0.5 4 0.84

Non-ESBLs E.

Coli 40 - 40 40 40 40 40 39 33 21 0 0 40 0.25~2 0.25 1 0.39

Bactericidal effect of antimicrobial peptide PL-5 and imipenem cilastatin sodium on Escherichia coli in vitro

MBC50

(μg/ml) MBC90

(μg/ml)

64 32 16 8 4 2 1 1/2 1/4 1/8 Solvent Culture

medium

PL-5

ESBLs E. Coli

(ATCC 35218） 1 1 1 1 1 1 0 0 0 - 0 0 1 4 4 4

Non-ESBLs E.

Coli (ATCC

25922） 1 1 1 1 1 1 0 0 0 - 0 0

1

4 4 4

ESBLs E. Coli 79 74 70 62 50 40 13 0 0 - 0 0 79 2~＞64 4 64

Non-ESBLs E.

Coli 40 35 32 30 25 10 1 0 0 - 0 0 40 2~＞64 8 >64

ESBLs E. Coli

(ATCC 35218） 1 - - 1 1 0 0 0 0 0 0 0 1

8 8 8

Non-ESBLs E.

Coli (ATCC

25922） 1 - - 94 81 1 1 1 1 0 0 0

1

0.5 0.5 0.5

ESBLs E. Coloi 79 - - 94 81 61 46 37 23 12 1 0 79 <0.125~>16 2 ＞16

Non-ESBLs E.

Coli 40 - - 89 77 12 12 8 3 1 0 0 40 0.25~>16 16 ＞16

Cumulative antibacterial percentage of antimicrobial peptide PL-5 and imipenem cilastatin sodium on Escherichia coli in vitro

Cumulative antibacterial percentage of different concentrations (μg/ml) of tested drugs (%) MBC50/MIC50 MBC90/MIC90

64 32 16 8 4 2 1 0.5 0.25 0.125

PL-5 ESBLs E. Coli 79 94 92 90 89 77 29 0 0 - - 1 2

Non-ESBLs E. Coli 40 93 83 83 83 45 5 0 0 - - 1 >1

ESBLs E. Coli 79 - 97 97 92 92 77 68 59 38 3 4 ＞4

Non-ESBLs E. Coli 40 - 100 100 100 100 100 98 83 53 0 64 >16

Cumulative bactericidal percentage of antimicrobial peptide PL-5 and imipenem cilastatin sodium on Escherichia coli in vitro

Sample Bacteria Strains

Number of Strains

Cumulative bactericidal percentage of different concentrations (μg/ml) of tested drugs (%)

64 32 16 8 4 2 1 0.5 0.25 0.125

PL-5

Enterococcus

faecalis 79 94 89 78 63 51 16 0 0 - -

Enterococcus

faecium 40 88 80 75 63 25 3 0 0 - -

Enterococcus

faecalis 79 - - 90 80 77 58 47 29 15 1

Enterococcus

faecium 40 - - 88 45 30 30 20 8 3 0