Supplementary Materials for







Full text


Supplementary Materials for

A polymeric approach toward resistance-resistant antimicrobial agent with

dual-selective mechanisms of action

Silei Bai, Jianxue Wang, Kailing Yang, Cailing Zhou, Yangfan Xu, Junfeng Song, Yuanxin Gu, Zheng Chen, Min Wang, Carolyn Shoen, Brenda Andrade, Michael Cynamon, Kai Zhou, Hui Wang, Qingyun Cai, Eric Oldfield,

Steven C. Zimmerman, Yugang Bai*, Xinxin Feng*

*Corresponding author. Email: (X.F.); (Y.B.) Published 27 January 2021, Sci. Adv.7, eabc9917 (2021)

DOI: 10.1126/sciadv.abc9917 The PDF file includes:

Supplementary methods Figs. S1 to S9

Table S1

Other Supplementary Material for this manuscript includes the following:

(available at


Supplementary Methods

MIC Determination

Screening assay: For all the bacteria strains and isolates except M. tuberculosis (provided separately in Supplementary Material), an overnight starter culture of bacteria in CAMHB was diluted 1000-fold in fresh media, and grown at 37 °C to reach an OD600 of approximately 0.3. This

log-phase culture was diluted in fresh media to generate the working solution containing 5×105 CFU/mL bacteria. 180 µL of this working solution was transferred into every well of a flat-bottom 96-well plate, except for the first column (which was reserved for CAMHB as negative control) and the first row (reserved for initial compound solutions). Compounds were added at specific starting concentrations (32 μg/mL for oligomers and 128 μg/mL for standard antibiotics, in working solution) with a total volume of 360 µL into the first row. The compounds were then sequentially diluted twofold across 8 wells with working solution. Plates were incubated at 37 °C, shaking at 220 rpm, for 16-24 h. The OD600 values were measured to determine MIC values. Experiments

were carried out in triplicate or more. Studies using FBS-containing media and whole blood were conducted likewise (see below). For M. smegmatis, the media used was 7H9 supplemented with glycerol and 10% ADC, and the incubation time was 36-48 h.

Growth Inhibition Assay in 50% FBS or Whole Sheep Blood: A clinical isolate of A. baumannii

(A. b-1 in Table 1) or S. aureus (S. a-1 in Table 1) was added to sheep blood or CAMHB containing 50% FBS, to a final concentration of 5 × 105 CFU/mL. Samples were then treated with oligomer 3

or ciprofloxacin, and were incubated for 24 h at 37 ℃. Bacteria loading was determined by plating an aliquot of sample with different serial dilutions onto an agar plate, and CFUs were counted after a 12 h incubation at 37 ℃.

MTT Cytotoxicity Assay

Cells were seeded into 96-well plates (BIOFIL, catalog number TCP-011-096) at 1 × 104 cells per

well and incubated for 12 h at 37 °C in 5% CO2 to facilitate attachment. Cells were treated with


with no compounds added served as controls. After incubation, old media was removed, and cells were washed with PBS once before cell media was replaced with 120 μL of fresh media with MTT (0.5 mg/mL). Cells were incubated for another 1.5 h at 37 °C in 5% CO2. Next, the media was

replaced with 100 μL of DMSO and cell viability was determined by measuring the absorbance at 595 nm. Cell viability values were expressed as percentages and calculated as follows: Viability % = [Abs595 nm of treated sample) / [Abs595 nm of control] × 100%.

Hemolysis Assay

Fresh sheep blood was subjected to 25-fold dilution with PBS buffer to reach a concentration of 4% blood. 300 μL of PBS solution containing oligomers at various concentrations was placed in a 1.5 mL microfuge tube, followed by addition of an equal volume (300 μL) of red blood cell suspension. The mixture was incubated at 37 °C for 1 h (in HC10 or HC50 determination) or 24 h (in

hemolytic kinetics) to allow hemolysis to take place. At the end of the incubation period, non-hemolyzed red blood cells were separated by centrifugation at 100 g for 5 min. Aliquots (100 μL) of the supernatant were transferred to a 96-well plate, and hemoglobin release was measured by absorbance at 576 nm using a microplate reader (TECAN, Switzerland). Two controls were used: an untreated red blood cell suspension in PBS as the negative control; a solution containing red blood cells lysed with 1% Triton-X100 as the positive control. Percent hemolysis was calculated by using the following formula: Hemolysis (%) = [(OD576 nm of the treated sample − OD576 nm of the

negative control)/(OD576 nm of positive control − OD576 nm of negative control)]×100%.

In vitro Analysis Using M. tuberculosis

Note: Experiments involving the use of M. tuberculosis must be performed in a laboratory that meeting the standards of Biosafety Level 3 (BL3) or higher.

Drugs and compounds: Isoniazid was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO), and was dissolved in 100% dimethyl sulfoxide (DMSO) at 1 mg/mL, aliquoted into several freezer tubes. The drugs and compounds were frozen at -20 oC until use, avoiding refreezing drugs after use.


Isolate: M. tuberculosis Erdman (ATCC 35801) and M. tuberculosis H37Rv (ATCC 27294) were grown in modified 7H9 broth with 0.1% Tween-80 supplemented with 10% Middlebrook albumin-dextrose-catalase (ADC) enrichment on a rotary shaker at 37oC for 7-10 d.

In vitro susceptibility testing: On the day of in vitro testing, the compounds to be tested were thawed and diluted in 7H9 Broth to 4-times the maximum concentration to be tested. The test range for the oligoguanidine was 100 g/mL to 0.098 g/mL, and isoniazid was 8 g/mL to 0.008 g/mL. On the day of in vitro testing, the bacteria were diluted in 7H9 broth to a final concentration of 105 CFU/mL (in vitro inoculum). Polystyrene 96-well round-bottom plates (Corning Inc., Corning, NY) were prepared with 50 µL of 7H9 broth per well. The compounds were added to the first well prior to being serially (2-fold) diluted throughout the row, leaving the last well with 7H9 broth only (positive growth control). 50 µL of the in vitro inoculum was added to each well. Plates were sealed and incubated at 37 oC in ambient air for 14 d prior to reading. The minimal inhibitory concentration (MIC) was determined visually and is defined as the minimum concentration of compound required to inhibit 90% growth of M. tuberculosis compared to the positive control. The MIC assays were run in duplicate.

The actual inoculum used was measured by titration in saline with Tween-80 and plating on 7H10 agar plates supplemented with 10% OADC (Becton Dickinson, Sparks, MD). The plates were incubated in ambient air at 37 oC for 4 weeks.

Transcriptome Study

The transcriptomic dataset has been deposited to NCBI Sequence Read Archive with the identifier PRJNA609956.

1. Sample preparation

An overnight starter culture in CAMHB broth of A. b -1(clinical isolate) was diluted 200-fold in fresh CAMHB media. The control group and treatment group (2 μg/mL of 3) were allowed to grow for 5 h and 8 h, respectively, to reach log phase. Cells were harvested by centrifuging at 3200 g for 20 min. Samples were then frozen in liquid nitrogen. The wet sample weights were 184 mg, 153


mg, 132 mg for the control group, and 181 mg, 214 mg and 174 mg for the treatment group.

2. RNA quantification and qualification

Total RNAs obtained from replicate samples were extracted by using commercial kits, according to the manufacturer’s instructions. RNA degradation and contamination were monitored on 1% agarose gels. RNA concentrations were measured using Qubit 2.0 (Thermo Fisher Scientific, MA, USA) and Nanodrop One (Thermo Fisher Scientific, MA, USA), at the same time. RNA integrity was determined by using an Agilent 2100 system (Agilent Technologies, Waldbron, Germany).

3.Library preparation

Whole mRNAseq libraries were generated using NEB Next® UltraTM Directional RNA Library Prep

Kit for Illumina® (New England Biolabs, MA, USA), following the manufacturer’s

recommendations. Briefly, bacterial and archaeal 16S and 23S rRNA transcripts in total RNA samples were reduced by using a Ribo-zero rRNA Removal Kit. Fragmentation was carried out using NEB Next First Strand Synthesis Reaction Buffer. The first strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). During the synthesis of the second strand of cDNA, a chain-specific library was constructed by replacing dTTP with dUTP, to improve accuracy. Remaining overhangs were converted into blunt ends via exonuclease/polymerase reactions. After adenylation of the 3’ ends of DNA fragments, NEB Next Adaptor with a hairpin loop structure were ligated to be ready for hybridization. In order to select cDNA fragments of ~150-200 bp length, fragments were selected with AMPure XP beads (Beckman Coulter, Beverly, USA). Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified with AMPure XP beads and library insert size assessed by using an Agilent 2100 system (Agilent Technologies, Waldbron, Germany).

4. Transcriptome sequencing

The clustering of the index-coded samples was performed on a cBot Cluster Generation System. After cluster generation, the library was sequenced on an Illumina Hiseq Xten platform and 150 bp paired-end reads were generated.


5. Data analysis

Quality control. Raw data in fastq format were processed by Trimmomatic (v.0.36, to acquire clean data (clean reads). Clean reads were mapping to NCBI Rfam databases, to remove the rRNA sequence, by using Bowtie2 (v2.33,

Reads mapping to the reference genome. Reference genome and gene model annotation files were downloaded from the NCBI genome website directly ( genome/?term=Acinetobacter+baumannii). The remaining mRNA sequences were mapped to the

A. baumannii reference genome by using Hisat2 (version 2.1.0,

Transcript quantification and sample relationship analysis. HTSeq-count (v0.9.1, was used to obtain the read count and function information of each gene, based on the mapping results. In order to make the expression levels of genes be comparable among different genes and different experiments, the RPKM of each gene was calculated. RPKM, Reads Per Kilobase of transcript, per Million mapped reads, is a normalized unit of transcript expression and takes into account the effects of sequencing depth and gene length for the read count, and is currently the most commonly used method for estimating gene expression levels. PCA (principal component analysis), correlation coefficient heat maps and expression heat maps were then used to reveal transcription relationships between all samples.

Differential expression analysis. Read count of each gene obtained from HTSeq-count was used for differential expression analysis. Differential expression analysis of gene expression data was performed using edgeR (v3.16.5, edgeR.html) which takes the length and number of genes into account. The resulting P-values were adjusted by using the Benjamini and Hochberg approach for controlling the false discovery rate (FDR). Genes with FDR ≤ 0.05 and |log2(fold change)| ≥ 1 were taken as differentially expressed

genes, and these were used for heatmap construction.

Bioinformatic analysis. GO (Gene Ontology, annotation analysis of differentially expressed genes were implemented by using clusterProfiler (v3.4.4, Searching of


sequence homologs, secondary structure prediction and protein 3D structure prediction of gene products were performed with the Protein Basic Local Alignment Search Tool (BLAST, see J. Mol. Biol.1990, 215, 403-410), TMHMM (see J. Mol. Biol. 2001, 305, 567-580) and Phyre2 (Nat. Protoc.

2015, 10, 845-858) servers, respectively.

Generation of the colocalization histograms

The DAPI channel and FM4-64 channel of the confocal images were combined to obtain a membrane + DNA distribution pattern for the bacteria or eukaryotic cells (referred to as “FI of DAPI + FM4-64”). Since the FM4-64 channel is empty for NIH/3T3 cells, “FI of DAPI + FM4-64” for 3T3 only represented the signal of DAPI. Then the newly combined channel “FI of DAPI+FM4-64” was compared to oligomer 3’s channel using the Coloc 2 plug-in (in ImageJ), to obtain a histogram and a corresponding Pearson’s R value for each confocal image. This generated the histograms like the one in Figure 3B.

For each group of samples (M. smegmatis, B. subtilis, E. coli and NIH/3T3), the experiments were repeated independently for at least three times, and obtained many sets of confocal images for each group. These images were analyzed as above to give multiple histograms and Pearson’s R values for each group. Figure 3C represented the average values and standard deviations of Pearson’s R values from different independent experiment for each group.

Generation of the box plots

R codes used to generate the box plots. The codes for making the box plots (Fig. 5 of the main article) are shown below:


setwd("/Users/songjunfeng/Desktop") table1 <- read.csv("survival.csv",header = T) df1<-table1


p <- ggboxplot(df1, x="dose", y="len", color = "dose", palette = c("#00AFBB", "#E7B800", "#FC4E07"),

add = "jitter", shape="dose") p

my_comparisons <- list(c("OLIGO3", "PBS"),c("OLIGO3","CIP"),c("PBS","CIP")) p+stat_compare_means(comparisons = my_comparisons)+


Supplementary Figures and Tables

Supplementary Figures

Fig. S1. MALDI-TOF mass spectrometric characterization of oligoamidine 3. Polymers and oligomers usually show several peak series in MALDI-TOF analyses, resulted from different fragmentation pathways. The distance between peaks between peaks denotes the molecular weight of the repeating unit of the analyte. The repeating unit of oligoamidine 3 has a m/z of 328.24, which matches well with the distance between peaks (328.2-328.3 Da) in the above figure. The raw data of this MALDI-TOF analysis is provided in Data File S1.

Detailed analysis of the spectrum:

There are three possible end-group combinations for oligomer 3.

(1) two ethyl esters (the product from the hydrolysis of unreacted terminal ethyl imidate groups during dialysis); (2) ethyl ester and free amine; (3) two amines.

Each combination gives their own peak series, but distance between peaks within one series remains the same (328 Da), which equals to the molecular weight of the oligomer’s repeating unit. Illustrations on the fragmentation pathways are provided below:


Fragmentation pathways

Several fragmentation pathways are commonly seen for aromatic esters: (a) Molecular ion of an aromatic ester easily undergo McLafferty rearrangement and give peak for the corresponding aromatic acid (ArCOOH+). (b) Molecular ion of an aromatic ester easily eliminates COOR radical to give Ar+ peak. (c) Terephthalic esters (ethyl or longer) typically give M - R peak and R peak due


Long-chain aliphatic amine usually shows weak or indiscernible molecular ion peak, and a series of lower intensity peaks.

Assignment of peak series

Blue series: Diester oligomer undergoes McLafferty rearrangement on one end, forming an ArCOOH+ termini.

Gray series: Fragments of the blue series undergo C-O cleavage to remove a CH3CH2+ cation.

Alternatively, this peak series may be from diester oligomer having one of its ends undergoing C-O cleavage to remove •CH2CH3, followed by the elimination of an ethylene on another end.

Black series: Diester oligomer removes CH3CO to give a protonated aldehyde on one end

(ArCHOH+). Mechanism is unknown.

Green series: Diester oligomer removes •COOEt radical to form an Ar+ termini. Alternatively, this peak series may come from the decarboxylation of the fragments responsible for the blue series. Pink series: Molecular ions of the monoester (monoamine) oligomer.

Red series: Monoester (monoamine) oligomer undergoes McLafferty rearrangement on its ester end, forming an ArCOOH+ termini.

Cyan series: Monoester (monoamine) oligomer removes CH3CO to give a protonated aldehyde on

its ester end (ArCHOH+). Mechanism is unknown.

Yellow series: Molecular ions of the diamine oligomer.

The MALDI instrument has an error of approximately 0.7 Da (<0.1%) in the measurement of absolute m/z values. All the measured mass in Da are larger than the theoretical value by approximately 0.7.


Fig. S2. Oligoamidine 3’s interaction with bacteria and red blood cells under different settings.

(A) Antimicrobial efficacy of 3 in environments containing high concentration of serum. (B) Killing kinetics of oligomer 3 against MDR clinical isolate, A. b -1. The figure on the right is an area enlarge (0 – 16 min) of the left one. (C) Killing kinetics of oligomer 3 against M. smegmatis. (D) Hemolytic activity of oligomer 3 over 24 h. At the concentrations around 3’s MICs, 3 did not show significant hemolytic activity in a much longer period of time (in most reports, the HC50 values of antimicrobial


Fig. S3. Additional evidences for 3’s membrane disruption mechanism. (A) SEM characterization of M. smegmatis before (top) and after (bottom) a 1.5 h treatment of oligomer 3

(20 µg/mL). (B) Results from the cytoplasmic membrane depolarization assay. Apparent cell membrane disruption by addition of 3 could be observed. DiSC3(5) dye was used as the fluorescence probe. (C) Studies on the ROS response from E. coli upon treatment of 3 of different concentrations. DCFH-DA was used as the ROS probe. (D) Inhibition effect of LPS on 3’s antimicrobial efficacy against A. baumannii. (E) Zeta potential changes of bacteria and mammalian cells after being treated with 3 of different concentrations. (F) The necessity of multivalence brought by the polymeric approach. DB213, a structural analog of 3’s repeating unit, showed much weaker binding to membranes of S. aureus and A. baumannii than 3 did, as shown in these zeta potential analyses. (G)


Fig. S4. Additional evidences for 3’s dual mechanisms. (A) Confocal microscope images showing that oligomer 3 disrupts the membranes of M. smegmatis, leading to the uptake of PI. In contrast, kanamycin kills bacteria without changing membrane permeability, since there is no (red) PI fluorescence signal observed with the kanamycin-treated sample. (B) Additional PI-based flow cytometry study showing the membrane disruptive phenomenon of a quaternary amine-based oligomer. Oligomer 3, on the other hand, was much more eukaryotic cell-friendly. (C) Dynamic light scattering results show that oligomer 3 binds to A. baumannii’s genomic DNA to form aggregates. (D) Binding of oligomer 3 with plasmid DNA and linear DNA using dynamic light scattering. The plasmid used for this study was pCDFDuet-GFP, and the linear DNA used was the cleaved product from pCDFDuet-GFP. (E) Docking result using a fragment of oligomer 3 and

dsDNA (CGCGAATTCGCG)2 (PDB: 4U8A). (F) Gel retardation assay showing the complexation

of a 6500-bp plasmid DNA (Pcold-ctxm-15) and oligomer 3. DNA was fluorescently labeled by Gel-Red, an intercalator-type dye.


Fig. S5. Additional confocal microscopic images showing 3 stained both bacterial membranes and DNA in various bacterial strains. From top to bottom are images of M. smegmatis, B. subtilis,


Fig. S6. Evidences for 3’s exclusion from the mammalian cell nucleus and mitochondria. (A) Additional confocal microscopic images showing that 3 was excluded from the mammalian cell (NIH/3T3) nucleus. Scale bar = 20 µm. (B) Colocalization study using rhodamine-labeled oligomer

3 and Mitotracker Green in NIH/3T3 cells. The 2D intensity histogram was plotted using Mitotracker fluorescence against 3’s fluorescence. The calculated R value (Mitotracker channel vs

oligomer 3 channel) indicates that 3 does not target mitochondria in mammalian cells. Scale bar = 20 µm.


Fig. S7. Resistance generation of E. coli using DNA-binding small molecules. Hoechst and DAPI binds dsDNA in a non-covalent manner while mitomycin alkylates dsDNA. It can be clearly seen that unlike 3, these small molecules develop resistance in a short period of time. It is likely that the polymeric (multivalent) structure of 3 plays a key role in the resistance-resistant feature.


Fig. S8.Additional confocal microscopic images of the intracellular killing study. RAW 264.7 cells were infected by M. smegmatis, and the effectiveness of treatments by different antimicrobial agents could be observed and compared. Scale bar = 10 μm. In “Infected RAW 264.7 model cells (Right after infection)”, M. smegmatis was stained with Syto 84 dye before infection for better visualization.


Fig. S9.Additional animal study results. (A) Toxicity evaluation of oligomer 3 at its treatment dosage (10 mg/kg) in the cutaneous abscess model study. No apparent weight loss was observed for the mice. (B) Immunohistology section images of multiple organs of the mice after the above toxicity evaluation. (C) Representative images of the agar plates for CFU determination in the mice excision wound model study, after the treatment of oligomer 3, ciprofloxacin or PBS. Despite that the surface bacteria were eliminated for the treatment group, some bacteria may still escape the antimicrobial agents by disseminating into mice organs, leading to fatal systemic infections. (D) Healing curve of the wounds in the mice excision wound model study. The relative percentage of


wound size was plotted against time. (E) Statistics on the days needed for the infected wounds to heal 50% under different treatments in the mice excision wound model study. N = 10, 5 and 11 for ciprofloxacin, PBS and oligomer 3 group, respectively.


Supplementary Tables

Table S1. Antimicrobial efficacy and hemolytic ability of the screened oligoamidinesa.

Diamine Structure

Structure of the Corresponding Oligomer or Other Compounds



Minimal inhibitory concentration (MIC, μg/mL) E. faecalis S. aureus E. coli B. subtilis 44 1 2 1 1 165 2 1 2 1

Does not polymerize

with the bisimidate ester ND

79 1 0.25 0.5 1.6

220 2 2 2 2

42 2 0.5 1 2


>5000b 4 0.5 2 4

103 4 2 4 4

178 4 0.25 0.5 1

98 2 2 2 2

ND (product polymer is unstable)

NA (PHMB) 102c 2 1 2 ND NA (DB213) ND >128 >128 >128 >128

a All the oligomers had similar molecular weight (ca. 2 kDa). b The HC

10 value of this compound was also >5000

μg/mL. c The HC





Related subjects :