The antimicrobial and biofilm disruption activity of novel

James Madison UniversityJMU Scholarly Commons

Masters Theses The Graduate School

Spring 2017

The antimicrobial and biofilm disruption activity ofnovel amphiphilesElizabeth A. RogersJames Madison University

Follow this and additional works at: https://commons.lib.jmu.edu/master201019Part of the Pathogenic Microbiology Commons

This Thesis is brought to you for free and open access by the The Graduate School at JMU Scholarly Commons. It has been accepted for inclusion inMasters Theses by an authorized administrator of JMU Scholarly Commons. For more information, please contact [email protected].

Recommended CitationRogers, Elizabeth A., "The antimicrobial and biofilm disruption activity of novel amphiphiles" (2017). Masters Theses. 497.https://commons.lib.jmu.edu/master201019/497

https://commons.lib.jmu.edu/?utm_source=commons.lib.jmu.edu%2Fmaster201019%2F497&utm_medium=PDF&utm_campaign=PDFCoverPages

https://commons.lib.jmu.edu/master201019?utm_source=commons.lib.jmu.edu%2Fmaster201019%2F497&utm_medium=PDF&utm_campaign=PDFCoverPages

https://commons.lib.jmu.edu/grad?utm_source=commons.lib.jmu.edu%2Fmaster201019%2F497&utm_medium=PDF&utm_campaign=PDFCoverPages

https://commons.lib.jmu.edu/master201019?utm_source=commons.lib.jmu.edu%2Fmaster201019%2F497&utm_medium=PDF&utm_campaign=PDFCoverPages

http://network.bepress.com/hgg/discipline/52?utm_source=commons.lib.jmu.edu%2Fmaster201019%2F497&utm_medium=PDF&utm_campaign=PDFCoverPages

https://commons.lib.jmu.edu/master201019/497?utm_source=commons.lib.jmu.edu%2Fmaster201019%2F497&utm_medium=PDF&utm_campaign=PDFCoverPages

mailto:[email protected]

The Antimicrobial and Biofilm Disruption Activity of Novel Amphiphiles

Elizabeth Anne Rogers

A thesis submitted to the Graduate Faculty of

JAMES MADISON UNIVERSITY

In

Partial Fulfillment of the Requirements

for the degree of

Master of Science

Department of Biology

May 2017

FACULTY COMMITTEE:

Committee Chair: Dr. Kyle Seifert

Committee Members:

Dr. Kevin Caran

Dr. James Herrick

ii

Dedication

This thesis is dedicated to my friends and family who have supported me throughout my

time at James Madison University. In particular, I would like to dedicate this work to my

mother, Mary Rogers, and my father, Malcolm Rogers, for instilling in me a strong sense

of curiosity and a desire to learn. I would also like to thank Scott Oslin for being a

constant friend and confidant for the past six years and offering endless encouragement

when things got hard.

iii

Acknowledgments

I would like to thank my advisor, Dr. Kyle Seifert, for being a supportive mentor and role

model for me during my time at JMU, and for helping me to develop my scientific

research and communication abilities. He has spent the last two years encouraging me to

improve as a student, a teacher, and a scientist. I would also like to thank my committee

members, Dr. Kevin Caran and Dr. James Herrick, for helping me to determine the best

path for my research project. They have contributed significant feedback and support to

help me improve my work.

The undergraduate members of the Seifert lab have contributed massive amounts of time

and effort to this project, as have the students working in the Caran lab, and I would like

to thank all of them for everything they have added to this research. I would also like to

thank my fellow graduate students, especially Stephanie Sharpes, for their feedback and

support from start to finish. This thesis would not have been possible without all of them.

I would also like to acknowledge all of the faculty members who have contributed to this

research project and have supported me during my time in this program. In particular, I

would like to thank Dr. Kristopher Kubow for offering his time to help us with confocal

microscopy.

Finally, I would like to thank James Madison University for their financial support of this

project. Through them, I have been able to present our research at multiple conferences

during my time as a graduate student.

iv

Table of Contents

Dedication…………………..……………………………………………………………ii

Acknowledgements…………………………………………………………………...…iii

List of Tables…………………………………………………………………..……......vi

List of Figures……………...…………………...………………………………...…….vii

Abstract………………………………………………..………………………...……....ix

Chapter 1. Introduction………………………………………………………………......1

1.1. Resistant Bacteria

1.2. Biofilms and Transmission – Implications for Nosocomial Infection

1.3. Antibacterial Development

1.4. Amphiphiles

1.5. Amphiphiles Used in the Current Study

Chapter 2. The Effect of Tail Length Variation and Head Group Substitution on

Minimum Inhibitory Concentration and Biofilm Disruption.............…………………..16

2.1. Introduction

2.2. Results and Discussion

Critical aggregation concentration

Minimum inhibitory concentration

Biofilm disruption

2.3. Conclusions

2.4. Methods

Colloidal properties

Bacterial strains and growth conditions


Biofilm disruption

Chapter 3. The Effect of Hofmeister Series Counterion Exchange on Minimum

Inhibitory Concentration of Single- and Double-Tailed Amphiphiles……….....……...30

3.1. Introduction



v


3.3. Methods

Synthesis of M-1,1

Synthesis of M-1,1,18

Counterion exchanges of M-1,1,18




Chapter 4. The Effect of Spacer Variation on the Minimum Inhibitory Concentration of

Bis- and Tetra-Cationic Double-Tailed Amphiphiles……........................……………..39

4.1. Introduction


4.3. Methods



Chapter 5. Conclusions and Future Studies…………………………………..………...48

5.1. Conclusions

5.2. Future Research

10-14-AO – Fluorescent compound

Activity against other E.S.K.A.P.E. pathogens

Cytotoxicity

Synergistic combinations and pre-treatment

References………………………………………………………………………………55

vi

List of Tables

Table 1. MIC values for tris-cationic amphiphiles of different tail lengths, and

benzalkonium chloride and tobramycin….......................................................................21

Table 2. CAC values of M-1,1,18 counterion exchange series…...................................32

Table 3. MIC values of M-1,1,18 counterion exchange series…....................................33

Table 4. MIC values of M-1,12,12 counterion exchange series…..................................35

Table 5. MIC values of bis-cationic ortho-, meta-, and para- orientation series........…41

Table 6. MIC values of tetra-cationic ortho-, meta-, and para- orientation series…......43

Table 7. MIC values of the M-(2,n)3 series….................................................................45

vii

List of Figures

Figure 1. SEM image of Staphylococcus aureus biofilm…………………………...……3

Figure 2. Type distribution of reported nosocomial infection in the United States…...…5

Figure 3. Timeline of novel drug discovery and development…………………......……7

Figure 4. Generic amphiphile structure………………………………….......…………...9

Figure 5. Structure of tris-cationic, double-tailed amphiphile series (M-E,n,n; M-

DMAP,n,n; M-IQ,n,n; and M-4PP,n,n)……………….........……………………….…..11

Figure 6. Structure of Hofmeister counterion exchange series amphiphiles

(M-1,1,18 and M-1,12,12)……………………………………………....………......…..12

Figure 7. Structure of bis-cationic ortho-, meta-, and para- orientation series

(oX-n,n; mX-n,n; and pX-n,n)………………………………..…………………....……13

Figure 8. Structure of tetra-cationic ortho-, meta- and para- orientation series

(oX-(2,n)2; mX-(2,n)2; and pX-(2,n)2)……………………………….....……………….14

Figure 9. Structure of the M-(2,n)3 series…………………...…………………………..15

Figure 10. Structure of the fluorescent amphiphile, 10-14-AO…………………..…….15

Figure 11. CAC values for the M-E,n,n series……………………………..…………...19

Figure 12. MIC values for the M-E,n,n; M-DMAP,n,n; M-IQ,n,n; and M-4PP,n,n series

against all bacterial species……………………………..………….………………...…23

Figure 13. P. aeruginosa biofilm disruption for M-E,12,12; M-DMAP,12,12; M-

IQ,12,12; M-4PP,12,12; tobramycin and benzalkonium chloride………….………..…25

Figure 14. Selected Hofmeister counterions ordered from strongly hydrated kosmotropes

to weakly hydrated chaotropes….....................................................................................30

Figure 15. MIC values for the oX-n,n; mX-n,n; and pX-n,n series against all bacterial

species…..........................................................................................................................42

Figure 16. MIC values for the oX-(2,n)2; mX-(2,n)2; and pX-(2,n)2 series against all

bacterial species…...........................................................................................................44

Figure 17. MIC values for the M-(2,n)3 series against all bacterial species…................45

Figure 18. Excitation and emission spectra for 10-14-AO..........................................…51

viii

Figure 19. Streptococcus agalactiae cells incubated with 50 µM 10-14-AO and imaged at

60X magnification using confocal microscopy…............................................................51

ix

Abstract

Antibiotic resistant infections are responsible for approximately 23,000 deaths every year

in the United States alone. The formation of bacterial biofilms makes resistant bacteria

difficult to eliminate completely using chemical treatment. Therefore, novel antimicrobial

compounds such as amphiphiles are essential to slow or stop the spread of resistant

bacteria. Several novel series of amphiphiles were synthesized, and discrete aspects of

their chemical structure were altered to investigate the relationship between structure and

antibacterial activity. Minimum inhibitory concentration (MIC) assays were used to

measure antibacterial activity against two Gram-negative and five Gram-positive

bacteria, and the most effective compounds were tested for biofilm disruption activity

against Pseudomonas aeruginosa using a Crystal Violet assay. For the tris-cationic M-

E,n,n; M-DMAP,n,n; M-IQ,n,n; and M-4PP,n,n series, twelve carbons per tail was the

ideal tail length for antibacterial activity, having MIC values as low as 4-8 µM against

Gram-negative species and 2-4 µM against Gram-positive species. The 12-carbon

derivatives also disrupted up to 70% of established P. aeruginosa biofilms at

concentrations comparable to tobramycin and benzalkonium chloride. Hofmeister

counterion substitution of one single-tailed and one double-tailed compound (M-1,1,18

and M-1,12,12, respectively) resulted in a notable 8-fold decrease in the MIC against P.

aeruginosa when the bromide counterion was replaced with chloride, and a 16-fold

decrease when the counterion was substituted with iodide. Finally, in order to determine

the effect of charge distribution in the head group, three bis-cationic (oX-n,n; mX-n,n;

and pX-n,n) and three tetra-cationic series (oX-(2,n)2; mX-(2,n)2; and pX-(2,n)2) of

double-tailed amphiphiles of varying tail lengths were synthesized and tested for their

x

MIC values. For all three series of bis-cationic amphiphiles, as well as the mX-(2,n)2 and

pX-(2,n)2 series, the 12-carbon derivatives had the lowest MIC values. However, the oX-

(2,n)2 series had an ideal tail length of 8 carbons per tail. This further understanding of

the relationship between structure and function of antimicrobial amphiphiles can be used

to create more effective disinfectants and antibiotics. Continuing research into novel

antibacterial compounds and biofilm disrupting agents is essential to combat the growing

problem of antibiotic resistance.

Chapter 1. Introduction

1.1 Antibiotic- and Disinfectant-Resistant Bacteria

Bacteria that are resistant to commonly used antibiotics and disinfectants cause over 2

million illnesses and 23,000 deaths each year in the United States alone (CDC, 2013).

Several strains of resistant bacteria have emerged since the development of antibiotics.

Staphylococcus aureus is well-known for its ability to resist killing by antibiotics, and

Methicillin-resistant strains of S. aureus (MRSA) have been implicated in community-

acquired infections after decades of being historically limited to a healthcare settings

(Chambers and DeLeo, 2009). Gram-negative bacteria can resist antibiotics by, among

other methods, altering the makeup and permeability of their outer membrane (Sawai et

al. 1979). Pseudomonas aeruginosa is a ubiquitous organism that has demonstrated

resistance to certain compounds partly due to its ability to enzymatically break down

these compounds after they enter the cell (Ali et al. 2015; Ma et al. 1998). Bacteria such

as Escherichia coli further resist centimide and other antibiotics by forming highly

resilient biofilms (Evans et al. 1990).

The development of resistance can be affected – and often accelerated – by human

activity. The overuse of antibiotics has been shown to lead to an increase in the incidence

of highly resistant organisms, particularly in hospitals (Dancer et al. 2009). Resistance to

some of the most effective antibiotics such as vancomycin and colistin, often used to treat

highly resistant infections, has also recently begun to emerge (Liu et al. 2016; Cardile et

al. 2015; Schrenk et al. 2015). Despite this increasing emergence of resistant infections,

antibiotics continue to be overprescribed in the United States and worldwide. The

consumption of antibiotic drugs increased 36% worldwide between 2000 and 2010,

2

including increases in the prescription of last-resort antibiotics such as polymixins and

carbapenems (van Boeckel et al. 2014). Additionally, as many as 60% of the antibiotics

prescribed in United States intensive care units may not be the optimal agent for

eradication of the infection that is being treated, and the choice of treatment may be

incorrect in as many as 50% of cases (CDC, 2013; Luyt et al. 2014). In fact, in many

cases of infection, the causative agent is not positively identified at all (Bartlett et al.

2013), increasing the risk of inappropriate therapy contributing to the development of

resistance.

The majority of antibiotics sold in the United States, however, are used in agriculture

rather than in medicine in order to increase the growth and yield of domesticated animals

(Bartlett et al. 2013). The first case of plasmid-mediated resistance to the last-resort

antibiotic, colistin, carried on the mcr-1 gene, was traced back to the antibiotic’s use in

Chinese pig farms as a growth promoting treatment (Liu et al. 2016). These resistant

bacteria can reach consumers through animal products such as meat (Bartlett et al. 2013),

but can also be dispersed through secretion in urine and stool into groundwater and

fertilizer (Bartlett et al. 2013; CDC 2013). The same gene has been found in multiple

pathogenic bacteria such as Klebsiella pneumoniae (Nicolet et al. 2016) and E. coli

(Poirel et al. 2016). The dispersal of resistance genes between different species and even

genera or families leaves some pathogens unable to be treated with any known antibiotic.

The widespread and inappropriate use of antibiotics has led to an increase the demand

for novel antimicrobial compounds, particularly in hospital settings where resistant

infections can spread rapidly within immunocompromised populations. These healthcare-

associated, or nosocomial infections, are often associated with invasive medical devices

3

such as ventilators and catheters, and can be extremely difficult to treat due to several

bacterial resistance genes and virulence factors that decrease susceptibility to common

antibacterial agents (CDC, 2014).

1.2 Biofilms and Transmission of Resistant Bacteria – Implications for Nosocomial

Infection

Many diverse virulence factors may contribute to the resistance of pathogenic

bacteria to eradication by standard antimicrobials, but one that is particularly significant

to the spread of infections within hospitals is the formation of biofilms. A biofilm is a

collection of bacterial cells that have adhered irreversibly to a surface within a

heterogeneous extracellular polysaccharide matrix (Figure 1), which may compose as

much as 90% of the organic material of the biofilm. Although the physical characteristics

of the biofilm itself are highly variable, bacteria living within a biofilm tend to have a

decreased metabolic rate, as well as

increased resistance to antibacterial

compounds and the host immune

response (Donlan, 2002; Pawar et al.

2015). In addition, bacteria within

biofilms have also been shown to

acquire new resistance at a faster

rate than planktonic cells (Gillings et

al. 2009).

Biofilm formation begins when bacteria attach at first reversibly, and later

irreversibly, to a surface. At this stage, characteristics of the surface itself have significant

Figure 1. Scanning electron microscope image of

Staphylococcus aureus biofilm. Bacteria are surrounded by a

web-like extracellular matrix which protects them from

desiccation and chemical stressors. (Photo from the CDC Public

Health Image Library)

4

influence on the rate of biofilm development, with rougher and more hydrophobic

materials generally fostering more rapid growth (Fletcher et al. 1979; Quirynen et al.

2000). Characteristics such as the presence of fimbriae (Rosenberg et al. 1982) or flagella

(Korber et al. 1989) also affect the initial attachment of bacteria to surfaces during

biofilm formation. Irreversibly attached cells form highly hydrated extracellular

polysaccharides, forming a heterogeneous matrix that includes filter-like channels which

transport nutrients throughout the biofilm (Lewandowski, 2000).

Established biofilms are difficult to remove using established chemical cleaning

protocols, often requiring extensive physical means of dispersal or harsh chemical

sterilization for complete removal (Vickery et al. 2004, 2012; O’Donnell et al. 2005).

Failure to completely remove these biofilms from hospital equipment such as catheters

can contribute to nosocomial infection by providing a route of infection to an

opportunistic pathogen. Cancer patients, for example, are particularly susceptible to

Staphylococcus infection through semi-permanent catheters such as peripherally inserted

central catheter (PICC) lines (Schrenk et al. 2015). Endoscopes serve as a major route of

nosocomial infection due to biofilm adherence; P. aeruginosa is often transmitted

between patients following incomplete cleaning of endoscopes between procedures.

However, although human error may contribute to a large portion of infection cases, even

strict compliance with cleaning protocol has been found to result in roughly a 2%

bacterial contamination rate in endoscopes deemed ready for use in patients (Vickery et

al. 2004). Therefore, current decontamination procedures are unable to completely

eradicate all biofilms or prevent transmission of infection between patients, even without

the addition of human error.

5

P. aeruginosa and S. aureus, two opportunistic pathogens with high affinity for

biofilm formation, are also associated with additional complications following surgery.

Bacteria with higher capacity for biofilm formation tend to be associated with longer

recovery time and more complications following endoscopic surgical procedures

(Bendouah et al. 2006). S. aureus has also been implicated in cases of septic arthritis and

graft failure following ligament reconstruction surgery, as well as surgical site and bone

infections (Hiller et al. 2015; Wright and Nair, 2010; Humphreys et al. 2016). It is

important to note, however, that S. aureus and even P. aeruginosa have been observed as

part of the normal skin and nasal flora in healthy individuals, suggesting that other factors

influence virulence in addition to high affinity for biofilm formation (Al-Shemari et al.

2007).

P. aeruginosa is a prominent pathogen in hospital settings, and is responsible for

most cases of hospital-associated pneumonia (Brewer et al. 1996, Richards et al. 1999).

Pneumonia, along with

surgical site infection, is one

of the leading causes of

morbidity and mortality

associated with nosocomial

infection (CDC, 2014; Figure

2). Additionally, P.

aeruginosa is highly resistant

to many disinfectant

compounds largely due to its

Pneumonia 157,500

GI infection 123,100

UTIs 93,300

Bloodstream

Infections 71,900

Surgical site infections 157,500

Other 118,500

Figure 2. Type distribution of reported nosocomial infections in the

United States (CDC, 2014).

6

production of efflux pumps and β-lactamases (Li et al. 1994). Contamination of hospital

equipment such as ventilators and endoscopes can increase hospital-associated morbidity

and mortality by fostering the spread of bacteria between patients, causing infections

such as ventilator-associated pneumonia (VAP; Brewer et al. 1996).

Another prominent cause of nosocomial infection is Escherichia coli, which,

along with P. aeruginosa and Enterococcus species such as E. faecalis and E. faecium, is

one of the most common causes of bloodstream infections (Peleg, 2010, Olawale et al.

2011). E. coli is also the most common Gram-negative species implicated in urinary tract

infections associated with urethral catheters (Hidron et al. 2008), and uropathogenic

strains possess a variety of virulence factors aiding in persistence and infection, including

several associated with biofilm formation (Jacobsen et al. 2008).

Streptococcus agalactiae, also commonly known as Group B streptococcus, is

associated with severe meningitis in newborns, with onset possible as early as within one

week of birth, or as late as 3 months of age (Guiltbert et al. 2010). Although nosocomial

transmission has been mostly attributed to limited contamination by medical workers,

rare outbreaks have occurred where no common health care workers could be identified,

and were linked to environmental reservoirs within the neonatal intensive care unit

(MacFarguhar et al. 2010).

1.3 Antibacterial Development

Although the demand for antibiotic drugs is high, mof the world’s most prominent

scientific funding agencies allocate very little grant money to antibiotic discovery and

development (Speck, 2013). Funding institutions in the United Kingdom, for example,

7

allocate only approximately 3.9% of total funds to antimicrobial resistance research

(Head et al. 2013). In the United States, the NIH allocated slightly over 1% of its budget

to antimicrobial resistance research, less than a tenth of what was allocated to

biotechnology and a seventh of what was allocated to research into aging (NIH, 2016).

Additionally, the development of antibiotics with novel targets and mechanisms

has slowed considerably. Two truly novel antibiotic classes with new microbial targets –

daptomycin and linezolid – have been introduced in the past decade (IDSA, 2010; 2011).

This is due in large part financial limitations, as research into antibiotics – which are

relatively cheap and most often used short-term – is not viewed as economically

profitable for pharmaceutical companies when compared to research into other drugs for

chronic health conditions (Piddock, 2012; Bartlett et al. 2013). Additionally, regulatory

barriers prevent the rapid delivery of novel drugs to market. Implementation of new drugs

can take up to 2 decades from drug discovery to use in the general public (Figure 3). This

is in part because human trials comparing the efficacy of new compounds to a placebo

are often considered unethical, and so studies must rely on comparisons to existing

antibiotics, necessitating large sample sizes and therefore high cost (Wright and Nair,

2014).

Figure 3. Timeline of novel drug discovery and development. Novel drug development can take up to

2 decades to complete, and involve proving safety and efficacy in humans before reaching the

majority of patients in need.

8

One promising area of study is in the activity of antimicrobial peptides (AMPs),

which are short, positively charged, amphiphilic amino acid chains produced by a wide

array of organisms as part of the innate immune response (Jenssen et al. 2006). These

peptides have broad-spectrum antibacterial activity, affecting the bacterial cytoplasmic

membrane and causing depolarization and cell lysis (Hancock and Rozek, 2002). The

bactericidal activity of these peptides relies on both the charge of the molecule and

hydrophobic interactions for insertion into the membrane (Dathe and Wieprecht, 1999).

Many bacteria remain sensitive to killing by AMPs despite frequent exposure to these

peptides, likely due to the fact that many mutations conferring resistance to AMPs require

a high metabolic cost (Nizet, 2006). Despite the broad spectrum of efficacy of AMPs, the

widespread implementation of AMPs in a clinical setting is limited by the high cost of

large-scale AMP production (Marr et al. 2006; Findlay et al. 2010). The development of

wholly synthetic AMPs provides a potential way to circumvent the high cost of AMP

production, and amphiphilic AMP mimics lacking natural amino acids still have broad-

spectrum antibacterial activity against both Gram-negative and Gram-positive organisms

(Mowery et al. 2007; Scott et al. 2008). Quaternary ammonium compounds are simple

amphiphilic mimics of AMPs that are easier to produce, but still retain broad-spectrum

antibacterial and anti-biofilm activity (Jennings et al. 2014).

1.4 Amphiphiles

Amphiphiles are compounds that possess both a hydrophobic and a hydrophilic

portion on a single molecule (LaDow et al. 2011; Figure 4). The hydrophilic portion of

the molecule is charged, and cationic (positively-charged) amphiphiles have been the

focus of study for purposes of biofilm disruption due to the potential for interference with

9

the negatively charged extracellular

matrix produced by bacteria (Jennings

et al. 2014). Such interactions would

likely be species-independent and

broad-spectrum, allowing for a wide

range of uses. Additionally the sheer

number of possible structures available would promote a higher diversity of effective

compounds to protect against the development of resistance resulting from the overuse of

any single compound.

The antibacterial activity of these types of compounds was first noted in 1935,

and they are still commonly used as disinfecting agents and detergents (Gerhard, 1935).

Amphiphiles have a wide array of possible structures, and some of the most promising

are quaternary ammonium compounds (Forman et al, 2016). Ample research exists

investigating the relationship between amphiphile structure and antibacterial activity

(LaDow et al. 2011; Marafino et al. 2015; Song et al. 2011; Palermo et al. 2009).

Previous studies have provided information about the effect of cationic head group

substitution and hydrophobic hydrocarbon chain length on the minimum inhibitory

concentration (MIC) of certain compounds on bacteria including P. aeruginosa and S.

aureus. Structural changes such as hydrocarbon tail length variation, tail symmetry, and

changes in the charge and structure of the hydrophilic region have been studied for their

effects on antibacterial activity (Marafino et al. 2015; LaDow et al. 2011; Paniak et al.

2014). In particular, the size of the hydrophobic region appear to be the strongest

indicator of membrane permeability and antibacterial activity, with changes in the charge

Hydrophobic Hydrophilic

Figure 4. Generic amphiphile structure, including the

hydrophilic head group (blue) and the hydrophobic

hydrocarbon tail group (red).

10

and size of the hydrophilic region having less of an effect on the effectiveness of QACs

(Paniak et al. 2014).

Cationic amphiphiles interact strongly with the negatively charged phospholipids

that make up 20-25% of the membrane (Glukov et al. 2005). The primary mechanism of

action of these amphiphiles is disruption of the bacterial cell membrane, leading to

disruption of transport proteins and proton motive force, cell lysis and death (Hoque et al.

2012; Thiyagarajan et al. 2014; Ladow et al. 2011). As the lipid membrane is ubiquitous

and energetically costly to repair, QACs offer broad-spectrum efficacy and little potential

for the development of total resistance (Vudumula et al. 2012). This is particularly

promising for the treatment of Gram-negative pathogens, whose additional outer

membrane can decrease their susceptibility to several antibiotics unable to pass through

the additional barrier (Bola et al. 2011).

The biofilm disruption capabilities of amphiphilic compounds have also been the

focus of a large amount of research. Previous work has suggested that amphiphilic

polymers containing amide side chains are not only effective bactericidal agents against

antibiotic-resistant strains of bacteria such as MRSA and vancomycin-resistant

Enterococcus faecium (VRE), but were also more effective at disrupting both non-

resistant and multidrug-resistant Acinetobacter baumanii biofilms than established

antibiotics such as erythromycin, tobramycin, and colistin (Uppu et al. 2016).

Amphiphiles with different structures, including QACs, have also exhibited biofilm-

disruption activity. The likely mechanism of action of biofilm disruption by these

compounds involves biofilm dispersal by electrostatic interactions which allow the

11

compounds to lyse the planktonic cells released from the biofilm, leading to biofilm

disruption due to the death of bacterial cells (Jennings et al. 2014).

1.5 Amphiphiles Used in the Current Study

Head group substitution and hydrocarbon tail length variation

Four series of double tailed tris-

cationic amphiphiles were

synthesized by the Caran lab (James

Madison University). For each

series, the amphiphiles consisted of a

mesitylene core (M) with three

attached positively charged head

groups. Two of these head groups

were dimethylalkylammonium

groups with attached hydrocarbon

tails. The third head group was either

dimethylethanolammonium for the

M-E,n,n series,

dimethylaminopyridinium for the M-

DMAP,n,n series, isoquinolinium for

the M-IQ,n,n series, and 4-propanol pyridinium for the M-4PP,n,n series. The

hydrocarbon tails were linear and symmetrical, and consisted of 8-16 carbons each for the

M-E,n,n series

n = 8,10,12,14,16

M-DMAP,n,n series

n = 10,12,14,16

M-IQ,n,n series

n = 10,12

M-4PP,n,n series

n = 10,12,14

Figure 5. Structure of amphiphiles. M represents the Mesitylene

core; n represents the number of carbons in each symmetrical

hydrocarbon tail. E = dimethylethanolammonium, DMAP =

dimethylaminopryidinium, IQ = isoquinolinium, and 4PP = 4-

propanol pyridinium.

12

M-E,n,n series, 10-16 carbons each for the M-DMAP,n,n series, 10-12 carbons each for

the M-IQ series, and 10-14 carbons each for the M-4PP,n,n seres (Figure 5).

Counterion exchange

Two tris-cationic amphiphiles were synthesized by the Caran lab in order to determine

the effects of counterion substitution on antibacterial activity. One single-tailed

compound (M-1,1,18) and one double-tailed compound (M-1,12,12), which have both

been previously studied (Marafino et al 2015), were used. Both compounds consisted of

three positively charged dimethylalkylammonium residues attached to a mesitylene core,

with a linear 18-carbon chain attached to one of these groups in M-1,1,18, and a linear

12-carbon chain attached to each of two of the groups in M-1,12,12 (Figure 6). The

standard bromide counterions

were substituted with

carbonate, acetate, nitrate,

chloride, or iodide for M-

1,1,18, and substituted with

acetate, chloride, or iodide for

M-1,12,12. The structure of the

amphiphiles themselves was

not changed with these

substitutions.

Spacer variation and hydrocarbon tail length variation

M-1,1,n series +

Hofmeister

counterion

n = 18

M-1,n,n series +

Hofmeister

counterion

n = 12 Figure 6. Structure of amphiphiles. M represents the Mesitylene core;

n represents the number of carbons in each symmetrical hydrocarbon

tail. X- represents the Hofmeister counterion in solution with the

amphiphile (bromide, carbonate, acetate, nitrate, chloride, or iodide).

13

Three series of bis-cationic and three series of tetra-cationic double-tailed amphiphiles

were synthesized by the Caran lab in order to determine the effects of varied placement

of the headgroups and tails on the central mesitylene ring. The bis-cationic oX-, mX-, and

pX-n,n series consisted of two dimethylalkylammonium residues attached to the central

ring in either the ortho (oX-n,n series), meta (mX-n,n series) or para orientation (pX,n,n

series). One linear hydrocarbon chain was attached to each of the two

dimethylalkylammonium groups, and contained 8-14 carbons for the oX-n,n series, 8-12

carbons for the mX-n,n series, and 8-16 carbons for the pX-n,n series (Figure 7).

The tetra-cationic amphiphile series were structured similarly, however two

dimethylalkylammonium head groups were attached to each of the same positions on the

central ring, connected by a 2-carbon linker. The linear hydrocarbon tail connected to the

head group in each of the tetra-cationic series contained 8-12 carbons for the oX-, mX-,

and pX-(2,n)2 series (Figure 8).

oX-n,n series

n = 8,10,12,14

mX-n,n series

n = 8,10,12

pX-n,n series

n = 8,10,12,14,16

Figure 7. Structure of bis-cationic amphiphiles. n represents the number of

carbons in each symmetrical hydrocarbon tail. oX = ortho-orientation; mX

= meta-orientation; pX = para-orientation.

14

Additional compounds

One series of amphiphiles was synthesized by the Caran lab including 6 total

dimethylalkylammonium head groups and three linear hydrocarbon tails of 8, 10, or 12

carbons in length (Figure 9). The hexa-cationic M-(2,n)3 series was tested for its

antibacterial activity with tail length variation.

The final compound synthesized by the Caran lab was 10-14-AO, a mono-cationic

fluorescent compound derived from acridine orange that included a single 14-carbon tail

(Figure 10).

Figure 8. Structure of tetra-cationic amphiphiles. n represents the number of carbons in

each symmetrical hydrocarbon tail. oX = ortho-orientation; mX = meta-orientation; pX =

para-orientation.

oX-(2,n)2 series

n = 10,12,14

mX-(2,n)2 series

n = 10,12,14

pX-(2,n)2 series

n = 10,12,14

15

Figure 9. Structure of the 6-headed,

triple-tailed M-(2,n)3 series. n

represents the number of carbons per

tail (8,10,12).

Figure 10. Structure of the fluorescent

acridine orange derivative, 10-14-AO.

16

Chapter 2: The effect of tail length variation and head group substitution on

minimum inhibitory concentration and biofilm disruption.

2.1 Introduction.

Bacteria that are resistant to commonly used antibiotics and disinfectants cause

over 2 million illnesses and 23,000 deaths each year in the United States alone (CDC,

2013). The development of resistance can be affected – and often accelerated – by human

activity. The overuse of frequently used antibiotics has led to an increase in the incidence

of highly resistant organisms, particularly in hospitals (Dancer et al. 2009). Resistance to

powerful antibiotics such as vancomycin and colistin, often used to treat highly resistant

infections, has also recently emerged (Liu et al. 2016; Cardile et al. 2015; Schrenk et al.

2015).

The accelerated development of resistance in ubiquitous organisms has

contributed to the increased prevalence of nosocomial infections, which spread rapidly

within immunocompromised populations often found in hospitals and nursing homes

(Johnston and Bryce, 2009). Nosocomial infections such as pneumonia, bloodstream

infections, and urinary tract infections are responsible for approximately 75,000 patient

deaths every year (Magill et al. 2014) and place a significant economic burden on the

United States healthcare system (CDC, 2013). Gram-negative bacteria can further resist

antibiotics by altering the makeup and permeability of their outer membrane in addition

to having other adaptations for resistance (Sawai et al. 1979). Pseudomonas aeruginosa is

a ubiquitous organism that has demonstrated resistance to certain compounds partly due

to its ability to enzymatically break down these compounds after they enter the cell (Ali

et al. 2015; Ma et al. 1998). Bacteria such as Escherichia coli resist centimide and other

17

antibiotics by forming biofilms (Evans et al. 1990). The development of novel

antibacterial compounds is essential to curb the spread of resistant infections between

patients and to reduce hospital-associated morbidity and mortality.

Amphiphiles are a diverse class of compounds with well-documented

antibacterial effects (Marafino et al. 2015; Ladow et al. 2011; Goswami et al. 2015;

Thiyagarajan et al. 2014; Song et al. 2011). The antibacterial activity of these compounds

was first noted in 1935, and they are still commonly used as disinfecting agents and

detergents (Gerhard, 1935). Amphiphiles have a wide array of possible structures, and

some of the most promising are quaternary ammonium compounds, which are molecules

containing positively charged nitrogen residues (Forman et al. 2016). In addition to the

charged hydrophilic head group, amphiphiles also contain a hydrophobic portion, usually

consisting of one or more hydrocarbon tails. Structural changes such as hydrocarbon tail

length variation, symmetry, and changes in the charge and structure of the hydrophilic

region have been studied for their effects on antibacterial activity (Marafino et al. 2015;

Ladow et al. 2011; Paniak et al. 2014). In particular, the size of the hydrophobic region

appear to be the strongest indicator of membrane penetration and antibacterial activity,

with changes in the charge and size of the hydrophilic region having less of an effect on

the effectiveness of QACs (Paniak et al. 2014). Increased understanding of the effects of

amphiphile structure on bactericidal activity is essential for the development of more

effective antibacterial compounds.

Four novel series of amphiphiles were synthesized, and the antibacterial effect of

changes in head group structure and tail length was determined. All four series consisted

of three cationic head groups connected to a mesitylene core. For each series, two of the

18

head groups were dimethylalkylammonium groups, each connected to a linear

hydrocarbon tail. The hydrocarbon tails were symmetrical and ranged in length from 8-16

carbons for the M-E series, 10-16 carbons for the M-DMAP series, 10-12 carbons for the

M-IQ series, and 10-14 carbons for the M-4PP series. The third head groups were an

dimethylethanolammonium (M-E series), dimethylaminopyridinium (M-DMAP series),

isoquinolinium (M-IQ series), or 4-propanol pyridinium (M-4PP series; Figure 5).

2.2 Results and Discussion.

Critical Aggregation Concentration

The critical aggregation concentration (CAC) refers to the concentration at which

the molecules aggregate in solution. Below the CAC, the amphiphile tends to behave as a

monomer, while above the CAC, the amphiphile behaves as a micelle. The CAC was

determined for the 10-16 carbon chain length derivatives of the M-E series. Consistent

with previous studies of amphiphiles with similar structures (Marafino et al. 2015), the

CAC decreases with increasing tail lengths, suggesting that a longer pair of hydrophobic

hydrocarbon tails encourages aggregation into micelles at lower concentrations (Figure

11).

19

Information about the CAC can also be used to determine more about the

mechanism of action of these compounds, namely whether they act as monomers to exert

a bacteriocidal effect, or if they act more similarly to detergents (as micelles). Because

the MIC of all of the M-E series derivatives tested was below the CAC (Table 1, Table

2), it is likely that these compounds are acting as monomers at bactericidal

concentrations, and do not have a mechanism of action similar to detergents. This

supports previous research on similar compounds, where tris-cationic double-tailed

amphiphiles with trimethylammonium or pyridinium head groups also had MICs that fell

far below their respective CAC values (Marafino et al. 2015).

Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) was determined for two Gram-

negative bacterial species (P. aeruginosa and E. coli) and five Gram-positive species (S.

aureus, E. faecalis, S. agalactiae, B. subtilis, and B. anthracis; Table 1, Figure 12). For

-1.5

-1.0

-0.5

0.0

0.5

1.0

18 20 22 24 26 28 30 32 34

log

[CA

C(m

mo

l/L

)]

Total Carbons in Hydrophobic Tails

Figure 11. Critical aggregation concentration for the 10, 12, 14, and 16 carbon

tail length derivatives of the M-E,n,n series.

20

all series (with only one exception) the MIC was lowest at a tail length of 12 carbons per

tail. Above or below this optimal tail length, the MIC increased. This trend was observed

for both Gram-negative and all Gram-positive bacteria tested. A similar trend was

observed for all head group substitutions, with 12 carbons being the ideal tail length for

structures with an dimethylethanolammonium, dimethylaminopyridinium,

isoquinolinium, or 4-propanol pyridinium head group (Figure 12).

The 12-carbon compound M-E,12,12 had MIC values ranging from 2-4 μM for

Gram-positive bacteria, and 4-8 μM for Gram-negative bacteria. M-DMAP,12,12 had an

MIC of 8 μM for both Gram-negative species, and MIC values ranging from 2-8 μM for

Gram-positive species. MIC values for M-4PP,12,12 ranged from 2-4 μM for all bacteria

tested, and those for M-IQ,12,12 ranged from 4-8 μM. Both M-4PP,12,12 and M-

IQ,12,12 were able to effectively inhibit the growth of P. aeruginosa at concentrations as

low as 4 μM, a concentration comparable to the antibiotic tobramycin, an aminoglycoside

widely used to treat chronic P. aeruginosa infection (Shawar et al. 1999).

The trends observed for these compounds are consistent with previous research on

structurally similar amphiphiles. Derivatives with two 12-carbon chains in the tail region

have previously been reported to have lower MIC values than those with longer or shorter

tails (Marafino et al. 2015).

21

Table 1. MIC values for all compounds, benzalkonium chloride (BZK), and tobramycin. Values were

confirmed with 2-3 independent experiments. X = head group (E = dimethylethanolammonium; DMAP =

dimethylaminopyridinium; IQ = isoquinolinium; 4PP = 4-propanol pyridinium) and n = the number of

carbons per tail. G+ = Gram-positive and G- = Gram-negative.

Replacing the hydrophilic dimethylethanolammonium residue with

dimethylaminopyridinium, isoquinolinium, or 4-propanol pyridinium did not

significantly change the trend in MIC. Other structurally similar 12-carbon derivatives

with different head groups (trimethylammonium and pyridiunium) studied previously

have had comparable antibacterial activity, further illustrating the effect of tail length on

the efficacy of these double-tailed amphiphiles (Marafino et al. 2015). Taken together,

these data suggest that substitution with equally charged head groups of comparable size

does not have a considerable effect on antibacterial activity, and that tail length has a

much larger effect on the ability of these compounds to inhibit bacterial growth.

Compound

(M-X,n,n)

Minimum Inhibitory Concentration (μM) P. aeruginosa

(G-)

E. coli

(G-)

E. faecalis

(G+)

S. aureus

(G+)

B. subtilis

(G+)

S. agalactiae

(G+)

B. anthracis

(G+)

M-E,8,8 >250 >250 63 250 250 125 63 M-E,10,10 250 125 4 125 16 4 4 M-E,12,12 8 4 2 4 4 2 2 M-E,14,14 125 8 4 8 8 4 8 M-E,16,16 125 63 63 63 >250 >250 >250

M-DMAP,10,10 63 63 8 63 16 8 4 M-DMAP,12,12 8 8 2 4 8 2 2 M-DMAP,14,14 16 16 4 4 8 4 4 M-DMAP,16,16 250 31 8 16 16 8 8 M-4PP,10,10 8 4 4 4 2 2 2 M-4PP,12,12 4 2 4 2 2 4 2 M-4PP,14,14 31 16 4 16 4 4 4 M-IQ,10,10 125 8 4 8 8 4 8 M-IQ,12,12 4 8 4 4 4 4 4

BZK 8 31 8 16 2

Tobramycin 4

22

Structurally, these compounds are similar to benzalkonium chloride (BZK), a

synthetic quaternary ammonium biocide with broad-spectrum antibacterial activity

(Carson et al. 2008; Fazlara and Ekhtelat, 2012). Interestingly, all of the 12-carbon

derivatives, as well as some 10- and 14-carbon derivatives tested have greater

antibacterial activity than BZK against Gram-negative and Gram-positive species

(Fazlara and Ekhtelat, 2012).

All four series of tris-cationic, double-tailed amphiphiles presented in this study

follow the previously illustrated trend suggesting a strong relationship between

hydrocarbon tail length and antibacterial activity. Amphiphiles with 12 carbons in each

symmetrical tail were the most effective at inhibiting all species of bacteria tested,

supporting the findings of previous work (Marafino et al. 2015).

23

Tail length (n for M-DMAP,n,n)

Figure 12. The effect of tail length on antibacterial activity of the M-E,n,n; M-DMAP,n,n; M-IQ,n,n;,

and M-4PP,n,n series. G- = Gram-negative and G+ = Gram-positive. Bacterial strain names are

indicated.

Tail length

(n)

0

0.5

1

1.5

2

2.5

3

8 13

log [

MIC

(µ

M)]

P. aeruginosa (G-)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

E. coli (G-)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

S. aureus (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

log [

MIC

(µ

M)]

B. subtilis (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

log [

MIC

(µ

M)]

Tail length (n)

S. agalactiae (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

B. anthracis (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

log [

MIC

(µ

M)]

E. faecalis (G+)

24

Biofilm disruption.

Established P. aeruginosa biofilms were exposed to two-fold dilutions M-

E,12,12; M-DMAP,12,12; M-IQ,12,12; and M-4PP,12,12 . The percentage of biofilm

disruption increased in a concentration-dependent manner from 2-63 µM for all

amphiphiles tested, but at concentrations above 63 µM, increasing the compound

concentration did not increase biofilm disruption. The amphiphiles tested disrupted a

maximum of approximately 65% of established biofilm, with the exception of M-

4PP,12,12, which had a maximum disruption of approximately 40% (Figure 13).

This disruption activity was compared to that of BZK and the aminoglycoside

tobramycin. At similar concentrations, tobramycin disrupted approximately 66% of

established biofilm, while BZK disrupted approximately 35-40% (Figure 13).

Bacterial biofilms are commonly associated with greatly increased resistance to

biocides due to the formation of a protective extracellular matrix and reduced metabolic

rate of biofilm-associated cells (van der Veen and Abee, 2010; Costerton et al. 1999).

Due to this increased resistance compared to planktonic cells, it is unsurprising that the

concentration of biocide necessary to attain maximum biofilm disruption is several

dilutions higher than the MIC, which was determined against planktonic cells.

25

Figure 13. P. aeruginosa biofilm disruption by (A) M-E,12,12; (B) M-DMAP,12,12;

(C) M-4PP,12,12; (D) M-IQ,12,12; (E) tobramycin and (F) BZK. Error bars represent

standard deviation of three independent trials.

0

20

40

60

80

0 100 200 300

% b

iofi

lm d

isru

pti

on

0

20

40

60

80

0 100 200 300

0

20

40

60

80

0 100 200 3000

20

40

60

80

0 100 200 300

% b

iofi

lm d

isru

pti

on

0

20

40

60

80

0 100 200 300

% b

iofi

lm d

isru

pti

on

Compound concentration (μM)

0

20

40

60

80

0 100 200 300

Compound concentration (μM)

A B

C D

E F

26

2.3 Conclusions.

These data further support the relationship between solubility and antibacterial

activity, suggesting that there is an ideal level of solubility that may maximize

bactericidal efficiency. Of particular note are the MIC values of M-E,12,12; M-

DMAP,12,12; M-4PP,12,12; and M-IQ,12,12. These compounds are effective at

inhibiting the growth of P. aeruginosa at concentrations as low as 4-8µM (Table 1). P.

aeruginosa is a prominent pathogen in hospital settings, and is responsible for most cases

of hospital-associated pneumonia (Brewer et al. 1996; Richards et al. 1999).

Additionally, P. aeruginosa is highly resistant to many disinfectant compounds due to its

semipermeable outer membrane and the production of efflux pumps and β-lactamases (Li

et al. 1994). Contamination of hospital equipment such as ventilators and endoscopes can

increase hospital-associated morbidity and mortality by fostering the spread of bacteria

between patients, causing infections such as ventilator-associated pneumonia (VAP;

Brewer et al. 1996). Four compounds mentioned effectively inhibit the growth of P.

aeruginosa at concentrations comparable to the MIC of tobramycin, an antibiotic used to

treat P. aeruginosa infection in cystic fibrosis patient (Oliver et al. 2000; Singh et al.

2000; Vidya et al. 2016). Given this, as well as the fact that the MIC values of the most

effective compounds were lower than those of the disinfectant, benzalkonium chloride,

these compounds may prove useful in a hospital setting as novel disinfectants to prevent

the spread of nosocomial infection due to P. aeruginosa. Because biofilm contributes to

the spread and pathogenesis of P. aeruginosa, he potential for synergistic interaction

between the compounds presented in this study and biofilm matrix-degrading enzymes

such as dispersin B warrants future study (Kaplan, 2010).

27

Further research is needed to understand the mechanism of action of these

compounds against bacterial biofilms, as it is uncertain whether they are actively

interacting with the extracellular biofilm matrix or merely penetrating the matrix to target

the bacterial cells embedded in the biofilm. The solubility and hydrophobicity of the EPS

is highly variable, and it likely possesses localized hydrophilic and hydrophobic regions,

making it unclear to what extent these amphiphiles are able to interact with the matrix

effectively (Sutherland, 2001).

Given the increasing incidence of hospital acquired infection, particularly by

highly resistant strains of bacteria, the need for novel antibacterial compounds is

paramount, and understanding structure-function relationships of bactericidal molecules

is necessary for the development of more effective disinfectants. Several compounds

presented in this study show potential as novel disinfectants, either alone or in synergistic

combination with other disinfecting agents or antibiotics. It is likely that these

compounds behave similarly to other amphiphiles against planktonic cells by targeting

the bacterial membrane (Goswami et al. 2015; Thiyagarajan et al. 2014; Song et al.

2011). Gram-negative bacteria are particularly difficult to eradicate due to the presence of

an outer membrane serving as a selectively permeable barrier to chemical stressors (Bolla

et al. 2011). Membrane-permeabilizing agents, therefore, may lead to enhanced

susceptibility to antibiotics not normally able to bypass this barrier. The results presented

in this work provide information on the relationship between amphiphile structure and

function, and may lead to the development of novel disinfecting agents that could

decrease hospital-associated morbidity and mortality as a result of bacterial transmission.

28

2.4 Methods

Colloidal properties

A conductivity probe was used to measure the critical aggregate concentration of

the amphiphile. The solution was heated to 37°C. The probe was mixed in the solution

and the measurements were collected. Ten percent of the solution was pipetted out and

replaced with nanopure water. The solution was mixed and the conductivity measurement

recorded. These steps were repeated for the entirety of the experiment.

Bacterial strains and growth conditions.

The two Gram-negative bacterial strains used were Escherichia coli ATCC®

25922™

and Pseudomonas aeruginosa ATCC®

27853™

, and the five Gram-positive

bacterial strains used in this study were Staphylococcus aureus subsp. aureus ATCC®

29213™

, Enterococcus faecalis ATCC® 29212

™, Bacillus subtilis, B. anthracis Sterne,

and Streptococcus agalactiae J48 (Seifert et al. 2006). All strains were grown in 1x

Mueller-Hinton Broth at 37° C for 12–24 h. For the MIC studies, bacterial suspensions

were prepared by diluting overnight cultures to 5x106 CFU/mL in 2X Mueller-Hinton

Broth, so that when amphiphile solutions were added the final broth strength was 1X.

Minimum inhibitory concentration.

The methods used to determine the MIC were performed as previously described

and followed standards of the Clinical and Laboratory Standards Institute (Ladow et al.

2011; CLSI, 2012). Briefly, compounds were serially diluted in sterile deionized water

and 100µL of each dilution were added to the wells of a 96-well flat-bottomed microtiter

plate in triplicate. After adding 100µL of the bacterial cell suspension (5x106 CFU/mL),

29

the plates were incubated at 37°C for 72 h. The MIC of the compound was defined as the

minimum concentration that resulted in visible inhibition of bacterial growth.

Biofilm disruption

Biofilm disruption was determined as previously described (O’Toole, 2011).

Briefly, 100µL of 5x106 CFU/mL P. aeruginosa suspended in Luria Broth (LB) were

added to a 96-well flat-bottomed microtiter plate. Plates were incubated at 37° C for 24 h

to allow the biofilm to form. Sterile dI H2O served as a negative control for biofilm

disruption, and 100µL of each dilution of the compounds were added in triplicate to the

remaining wells. Plates were incubated at 37° C for another 24 h, after which the plates

were emptied and rinsed with sterile dI water and allowed to dry. Wells were stained with

100µL crystal violet, rinsed with gently running water, and allowed to dry. Finally,

100µL of 95% ethanol was added to each well for 1 h. The ethanol-crystal violet solution

was transferred to a fresh 96-well microtiter plate, and the absorbance value at 570 nm

was determined.

30

Chapter 3: The effect of Hoffmeister series counterion exchange on minimum

inhibitory concentration of single- and double-tailed amphiphiles.

3.1 Introduction.

Counterions accompany charged molecules in solution in order to maintain

electric neutrality. These counterions can have an effect on several aspects of the

interaction between molecules in solution, most notably on micellization of surfactants

(Naskar et al. 2012) . The self-assembly of amphiphiles in solution is a

thermodynamically driven process that can be affected by differences in the hydrocarbon

tail length and the charge and size of the hydrophilic head group. Ions in solution interact

with the head group, and thereby have an effect on the assembly of monomers into

micelles (Rosholm et al. 2010). These interactions are of particular interest to medicinal

chemistry and drug discovery, since variation of the counterion paired with antibacterial

compounds may affect antibacterial activity.

Of particular interest are counterions from the well-studied Hofmeister series

(Figure 14). The Hofmeister series was originally observed by Franz Hofmeister in 1888,

and was reported to be a series of salts ordered according to their ability to precipitate

proteins out of solution

(Hofmeister, 1888; Kunz

et al. 2004; Lo Nostro et

al. 2012). More recently,

ions in the Hofmeister

series have been

correlated with several Figure 14. Selected counterions from the Hofmeister used in this study,

presented in order from strongly hydrated kosmotropes (left) to weakly

hydrated chaotropes (right).

31

chemical properties of amphiphiles in solution (Rosholm et al. 2010), as well as having a

documented effect on the growth of both Gram-positive and negative bacteria including

S. aureus and P. aeruginosa (Lo Nostro et al. 2005).

Interestingly however, relatively little research has been done on the effects of

Hofmeister series counterion substitution on the antibacterial activity of amphiphiles and

similar membrane-active compounds. Therefore, the relationship between the known

properties of Hofmeister series counterion exchange and amphiphile activity remains

largely unknown.

It was hypothesized that changes in micelle formation and aggregation due to

counterion substitution could lead to a change in MIC correlated with the chemical

changes associated with the Hofmeister series. Therefore, one single-tailed amphiphile

(M-1,1,18) and one double-tailed amphiphile (M-1,12,12) were selected for counterion

substitution (Figure 6). These amphiphiles were selected due to their low MIC values

compared to other compounds with different hydrocarbon chain lengths in their

respective series (Marafino et al. 2015). Counterions from the Hofmeister series were

substituted for bromide (3Br-) without altering the structure of either amphiphile in order

to determine whether counterion exchange on its own was sufficient to cause changes in

MIC.



CAC, predictably, increased with increasing chaotropicity for single-tailed

derivatives paired with different counterions in the Hofmeister series. When paired in

solution with iodide, M-1,1,18 formed micelles at a minimum concentration

32

approximately ten times lower than when paired with acetate, a more weakly hydrated

kosmotrope (CAC table).

Nitrogen-based cations such as those found in these amphiphiles have been categorized

previously as largely chaotropic. Chaotropes interact most strongly with other chaotropes

in solution, which may explain the decrease in CAC when M-1,1,18 was paired with

other more chaotropic Hofmeister ions in solution (Abezgauz et al. 2009).


Previously, all compounds tested in this study had bromide as the counterion;

however, the effects of counterion substitution with other Hofmeister series anions had

not been tested. Six Hofmeister anions (bromide, carbonate, acetate, nitrate, chloride, and

iodide) were selected for counterion exchange with the single-tailed compound, M-1,1,18

(Table 3). Substitution with carbonate, acetate, or nitrate resulted in slightly increased

MIC values, particularly against Gram-positive bacteria, suggesting that these

counterions may cause decreased bacteriocidal efficacy for this compound. Interestingly,

while acetate and carbonate are both more kosmotrophic than bromide in the Hofmeister

series, nitrate is more chaotrophic. It is likely that the change in antibacterial activity can

Counterion

(M-1,1,18 + X)

CAC

(mM)

CO32-

N/A

C2H3O2-

10.10

Cl-

5.96

Br-

3.13

NO3-

2.07

I-

1.03

Table 2. CAC values of M-1,1,18 paired with

six selected Hofmeister anions.

33

therefore not be attributed to the chemical attributes of the Hofmeister series alone when

these ions are paired with these antibacterial compounds.

Chloride and iodide were also investigated as possible counterions for these

amphiphiles. When paired with both of these anions, M-1,1,18 had much lower MIC

values compared to M-1,1,18 paired with the other anions studied. Most notably,

substitution with chloride and iodide resulted in a sixteen-fold and eight-fold decrease in

MIC compared to bromide for P. aeruginosa, respectively, and an eight-fold decrease in

MIC for S. aureus. While M-1,1,18 paired with bromide had an MIC of 125 µM against

P. aeruginosa, the same compound was able to inhibit the growth of P. aeruginosa at 8

µM when paired with chloride and at 16 µM when paired with iodide. Counterion

exchanged M-1,1,18 paired with chloride and iodide were, with very few exceptions,

more effective than M-1,1,18 paired with carbonate, acetate, or nitrate (Table 3).

Despite notable changes in MIC with counterion exchange, no clear trend

between Hofmeister series order and antibacterial activity was observed. The observed

increase in CAC with increasing chaotropicity was not an accurate predictor of

Counterion

(M-1,1,18 + X)


(G-)

E. coli

(G-)

E. faecalis

(G+)

S. aureus

(G+)

B. subtilis

(G+)

S. agalactiae

(G+)

B. anthracis

(G+)

3 Br-

125 16 4 16 4 2 4

1.5 CO32-

250 31 31 31 16 4 31

3 C2H3O2-

250 63 16 63 31 4 31

3 NO3-

250 63 16 31 16 4 31

3 Cl-

8 16 8 2 2 4 2

3 I-

16 8 16 2 16 4 8

Table 3. MIC values of M-1,1,18 paired with six selected Hofmeister series anions (X): bromide,

carbonate, acetate, nitrate, chloride, and iodide. Values were confirmed with 2-3 independent experiments.

G+=Gram-positive and G-=Gram-negative.

34

antibacterial activity. It is possible that other unknown factors, independent of Hofmeister

series attributes, may contribute to antibacterial activity for these counterion exchanged

amphiphiles, especially since somewhat different trends were observed for Gram-

negative versus Gram-positive species tested.

To determine whether similar trends existed with counterion exchange for double-

tailed amphiphiles, the compound M-1,12,12 was paired with four different Hofmeister

series anions: bromide, acetate, chloride, and iodide. Very little difference in MIC was

observed for Gram-positive species, although M-1,12,12 paired with iodide did have

lower MIC values compared with the compound paired with bromide. A four-fold

decease in MIC was observed against P. aeruginosa for the chloride- and iodide-paired

compound compared to the compound with bromide serving as the counterion.

Interestingly, M-1,12,12 paired with acetate had similar antibacterial activity when

compared with the compound with the chloride and iodide counterions, a trend that was

not observed for the single-tailed M-1,1,18 (Table 3, Table 4). Once again, no clear trend

relating the Hofmeister series to antibacterial activity of counterion exchanged

compounds was observed, suggesting that other factors may influence changes in

antibacterial activity than colloidal or chemical attributes of Hofmeister series anions

paired with antibacterial amphiphiles.

35

Previous research has suggested that more chaotropic ions may be more effective

at penetrating lipid monolayers than kosmotropic ions (Zhang and Cremer, 2006), and

may suggest that direct interactions between the ions and the bacterial membrane itself

may be at least partially responsible for the changes in MIC, in addition to interactions

between the counterions and the amphiphiles in solution. Dissolved Hofmeister anions

have also been shown to affect the absorption of cationic polyions onto hydrophobic

surfaces. The addition of more kosmotrophic salts such as NaCl increase electrostatic

screening and allow polycations to move closer to the interface with the hydrophobic

surface. Chaotropic salts such as NaI allow for significant polycationic absorption at low

concentrations, but the amount of polyion absorbed at the hydrophobic interface

decreases with increasing concentrations due to increased electrostatic screening of the

attraction between the anions and the polyelectrolytes (dos Santos and Levin, 2013).

Although these findings were limited to simulation, they support the conclusion that

chaotropic Hofmeister anions such as I- may increase the efficiency of the interaction

between the cationic hydrophilic head residue of these amphiphiles and the lipid

membrane of bacteria. It is also important to note that this does not account for the

Counterion (M-1,12,12 + X)


(G-)

E. coli

(G-)

E. faecalis

(G+)

S. aureus

(G+)

B. subtilis

(G+)

S. agalactiae

(G+)

B. anthracis

(G+)

3 Br-

16 8 4 4 4 8

3 C2H3O2-

4 4 4 4 4 8 2

3 Cl-

2 4 4 4 2 8 2

3 I-

2 8 2 2 2 2 2

Table 4. MIC values of M-1,12,12 paired with four selected Hofmeister series anions (X): bromide,

acetate, chloride, and iodide. Values were confirmed with 2-3 independent experiments. G+ = Gram-

positive and G- = Gram-negative.

36

hydrophobic tail residues, which have been shown to have a strong effect on antibacterial

activity. Therefore, it is likely that several aspects of structure contribute to the overall

antibacterial activity of these amphiphiles, and the ideal combination of head group, tail

length, counterion, etc. may differ depending on different attributes of the bacteria such

as membrane structure (Lo Nostro et al. 2005).

Continued research is necessary to fully understand how counterion exchange

affects the antibacterial activity of these amphiphiles. Although a clear trend was not

observed, chloride and iodide may have promise as counterions for future compounds,

especially those intended for use against P. aeruginosa. Further study of these effects is

necessary to fully understanding the mechanisms of this change in antibacterial activity,

as it is possible that counterion exchange with chloride and iodide may influence other

aspects of antibacterial activity, such as biofilm disruption.

3.3 Methods.

Synthesis of M-1,1

Preparation of M-1,1 was completed with a 1:1.8 molar equivalence of 1,3,5-

Trisbromomeythylbenzene (0.0056mol, 350mL) to trimethylamine (0.01009mol,

150mL) in acetone. Both solutions are chilled on ice for 15min. After chilling, the

trimethylamine solution was added drop wise to the 1,3,5-Trisbromomeythylbenzene

solution overnight, while on ice. The product of the reaction was then filtered and

recrystallized using a 100:3 solution of acetone to ethanol (100mL per 27mg M-1,1).

Synthesis of the product confirmed using 1HNMR.

37

Synthesis of M-1,1,18

Preparation of M-1,1,18 was completed using a 1:1.5 mole ratio of M-1,1 to

dimethyl-octadecylamine respectively. The reaction was completed in ethanol and left at

80°C under reflux overnight. The product was dried under N2 gas, re-suspended in

acetone, and filtered. The resulting solid was recrystallized using ethanol and acetone.

Synthesis of the product was confirmed using 1HNMR.

Counterion exchange of M-1,1,18

Monovalent counter ion exchange was done with a 1:3.6 molar equivalence of M-

1,1,18 to the corresponding AgX salt (X=NO3-, C2H3O2

-, I

-, and SCN

-). While divalent

counter ion exchange was done with a 1:1.8 molar equivalence of M-1,1,18 to the

corresponding Ag2X salt (X=CO32-

). The reaction was done in methanol (45mL per

250mg M-1,1,18), stirring overnight under light free conditions. The resulting product

was filtered through a celite packed filter and rinsed with 50 mL of excess methanol. The

filtrate was rotovapped down at temperatures below 50˚C. The resulting solid was

resuspended in 5mL methanol and centrifuged for 3min. A small partition of the

supernatant was combined with AgNO3- and the resulting precipitate was tested on the

PXRD. The absence of major peaks at 31 and 44.3 indicated that no remaining AgBr was

present in solution. The supernatant was then rotovapped down and the resulting solid

was put in the desiccator with P2O5 to remove any water present. The presence of the

correct ion exchanged compound was confirmed via HRMS and 1HNMR.


38

A conductivity probe was used to measure the critical aggregate concentration of

the amphiphile. The solution was heated to 37°C. The probe was mixed in the solution

and the measurements were collected. Ten percent of the current solution was pipetted

out and replaced with nanopure water. The solution was mixed and the conductivity

measurement recorded. These steps were repeated for the entirety of the experiment.



25922™ and Pseudomonas aeruginosa ATCC® 27853™, and the five Gram-positive


29213™, Enterococcus faecalis ATCC® 29212™, Bacillus subtilis, B. anthracis Sterne,

and Streptococcus agalactiae J48 (Seifert et al. 2006). All strains were grown in 1X



Broth, so that when amphiphile solutions were added the final broth strength was 1X.



(Ladow et al. 2011; CLSI, 2012). Briefly, compounds were serially diluted in sterile

dionized water and 100µL of each dilution were added to the wells of a 96-well flat-

bottomed microtiter plate in triplicate. After adding 100µL of the bacterial cell

suspension (5x106 CFU/mL), the plates were incubated at 37°C for 72 h. The MIC of the

compound was defined as the minimum concentration that resulted in visible inhibition of

bacterial growth.

39

Chapter 4: The effect of spacer variation on the antibacterial activity of bis- and

tetra-cationic double-tailed amphiphiles.

4.1 Introduction.

The relative size of the hydrophobic region of QACs relative to the hydrophilic

region is one of the greatest predictors of membrane permeability and antibacterial

activity (Paniak et al. 2014). While changing the length of the hydrocarbon tails alters the

size of the hydrophobic region relative to the charged head group and alter characteristics

of membrane insertion activity, this effect may also be achieved by changing the way the

hydrocarbon tails are positioned on the central ring of the amphiphile. It is therefore

possible that changing the number of carbons separating two symmetrical hydrocarbon

tails on a central Mesitylene ring may affect antibacterial activity.

Three series of bis-cationic amphiphiles and three series of tetra-catioic

amphiphiles were synthesized in order to determine differences in the orientation of the

hydrocarbon tails relative to one another on the central ring affects antibacterial activity.

The bis-cationic amphiphiles included one series with the symmetrical tails in an ortho

orientation (oX-n,n), one series with the tails in a meta orientation (mX-n,n), and one

series with the tails in a para orientation (pX-n,n; Figure 7). The oX-n,n series included

four tail length derivatives ranging from 8 to 14 carbons per tail. The mX-n,n series

included three tail length derivatives ranging from 8 to 12 carbons per tail, and the pX-

n,n series included five derivatives ranging from 8 to 16 carbons per tail.

Tetra-cationic amphiphiles each had two head groups, each containing two

dimethylammonium residues separated by a chain of two carbons. Each head group had a

single attached hydrocarbon tail. The two tails were symmetrical and varied in length

40

from 8-12 carbons for each of the three series: oX-(2,n)2, . mX-(2,n)2, and pX-(2,n)2

(Figure 8).

Additionally, one series of three triple-tailed amphiphiles was also tested for its

antibacterial activity. The M-(2,n)3 series consists of amphiphiles with six positive

charges and three tails, each of varying lengths (Figure 9)

41


The antibacterial activity of the bis-cationic series of amphiphiles followed the same

trend as previous amphiphile series according to tail length, with 12 carbon derivatives

having the lowest MIC values (Table 5, Figure 15). This trend was seen for all three

series, suggesting that, similar to series with changes in head group discussed in Chapter

1, the length of the hydrocarbon tails is the most accurate predictor of antibacterial

activity. Changing the spacer length between the tails on the central ring did not appear to

have a significant effect on which hydrocarbon tail length was most effective.

The 12-carbon derivatives of the bis-cationic series had broad-spectrum

antibacterial activity against both Gram-positive and Gram-negative bacteria.

Interestingly, oX-12,12 had the highest level of bacteriocidal activity against P.

aeruginosa, with an MIC of 2 μM, two- to four-fold lower than that of the Gram-positive

species tested. pX-12,12 had an MIC of 2-4 μM against Gram-positive bacteria, and 4

μM against both P. aeruginosa and E. coli (Table 5).

Compound Minimum Inhibitory Concentration (μM)

P. aeruginosa

(G-)

E. coli

(G-)

E. faecalis

(G+)

S. aureus

(G+)

B. subtilis

(G+)

S. agalactiae

(G+)

B. anthracis

(G+)

oX-8,8 >250 250 125 250 >250 250

oX-10,10 31 16 4 16 8 16 16

oX-12,12 2 4 8 8 4 8 4

oX-14,14 31 63 8 63 4 16 4

mX-8,8 250 >250 >250 >250 125 >250 125

mX-10,10 31 16 16 16 16 8 4

mX-12,12 8 8 8 8 8 4 4

pX-8,8 >250 >250 >250 >250 250 250 250

pX-10,10 8 16 8 8 8 8 8

pX-12,12 4 4 2 2 2 4 4

pX-14,14 31 31 16 31 16 4 8

pX-16,16 250 125 250 31 31 63

Table 5. Antibacterial activity of bis-cationic compounds in the oX-n,n, mX-n,n, and pX-n,n series with

different hydrocarbon tail lengths. oX = ortho orientation; mX = meta orientation; pX = para orientation

and n = the number of carbons per tail. G- = Gram-negative and G+ = Gram-positive.

42

Figure 15. Antibacterial activity against seven bacterial species of bis-cationic compounds in the oX-n,n

(diamonds), mX-n,n (squares), and pX-n,n (triangles) series with different hydrocarbon tail lengths. G- =

Gram-negative and G+ = Gram-positive.

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

log [

MIC

(µ

M)]

P. aeruginosa (G-)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

E. coli (G-)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

log [

MIC

(µ

M)]

E. faecalis (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

S. aureus (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

log [

MIC

(µ

M)]

B. subtilis (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

Tail length

S. agalactiae (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12 14 16

log [

MIC

(µ

M)]

Tail length

B. anthracis (G+)

43

Three series of tetra-cationic amphiphiles were also evaluated for their

antibacterial activity (MIC) at three different hydrocarbon tail lengths for three spacer

sizes (ortho, meta, and para orientation). For the compounds with the tails in the meta

and para orientations, the same trend was observed as the bis-cationic amphiphiles

discussed earlier. Compounds with the ideal 12 carbon chain length had the lowest MIC

values compared to other tail lengths in the series. However, for tetra-cationic

amphiphiles with the tails in the ortho orientation relative to one another, 8 hydrocarbons

per tail appeared to be the most effective, with oX-(2,8)2 having the lowest MIC values (4

μM against Gram-negative bacteria, and 2 μM against Gram-positive bacteria) compared

to oX-(2,10)2 and oX-(2,12)2 (Table 6, Figure 16).

Compound Minimum Inhibitory Concentration (μM)

P. aeruginosa

(G-)

E. coli

(G-)

E. faecalis

(G+)

S. aureus

(G+)

B. subtilis

(G+)

S. agalactiae

(G+)

B. anthracis

(G+)

oX-(2,8)2 4 4 2 2 2 2 2

oX-(2,10)2 125 31 16 16 4 2 4

oX-(2,12)2 250 63 250 125 250 125 250

mX-(2,8)2 >250 >250 250 >250 250 >250 125

mX-(2,10)2 16 16 16 16 4 4 4

mX-(2,12)2 8 8 2 2 4 4 4

pX-(2,8)2 >250 250 250 250 250 >250 125

pX-(2,10)2 16 16 16 16 4 4 4

pX-(2,12)2 16 4 2 2 4 4 4

Table 6. Antibacterial activity of tetra-cationic compounds in the oX-(2,n)2, mX-(2,n)2, and pX-(2,n)2

series with different hydrocarbon tail lengths. oX = ortho orientation; mX = meta orientation; pX = para

orientation and n = the number of carbons per tail. G- = Gram-negative and G+ = Gram-positive.

44

Figure 16. Antibacterial activity against seven bacterial species of tetra-cationic compounds in the oX-

(2,n)2 (diamonds), mX-(2,n)2 (squares), and pX-(2,n)2 (triangles) series with different hydrocarbon tail

lengths. G- = Gram-negative and G+ = Gram-positive.

0

0.5

1

1.5

2

2.5

3

8 10 12

log [

MIC

(µ

M)]

P. aeruginosa (G-)

0

0.5

1

1.5

2

2.5

3

8 10 12

E. coli (G-)

0

0.5

1

1.5

2

2.5

3

8 10 12

log [

MIC

(µ

M)]

E. faecalis (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12

S. aureus (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12

log [

MIC

(µ

M)]

B. subtilis (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12

Tail length

S. agalactiae (G+)

0

0.5

1

1.5

2

2.5

3

8 10 12

log [

MIC

(µ

M)]

Tail length

B. anthracis (G+)

45

A single triple-tailed series, M-(2,n)3, was also tested for its antibacterial activity.

This series of compounds contains six positive charges and three tails of equal length,

each with 8, 10, or 12 carbons. The compound with 10 carbons per tail (M-[2,10]3) had

the lowest MIC values of any of the three derivatives (Table 7. Figure 17). Similar to the

trend observed for the tetra-cationic oX-(2,n)2 series, the ideal tail length for the M-(2,n)3

series was not 12 carbons, as it was for the majority of the other compounds tested. The

8-carbon and 12-carbon tail length derivatives of this series both had higher MIC values

on the whole than the 10-carbon derivative.

Compound

M-(2,n)3


(G-)

E. coli

(G-)

E. faecalis

(G+)

S. aureus

(G+)

B. subtilis

(G+)

S. agalactiae

(G+)

B. anthracis

(G+)

M-(2,8)3 16 16 16 1 4 4 2

M-(2,10)3 8 2 2 1 1 4 1

M-(2,12)3 16 31 2 4 8 4 4

Table 7. Antibacterial activity of the triple-tailed M-(2,n)3 series. n = the number of carbons per tail. G- =

Gram-negative and G+ = Gram-positive.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

8 10 12

log(M

IC [μ

M])

Tail length (n for M-[2,n]2)

P. aeruginosa (G-)

E. coli (G-)

E. faecalis (G+)

S. aureus (G+)

B. subtilis (G+)

S. agalactiae (G+)

B. anthracis (G+)

Figure 17. Antibacterial activity of compounds in the M-(2,n)3 series with different

hydrocarbon tail lengths. G- = Gram-negative and G+ = Gram-positive.

46

The MIC of M-(2,10)3 was notably as low as 1 μM against several Gram-positive

species, including S. aureus, and was as low as 2 μM against the Gram-negative E. coli

and 8 μM against P. aeruginosa (Table 7). These MIC values are comparable to the MIC

values of 12-carbon derivatives of the double-tailed, triscationic series studied previously.

These data taken together suggest that the ideal length of the hydrocarbon tails of

these amphiphiles may change depending on other structural characteristics. Changes in

the distribution of charge within the head group of these molecules likely affect

electrostatic interactions between the amphiphiles and the largely negative lipid

membrane of bacteria. Because electrostatic interactions and hydrophobic interactions

both contribute to the overall antimicrobial mechanism of action (Ramos et al. 2007),

changing the distribution of charge in relation to the size of the hydrophobic region may

have significant effects on the ability of these molecules to insert into the membrane.

Because the oX-(2,n)2 series has four positive residues compared to two in the oX-(n,n)

series, this effect may be more pronounced, resulting in the change in the ideal size of the

hydrophobic tails for insertion into the bacterial membrane.

3.3 Methods.

Bacterial strains and growth conditions.


25922™

and Pseudomonas aeruginosa ATCC®

27853™

, and the five Gram-positive


29213™

, Enterococcus faecalis ATCC® 29212

™, Bacillus subtilis, B. anthracis Sterne,

and Streptococcus agalactiae J48 (Seifert et al. 2006). All strains were grown in 1x

47



Broth, so that when amphiphile solutions were added the final broth strength was1X.

Minimum inhibitory concentration.


(Ladow et al. 2011; CLSI, 2012). Briefly, compounds were serially diluted in sterile

dionized water and 100µL of each dilution were added to the wells of a 96-well flat-

bottomed microtiter plate in triplicate. After adding 100µL of the bacterial cell

suspension (5x106 CFU/mL), the plates were incubated at 37°C for 72 h. The MIC of the

compound was defined as the minimum concentration that resulted in visible inhibition of

bacterial growth.

48

Chapter 5: Conclusions and Future Research.

5.1 Conclusions

The minimum inhibitory concentration was determined for all amphiphiles

against two Gram-negative and five Gram-positive organisms. Tail length affects

antibacterial activity, with one tail length being optimal for each series. Consistent with

previous studies (Marafino et al. 2015), compounds with twelve carbons in each of two

symmetrical hydrocarbon tails are the most effective, compared to compounds with

longer or shorter tails. This trend was observed for four different amphiphile series, each

with a differently substituted third head group residue, suggesting that head group

substitution does not have as large of an effect on antibacterial activity as tail length,

provided that the head group residues are of comparable size and charge.

All of the amphiphiles tested (M-E,12,12; M-DMAP,12,12; M-IQ,1212; and M-

4PP,12,12) were capable of disrupting established biofilm. Despite having MIC values as

low as 4-8 µM, significant biofilm disruption was only observed at much higher

concentrations, approximately 31-63 µM. This was unsurprising, considering the fact that

bacteria in biofilms tend to be highly resistant to antibiotics and biocides (Lewis, 2001).

M-E,12,12; M-DMAP,12,12; and M-IQ,12,12 were able to disrupt a similar amount of

established P. aeruginosa biofilm as the antibiotic tobramycin at comparable

concentrations. The amphiphile M-4PP,12,12 disrupted a similar amount of biofilm as

benzalkonium chloride at comparable concentrations, though the amount of biofilm

removed by M-4PP,12,12 was lower than that removed by the other 12-carbon derivative

compounds tested. Due to their ability to remove established biofilms, these compounds

may have potential to be used as novel disinfectants, especially in combination with other

49

antimicrobial agents. Further research is necessary to determine the exact mechanism of

action of biofilm removal, and what aspects of amphiphile structure are most important

for variations in anti-biofilm activity.

Different counterions also affect antibacterial activity. Substitution of bromide

with different ions from the Hofmeister series led to changes in MIC, particularly against

P. aeruginosa. The single-tailed amphiphile M-1,1,18 and the double-tailed amphiphile

M-1,12,12 were more effective at inhibiting the growth of P. aeruginosa with chloride

and iodide counterions than with bromide. Other counterions such as acetate, carbonate,

and nitrate had little effect, or even increased the MIC for these amphiphiles. Despite

notable differences in the MIC for amphiphiles paired with different Hofmeister

counterions, no clear trend was observed between the MIC and any chemical attributes of

the Hofmeister series itself. Although use of different counterions such as chloride and

iodide appear to have potential to increase the efficacy of charged cationic antimicrobials,

particularly against Gram-negative pathogens such as P. aeruginosa, more research is

needed to determine the mechanism by which these different counterions increase the

efficacy of quaternary ammonium amphiphiles.

Finally, the orientation of tails relative to each other on the mesitylene core of

tetra-cationic amphiphiles also has an effect on the optimal tail length. The amphiphiles

with tails in the para or meta orientation on the central ring followed a similar trend to

the tris-cationic double-tailed amphiphiles, with 12 carbons being the ideal length per tail

for the most efficient antibacterial activity. However, amphiphiles with the two tails in

the ortho orientation on the central ring had an optimal tail length of 8 carbons per tail.

50

No shift in the optimal tail length was observed for the bis-cationic amphiphiles.

Bis-cationic amphiphiles with the two tails in the ortho, meta, and para orientation, all

had an ideal tail length of 12 carbons per tail.

The shift in optimal tail length specific to tetra-cationic amphiphiles bears further

study, particularly the possibility that compounds in the oX-(2,n)2 series with shorter

hydrocarbon tails may have even more efficient antibacterial activity. If 8 carbons is

indeed the ideal tail length for amphiphiles in this series, then it is more likely that the

MIC will increase with shorter tail lengths, similar to the trend observed for previously

tested compounds, whose ideal tail length is 12 carbons per tail.

All of these data can be used to synthesize more effective compounds in the

future. Understanding effects of tail length, counterion, and charge distribution on

antibacterial activity, biofilm disruption, and cytotoxicity will be useful for developing

amphiphilic compounds that may serve as novel antibiotics or disinfectants. The

development of novel compounds is essential to prevent the spread of resistant infections

in the population, particularly in people with compromised immune systems, such as the

elderly or those with chronic diseases, and especially in hospitals.

51

5.2 Future Reseach

10-14-AO – Fluorescent Compound

The fluorescent compound 10-14-AO was derived from acridine orange. A 50 μM

solution of 10-14-AO was incubated at room temperature for one hour with an overnight

liquid culture of Streptococcus agalactiae and visualized using a 60X objective on a

confocal microscope.

The compound 10-14-AO was

successfully taken up into the bacterial cells

and fluoresced at approximately 540 nm,

allowing for the visualization of intact S.

agalactiae cells (Figure 18, Figure 19). This

compound may have applications in the

determination of mechanism of action, or in

the study of the movement of amphiphiles

through bacterial biofilm over time. The

0

500

1000

1500

2000

2500

400 450 500 550 600 650

Inte

nsi

ty

Wavelength (nm)

Excitation

Emission

Figure 19. S. agalactiae incubated with

fluorescent compound 10-14-AO, visualized

at 60X magnification using confocal

microscopy.

Figure 18. Excitation and emission

spectra for 10-14-AO. Excitation

was determined by irradiating the

sample with light from 400-520 nm

and monitoring emitted light at 531

nm. Emission was determined by

irradiating the sample with light at

493 nm and monitoring emitted

light from 500-750 nm.

52

antibacterial activity of this compound has yet to be determined, and further studies will

determine the effects of structural changes on fluorescence and uptake into the cells, as

well as biofilm penetration.

Activity against other E.S.K.A.P.E. pathogens

E.S.K.A.P.E. pathogens refer to the most relevant antibiotic-resistant bacteria

(Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacteri baumanii, P.

aeruginosa, and Enterobacter species; Rice, 2008). Because of their promising level of

activity against prominent pathogens such as P. aeruginosa and S. aureus, these

amphiphiles should be tested for their ability to kill other as-yet-untested pathogenic

bacteria such as Acinetobacter baumannii and Klebsiella pneumoniae. While these

pathogens pose little risk to healthy people, they are common causes of healthcare-

associated wound and bloodstream infection, and are often resistant to many commonly

used antibiotics (CDC, 2010; 2015). K. pneumoniae, in particular, has been implicated in

the increasing spread of carbapenem-resistant Enterobacteriaceae (CRE) due to its ability

to produce a potent carbapenemase (CDC, 2015). Due to their broad spectrum of activity

against Gram-negative and Gram-positive pathogens notorious for having high levels of

resistance to antibacterial compounds, the compounds presented in this study may also be

effective against K. pneumoniae and A. baumannii, presenting an opportunity for the

development of new compounds to prevent the spread of these and other prominent

opportunistic pathogens.

53

Mechanism of action

Given ample previous research into the mechanism of action of amphiphilc AMPs

and structurally similar amphiphiles, it is likely that these compounds are membrane-

active, causing depolarization and lysis of bacterial cells (Dathe and Wieprecht, 1999;

Thiyagarajan et al. 2014; Goswami et al. 2015). Propidium iodide permeability assays

can be used in order to gain more information about the membrane-permeabilizing

activity of these compounds (Rieger et al. 2011). Further studies to determine the biofilm

disruption mechanism will be explored such as fluorescence microscopy.

Cytotoxicity

Because the mode of action of these compounds is likely membrane

permeabilization, depolarization and lysis, it is possible that these amphiphiles may not

be compatible with human cells. The cytotoxicity of amphiphiles is largely structure-

dependent, with some amphiphiles having cytotoxic concentrations many times higher

than the MIC (Goswami et al. 2015). It is also possible that fundamental differences

between the mammalian and bacterial cell membranes may increase the therapeutic

potential of these compounds. Namely, unlike the net negatively charged bacterial

membrane, the outer leaflet of the mammalian plasma membrane is neutral, with

negatively charged phospholipids largely localized to the cytosolic leaflet (Cooper,

2000). This may decrease the effects of electrostatic interactions between cationic

membrane-active compounds and the membrane by making hydrophobic interactions the

largest driving force behind insertion into the membrane. Determination of cytoxicity

54

through hemolysis assays and testing using epithelial cells is necessary to confirm the

safety of these compounds for therapeutic use.

Synergistic combinations and pre-treatment

Synergistic combinations of some similar amphiphiles have been observed in

prior studies (Marafino et al. 2015). Of particular interest is the potential for synergy

between antibacterial amphiphiles and sodium metaperiodate (NaIO4), which is used to

disrupt extracellular polysaccharides, particularly against established biofilms (Gawande

et al. 2007; Gutiérrez et al. 2013). Since these compounds likely disrupt bacterial biofilms

by infiltrating the matrix and lysing cells within the biofilm, without disrupting the matrix

itself directly, pre-treatment or co-incubation with NaIO4 may increase the biofilm

disruption ability of QACs.

Some research also suggests that synergistic interactions with conventional

antibiotics may increase bactericidal activity. Membrane permeabilizing agents have been

used with antibiotics such as erythromycin in order to make Gram-negative bacteria

susceptible to otherwise ineffective treatments by breaking down their outer membrane

(Goswami et al. 2015). Similar studies to characterize any synergistic combinations of

these compounds and a variety of antibiotics could further characterize the method of

action of the compounds being studied, as well as provide a proof of concept for these

compounds being used as adjuvants in treatment with conventional antibiotics.

55

References.

Abezgauz, L., K. Kuperkar, P. A. Hassan, O. Ramon, P. Bahadur, and D. Danino. 2010.

Effect of Hofmeister anions on micellization and micellar growth of the surfactant

cetylpyridinium chloride. J Colloid Interface Sci. 342: 83-92.

Al-Shmari, H., W. Abou-Hamad, M. Libman, M. Desrosiers. 2007. Bacteriology of the

sinus cavities of asymptomatic individuals after endoscopic sinus surgery. J Otolaryngol.

36: 43-48.

Ali, Z., N. Mumtaz, S. A. Naz, N. Jabeen, M. Shafique. 2015. Multi-drug resistant

Pseudomonas aeruginosa: a threat of nosocomial infections in tertiary care hospitals. J

Pak Med Assoc. 65: 1-7.

Bartlett, J. G., D. N. Gilbert, and B. Spellberg. 2013. Seven ways to preserve the miracle

of antibiotics. Clin Infect Dis. 56: 1445-1450.

Bendouah, Z., J. Barbeau, W. A. Hamad, M. Desrosers. 2006. Biofilm formation by

Staphylococcus aureus and Pseudomonas aeruginosa is associated with an unfavorable

evolution after surgery for chronic sinusitis and nasal polyposis. Otolaryngol Head Neck

Surg. 134: 991-996.

Bolla, J. M., S. Alibert-Franco, J. Handzlik, J. Chevalier, A. Mahamoud, G. Boyer, K.

Klec-Kononowicz, and J. M. Pages. 2011. strategies for bypassing the membrane barrier

in multidrug resistant Gram-negative bacteria. FEBS Letters. 585: 1682-1690.

Brewer, S.C., R.G. Wunderink, C.B. Jones, and K. V. Leeper, Jr. 1996. Ventilator-

Associated Pneumonia Due to Pseudomonas aeruginosa. Chest. 109: 1019-1029.

Cardile, A. P., C. Tan, M. B. Lustik, A. N. Stratton, C. S. Madar. 2015. Optimization of

time to initial vancomycin target trough improves clinical outcomes. SpringerPlus. 4:

364.

Carson, R. T., E. Larson, S. B. Levy, B. M. Marshall, and A. E. Aiello. 2008. Use of

antibacterial consumer products containing quaternary ammonium compounds and drug

resistance in the community. J Antimicrob Chemother. 62: 1160-1162.

Centers for Disease Control. 2010. Acinetobacter in Healthcare Settings.

Centers for Disease Control. 2013. Antibiotic resistance threats in the United States.

Centers for Disease Control. 2014. Healthcare-Associated Infections (HAI).

Centers for Disease Control. 2015. facility guidance for control of carbapenem-resistant

Enterobacteriaceae (CRE).

56

Chambers, H. F. and F. R. DeLeo. 2009. Waves of resistance: Staphylococcus aureus in

the antibiotic era. Nat Rev Microbiol. 7: 629-641.

Clinical and Laboratory Standards Institute. 2012. Methods for dilution antimicrobila

susceptibility tests for bacteria that grow aerobically, approved standard – ninth addition.

CLSI document M07-A9. Wayne, PA: Clinical and Laboratory Standards Institute.

Cooper, G. M. 2000. structure of the plasma membrane In M. A. Sunderland. The Cell: A

Molecular Approach. 2nd

edition. Sinauer Associates.

Costerton, J. W., P. S. Stewart, and E. P. Greenberg. 1999. Bacterial biofilms: a common

cause of persistent infection. Science. 284: 1318-1322.

Dancer, S. J., M. Coyne, C. Robertson, A. Thomson, A. Guleri, S. Alcock. 2009.

Antibiotic use is associated with resistance of environmental organisms in a teaching

hospital. J Hosp Infect. 62: 200-206.

Dathe, M. and T. Wieprecht. 1999. Structural features of helical antimicrobial peptides:

their potential to modulate activity on model membranes and biological cells. Biochem

Biophys Acta. 1462: 71-87.

Donlan, R. M. 2002. Biofilms: Microbial Life on Surfaces. Centers for Disease Control.

8.

dos Santos, A. P. and Y. Levin. 2013. Absorption of cationic polyions onto a

hydrophobic surface in the presence of Hofmeister salts. Soft Matter, 9: 10545-10549.

Evans, D. J., D. G. Allison, M. R. W. Brown, P. Gilbert. 1990. Growth rate and the

resistance of Gram-negative biofilms toward centrimide USP. J Antimicrob Chemother.

26: 473-478.

Fazlara, A. and M. Ekhtelat. 2012. the disinfectant effects of benzalkonium chloride on

some important foodborne pathogens. American-Eurasian J Agric & Environ Sci. 12: 23-

29.

Fletcher, M. and G. I. Loeb. 1979. Influence of substratum characteristics on the

attachment of a marine pseudomonad to solid surfaces. Appl Environ Microbiol. 37: 67-

72.

Findlay, B., G. G. Zhanel, and F. Schweizer. 2010. Cationic amphiphiles, a new

generation of antimicrobials inspired by the natural antimicrobial peptide scaffold.

Antimicrob Agents Chemother. 54: 4049-4058.

57

Forman, M. E., M. C. Jennings, W. M. Wuest, and K. P. Minbiole. 2016. Building a

better quaternary ammonium compound (QAC): branched tetracationic antiseptic

amphiphiles. ChemMedChem. 11: 1401-1405.

Gawande, P. V., K. LoVetri, N. Yakandawala, T. Romeo, G. G. Zhanel, D. G.

Cvitkovitch, and S. Madhyastha. 2008. Antibiofilm activity of sodium bicarbonate,

sodium metaperiodate and SDS combination against dental unit waterline-associated

bacteria and yeast. J Appl Microbiol. 105: 986-992.

Gerhard, D. 1935. A new class of disinfectants. Dtsch med Wochenschr. 61, 829-832.

Gillings, M. R., M. P. Holley, and H. W. Stokes. 2009. Evidence for dynamic exchange

of qac gene cassettes between class 1 integrons and other integrons in freshwater

biofilms. FEMS Microbiol Letters. 296: 282-288.

Glukhov, E., M. Stark, L. L. Burrows, and C. M. Deber. 2005. Basis for selectivity of

cationic antimicrobial peptides for bacterial versus mammalian membranes. J Bio Chem.

280: 33,960-33,967.

Goswami, S., D. Thiyagarajan, S. Samanta, G. Das, and A. Ramesh. 2015. A zinc

complex of a neutral pyridine-based amphiphile: a highly efficient and potentially

therapeutic bactericidal material. J Mater Chem B. 3: 7068-7078.

Guilbert, J., C. Levy, R. Cohen, Bacterial meningitis group, C. Delacourt, S. Renolleau,

and C. Flamant. 2010. Late and ultra late onset Streptococcus B meningitis: clinical and

bacteriological data over 6 years in France. Acta Paediatr. 99: 47-51.

Gutiérrez, D., P. Ruas-Mediedo, B. Martinez, A. Rodriguez, and P. Garcia. 2014.

Effective removal of staphylococcal biofilms by the endolysin LysH5. PLoS One. 9.

Hancock, R. E. W. and A. Rozek. 2002. Role of membranes in the activities of

antimicrobial cationic peptides. FEMS Microbiol Letters. 206: 143-149.

Head, M. G., J. R. Fitchett, M. K. Cookie, F. B. Wurie, R. Atun, A. C. Hayward, A.

Holmes, A. P. Johnson, and N. Woodford. 2013. Systemic analysis of funding awarded

for antimicrobial resistance research to institutions in the UK, 1997-2010. J Antimicrob

Chemother. 69: 548-554.

Hidron, A. I., J. R. Edwards, J. Patel, T. C. Horan, D. M. Sievert, D. A. Pollock, and S. K

Fridkin. 2008. NHSN annual update: antimicrobial-resistant pathogens associated with

healthcare-associated infections: annual summary of data reported to the National

Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006-

2007. Infect Control Hosp Epidemiol. 29: 996-1011.

58

Hiller, N. L., A. Chauhan, M. Palmer, S. Jain, J. G. Sotereanos. 2015. Presence of

bacteria in failed anterior cruciate ligament reconstructions. SpringerPlus. 4: 460.

Hofmeister, F. 1888. Zur Lehre von der Wirkung der Salze – Zweite Mittheilung. Archiv

f. experiment Pathol u Pharmakol. 24: 247-260.

Hoque, J., P. Akkapeddi, V. Yarlagadda, D. S. Uppu, P. Kumar, and J. Haldar. 2012.

Cleavable cationic antibacterial amphiphiles: synthesis, mechanism of action, and

cytotoxicities. Langmuir. 28: 12,225-12,234.

Humphreys, H., K. Becker, P. M. Dohmen, N. Petrosillo, M. Spencer, M. van Rijen, W.

Wechsler-Fӧrdӧs, M. Pujol, A. Dubouix, and J. Garau. 2016. Staphylococcus aureus and

surgical site infections: benefits of screening and decolonization before surgery. J Hosp

Infect. 94: 295-304.

Infectious Disease Society of America. 2010. The 10 x ’20 Initiative: pursuing a global

commitment to develop 10 new antibacterial drugs by 2020. Clin Infect Dis. 50: 1081-

1083.

Infectious Disease Society of America. 2011. Combating antimicrobial resistance: policy

recommendations to save lives. Clin Infect Dis. 52: S397-S428.

Jacobsen, S. M., D. J. Stickler, H. L. Mobley, and M. E. Shirtliff. 2008. Complicated

catheter-associated urinary tract infections due to Escherichia coli and Proteus mirabilis.

Clin Microbiol Rev. 21: 26-59.

Jennings, M. C., L. E. Ator, T. J. Paniak, K. P. C. Minibiole, W. M. Wuest. 2014.

Biofilm-eradicating properties of quaternary ammonium amphiphiles: simple mimics of

antimicrobial peptides. ChemBioChem. 15: 2211-2215.

Jenssen, H., P. Hamill, and R. E. W. Hancock. 2006. Peptide antimicrobial agents. Clin

Microbiol Rev. 19: 491-511.

Johnston, BL and E Bryce. 2009. Hospital infection control strategies for vancomycin-

resistant Enterococcus, methicillin-resistant Staphylococcus aureus, and Clostridium

difficile. Can Med Assoc J. 180: 627-631.

Kaplan, J. P. 2010. Biofilm dispersal: mechanisms, clinical implications, and potential

therapeutic uses. J Dent Res. 89: 205-218.

Korber, D. R., J. R. Lawrence, B. Sutton, and D. E. Caldwell. 1989. Effect of laminar

flow velocity on the kinetics of surface recolonization by Mot(+) and Mot(-)

Pseudomonas fluorescens. Microb Ecol. 18: 1-19.

59

Kunz, W., J. Henle, and B. W. Ninham. 2004. ‘Zur Lehre von der Wirkung der Salze’

(about the science of the effect of salts): Franz Hofmeister’s historical papers. Curr Opin

Colloid Interface Sci. 9: 19-37.

LaDow, J. E., D. C. Warnock, K. M. Hamill, K. L. Simmons, R. W. Davis. 2011.

Bicephalic amphiphile architecture affects antibacterial activity. Eur J Med Chem. 46:

4219-4226.

Lewandowski, Z. 2000. Structure and Function of Biofilms. In L. V. Evans (Ed),

Biofilms: Recent Advances in their Study and Control (pp. 1-17). Harwood Academic

Publishing.

Lewis, K. 2001. Riddle of biofilm resistance. Antimicrob Agents Chemother. 45: 999-

1007.

Li, X. Z., D. M. Livermore, and H. Nikaido. 1994. Role of efflux pump(s) in intrinsic

resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and

norfloxacin. Antimicrob. Agents Chemother. 38: 1732-1741.

Liu, Y., Y. Wang, T.R. Walsh, L.X. Yi, R. Zhang, J. Spencer, Y. Doi, G. Tian, B. Dong,

X. Huang, L. Yu, D. Gu, H. Ren, X. Chen, L. Lv, D. He, H. Zhou, Z. Liang, J. Liu, and J.

Shen. 2016. Emergence of a plasmid-mediated colistin resistance mechanism MCR-1 in

animals and human beings in China: a microbiological and molecular biological study.

Lancet Infec Dis. 16: 161-168.

Lo Nostro, P., B. W. Ninham, A. Lo Nostro, G. Pesavento, L. Fratoni, and P. Baglioni.

2005. Specific ion effects on the growth rates of Staphylococcus aureus and

Pseudomonas aeruginosa. Phys Biol. 2.

Luyt, C. E., N. Bréchot, J. L. Trouillet, and J. Chastre. 2014. Antibiotic stewardship in

the intensive care unit. Crit Care. 18: 480.

Ma, J. F., P. W. Hager, M. L. Howell, P. V. Phibbs, D. Hassett. 1998. Cloning and

characterization of the Pseudomonas aeruginosa zwf gene encoding glucose-6-phosphate

dehydrogenase, an enzyme important in resistance to methyl viologen (paraquat). J

Bacteriol. 180: 1741-1749.

MacFarguhar, J. K., T. F. Jones, A. M. Woron, M. A. Kainer, C. G. Whitney, B. Beall, S.

J. Schrag, and W. Schaffner. 2010. Outbreak of late-onset group B Streptococcus in a

neonatal intensive care unit. Am J Infect Control. 38: 283-288.

Magill, S., J. Edwards, W. Bamberg, Z. Beldava, G. Dumyati, M. Kainer, R. Lynfield, M.

Maloney, L. McAllister-Hollod, J. Nadle, S. Ray, D. Thompson, L. Wilson, and S.

60

Fridkin. 2014. Multistate point-prevalence survey of health care-associated infections. N

Engl J Med. 370: 1198-1208.

Marafino, J. N., T. M. Gallagher, J. Barragan, B. L. Volkers, J. E. LaDow. 2015.

Colloidal and antibacterial properties of novel triple-headed, double-tailed amphiphiles:

Exploring structure-activity relationships and synergistic mixtures. Bioorg Med Chem.

23: 3566-3573.

Marr, A. K., W. J. Gooderham, and R. E. W. Hancock. 2006. Antibacterial peptides for

therapeutic use: obstacles and realistic outlook. Curr Opin Pharmacol. 6: 468-472.

Mowery, B. P., S. E. Lee, D. A. Kissounko, R. F. Epand, R. M. Epand, B. Weisblum, S.

S. Stahl, and S. H. Gellman. 2007. Mimicry of antimicrobial host-defense peptides by

random copolymers. J Am Chem Soc. 129: 15,474-15,476.

Naskar, B., A. Dan, S. Ghosh, V. K. Aswal, and S. P. Moulik. 2012. Revisiting the self-

aggregation behavior of cetyltrimethylammonium bromide in aqueous sodium salt

solution with varied anions. J Mol Liq. 170: 1-10.

National Institutes of Health. 2016. Estimates of funding for various research, condition,

and disease categories (RDCD).

Nicolet, S., D. Goldenberger, T. Schwede, M. Page, and M. Creus. 2016. Plasmid-

mediated colistin resistance in a patient infected with Klebsiella pneumoniae. Lancet

Infec Dis. 16: 998-999.

Nizet, V. 2006. Antimicrobial peptide resistance mechanisms of human bacterial

pathogens. Curr Issues Mol Biol. 8: 11-26.

O’Donnel, M. J., C. M. Tuttlebee, F. R. Falkiner, D. C. Coleman. 2005. Bacterial

contamination of dental chair units in a modern dental hospital caused by leakage from

suction system hoses containing extensive biofilm. J Hosp Infect. 59: 348-360.

Olawale, K. O., S. O. Fadiora, and S. S. Taiwo. 2011. Prevalence of hospital-acquired

Enterococci infections in two primary-care hospitals in Osogbo, southwestern Nigeria.

Afr J Infect Dis. 5: 40-46.

Oliver, A., R. Cantón, P. Campo, F. Baquero, J. Blázquez. 2000. High frequency of

hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science. 288:

1251-1253.

O’Toole, G. 2011. Microtiter dish biofilm formation assay. JoVE. 47.

61

Palermo, E. F. and K. Kuroda. 2009. Chemical structure of cationic groups in

amphiphilic polymethacrylates modulates the antimicrobial and hemolytic activities.

Biomacromol. 10: 1416-1428.

Paniak, T. J., M. C. Jennings, P. C. Shanahan, M. D. Joyce, C. N. Santiago, W. M.

Wuest, and K. P. Minbiole. 2014. The antimicrobial activity of mono-, bis-, tris-, and

tetracationic amphiphiles derived from simple polyamine platforms. Bioorg Med Chem

Lett. 24: 5824-5828.

Pawar, V., U. Komor, N. Kasnitz, P. Bielecki, M. C. Pils, B. Gocht, A. Moter, M. Rohde,

S. Weiss, S. Häussler. 2015. In vivo efficacy of antimicrobials against biofilm-producing

Pseudomonas aeruginosa. Antimicrob Agents Chemother. 59: 4974-4981.

Peleg, A. Y. 2010. Hospital-acquired infections due to Gram-negative bacteria. N Eng J

Med. 362: 1804-1813.

Piddock, L. J. 2012. The crisis of no new antibiotics – what is the way forward? Lancet

Infect Dis. 12: 249-253.

Poirel, L., N. Kieffer, N. Liassine, D. Thanh, and P. Nordmann. 2016. Plasmid-mediated

carbapenem and colistin resistance in a clinical isolate of Escherichia coli. Lancet Infec

Dis. 16: 281.

Quirynen, M., M. Brecx, and D. van Steenberghe. 2000. Biofilms in the Oral Cavity:

Impact of Surface Characteristics. In L. V. Evans (Ed), Biofilms: Recent Advances in

their Study and Control (pp. 167-187). Harwood Academic Publishing.

Ramos, L., M. Schӧnhoff, Y. Luan, H. Mӧhwald, and G. Brezesinski. 2007. Electrostatic

interactions between polyelectrolyte and amphiphiles in two- and three-dimensional

systems. Colloids Surf., A. 303: 79-88.

Rieger, A. M., K. L. Nelson, J. D. Konowalchuk, and D. R. Barreda. 2011. Modified

annexin V/propidium iodide apoptosis assay for accurate assessment of cell death. J Vis

Exp. 24: 2597.

Rice, L. B. 2008. Federal funding for the study of antimicrobial resistance in nosocomial

pathogens: no ESKAPE. J Infect Dis. 197: 1079-1081.

Richards, M. J., J. R. Edwards, D. H. Culver, and R. P. Gaynes. 1999. Nosocomial

infections in medical intensive care units in the United States. National nosocomial

infections surveillance system. Crit. Care Med. 27: 887-892.

Rosenberg, M., E. A. Bayer, J. Delarea, and E. Rosenberg. 1982. Role of thin fimbriae in

adherence and growth of Acinetobacter calcoaceticus RAG-1 on hexadecane. Appl

Environ Microbiol. 44: 929-937.

62

Rosholm, K. R., A. González-Pérez, and O. G. Mouritsen. 2010. Self-assembly based on

hydrotrophic counterion – single-chain amphiphile ion pairs. Colloid Polymer Sci. 288:

1351-1357.

Sawai, T., K. Matsuba, A. Tamura, S. Yamagishi. 1979. The bacterial outer-membrane

permeability of β-lactam antibiotics. J Antibiot. 32: 59-65.

Schrenk, K. G., J. Frosinski, S. Scholl, S. Otto, P. La Rosee, A. Hochhaus, M. W. Pletz.

2015. Successful treatment of neutropenic MRSA bacteremia with septic superior vena

cava thrombus and cerebral embolism using high-dose daptomycin. Ann Hematol.95:

355-357.

Scott, R. W., W. F. DeGrado, and G. N. Tew. 2008. De novo designed synthetic mimics

of antimicrobial peptides. Curr Opin Biotechnol. 19: 620-627.

Seifert, KN, EE Adderson, AA Whiting, JF Bohnsack, PJ Crowley, and LJ Brady. 2006.

A unique serine-rich repeat protein (Srr-2) and novel surface antigen (ε) associated with a

virulent lineage of serotype III Streptococcus agalactiae. J Microbiol. 152: 1029-1040.

Shawar, R. M., D. J. MacLeod, R. L. Garber, J. L. Burns, J. R. Stapp, C. R. Clausen, and

S. K. Tanaka. 1999. Activities of tobramycin and six other antibiotics against

Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrob Agents

Chemother. 43: 2877-2880.

Singh, P. K., A. L. Schaefer, M. R. Parsek, T. O. Moninger, M. J. Welsh, E. P.

Greenberg. 2000. Quorum-sensing signals indicate that cystic fibrosis lungs are infected

with bacterial biofilms. Nature. 407: 762-764.

Song, A., S. G. Walker, K. A. Parker, and N. S. Simpson. 2011. Antibacterial studies of

cationic polymers with alternating, random, and uniform backbones. ACS Chem Biol. 6:

590-599.

Speck, P. 2013. Antibiotics: Avert an impending crisis. Nature. 496: 169.

Sutherland, I. W. 2001. Biofilm exopolysaccharides: a strong and sticky framework.

Microbiol. 147: 3-9.

Thiyagarajan, D., S. Goswami, C. Kar, G. Das, and A. Ramesh. 2014. A prospective

antibacterial for drug-resistant pathogens: a dual warhead amphiphile designed to track

interactions and kill pathogenic bacteria by membrane damage and cellular DNA

cleavage. Chem Commun. 50: 7434-7436.

Uppu, D. S., S. Samaddar, C. Ghosh, K. Paramanandham, B. R. Shome, J. Haldar. 2016.

Amide side chain amphiphilic polymers disrupt surface established bacterial bio-films

63

and protect mice from chronic Acinetobacter baumannii infection. Biomaterials. 74: 131-

143.

van Boeckel, T. P., S. Gandra, A. Ashok, Q. Caudron, B. T. Grenfell, S. A. Levin, and R.

Laxminaravan. 2014. Global antibiotic consumption 2000 to 2010: an analysis of national

pharmaceutical sales data. Lancet Infect Dis. 14: 742-750.

van der Veen, S. and T. Abee. 2010. Mixed species biofilms of Listeria monocytogenes

and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and

peracetic acid. Internat J Food Micriobiol. 144: 421-431.

Vidya, P., L. Smith, T. Beaudoin, Y. C. W. Yau, S. Clark, B. Coburn, D. S. Guttman, D.

M. Hwang, and V. Waters. 2016. Chronic infection phenotypes of Pseudomonas

aeruginosa are associated with failure of eradication in children with cystic fibrosis. Euro

J Clin Micriobiol Infec Dis. 35: 67-74.

Vickery, K., A. Pajkos, Y. Cossart. 2004. Removal of biofilm from endoscopes:

Evaluation of detergent efficiency. Am J Infect Control. 32: 170-176.

Vickery, K., A. Deva, A. Jacombs, J. Allan, P. Valente, I. B. Gosbell. 2012. Presence of

biofilm containing viable multiresistant organisms despite terminal cleaning on clinical

surfaces in an intensive care unit. J Hosp Infect. 32: 52-55.

Vudumula, U., M. D. Adhikari, O. Bimlesh, S. Goswami, G. Das, and A. Ramesh. 2012.

Tuning the bactericidal repertoire and potency of quinoline-based amphiphiles for

enhanced killing of pathogenic bacteria. RSC Adv. 2: 3864-3871.

Wright, J.A. and S. P. Nair. 2010. Interaction of Staphylococci with Bone. Int J Med

Microbiol. 300: 193-204.

Wright, G. D. 2014. Something old, something new: revisiting natural products in

antibiotic drug discovery. Can J Microbiol. 60: 147-154.

Zhang, Y. and P. S. Cremer. 2006. Interactions between macromolecules and ions: the

Hofmeister series. Curr Opin Chem Biol. 10: 658-663.

Documents

The antimicrobial and biofilm disruption activity of novel