Identification of the Markers of Antibiotic Resistance in Daptomycin Non-

Susceptible Strains of Staphylococcus aureus

By Riddhi Shantilal Shah

Submitted to the faculty of the Albany College of Pharmacy and Health Sciences
in partial fulfillment of the Degree of Master of Science in Pharmaceutical Sciences

August 2014

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Approved by:

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Meenakshi Malik, D.V.M., Ph.D. 8/7/2014
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Thesis Advisor Signature Date
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Jeffrey Voigt, Ph.D. 8/7/2014

Committee Member Signature Date

Amit Pai, Pharm.D. 8/7/2014

Committee Member Signature Date

Anthony Nicasio, Pharm.D. 8/7/2014

Committee Member Signature Date
 UMI Number: 1564316

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 ABSTRACT

Methicillin resistant Staphylococcus aureus (MRSA) is a major health care concern

worldwide and is responsible for a variety of nosocomial infections including skin and

soft tissue infections, bacteremia, endocarditis, and toxic shock syndrome. Antibiotic

resistance in MRSA has posed a major threat in recent years resulting in treatment

failures. Daptomycin was often used to treat MRSA infections but recent reports

indicated an increasing incidence of resistance to this antimicrobial agent. Studies have

shown that mutations in genes involved in regulation of transcription, cell wall

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biosynthesis, cell wall charge, autolysis and oxidative stress along with their altered

expression are principally responsible for induction of daptomycin resistance.

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We studied the gene expression profile associated with induction of daptomycin non-
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susceptibility in an isogenic pair of daptomycin non-susceptible S. aureus and

daptomycin susceptible S. aureus clinical isolates. We compared the expression of cell

membrane charge, cell wall synthesis, autolysis and penicillin binding protein (PBP)
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genes in DNSA and DSSA strains treated with daptomycin and ceftaroline individually or

in combination. In drug-free conditions, DNSA strain showed an upregulation of genes

responsible for cell membrane charge (mprF, dltABCD) and cell-wall synthesis (VraS

and femB) as compared to the DSSA strain whereas (atl and lytM) showed upregulation

in DSSA compared to DNSA strain. A combination therapy using daptomycin and

ceftaroline synergistically down-regulated these genes in DNSA strain and DSSA

respectively. Transcriptional analysis of PBPs revealed an upregulation of pbp2a gene in

DNSA strain which was downregulated when combination therapy was used. We also

looked at the mutation profile to look for the difference between the susceptible and non-
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susceptible strains. We tested several genes implicated in resistance and found mutations

in walR and mprF gene. Drug treatment both monotherapy and combination prevented

mutation in walR but not in mprF gene. Our results demonstrate that gene expression

profiles associated with the development of daptomycin non-susceptibility serve as

important markers in rapid differentiation of DNSA and DSSA strains and combination

of daptomycin and ceftaroline may have potential for use as therapy against DNSA

strains.

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 TABLE OF CONTENTS

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1.0 INTRODUCTION 1
1.1 Specific Aims 2

2.0 BACKGROUND AND SIGNIFICANCE 4

2.1 Methicillin resistant S. aureus 4
2.2 MRSA virulence factors 5
2.3 Resistance to beta-lactam antibiotics 7
2.4 Daptomycin 7
2.5 Factors leading to daptomycin non-susceptibility 8
2.6 Role of specific genes involved in daptomycin non- 11
susceptibility
2.7 Interplay of vancomycin resistance and subsequent 17

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Daptomycin non-susceptibility
2.8 Ceftaroline 18
2.9 Combinatorial therapyIE 18

3.0 MATERIALS AND METHODS 20

3.1 Bacterial strains 20
3.2 Antimicrobials 20
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3.3 Time kill assays 21
3.4 RNA isolation 22
3.5 cDNA synthesis and qRT-PCR 22
3.6 Statistical analysis 26
3.7 DNA isolation 26
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3.8 PCR 26
3.9 Purification of PCR products 28
3.10 DNA gel electrophoresis and sequencing 28

4.0 RESULTS 30

5.0 DISCUSSION 62

6.0 REFERENCES 69

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 LIST OF ABBREVIATIONS

ACME Arginine Catabolic Mobile Element

BLAST Basic Local Alignment Search Tool
CA Community Acquired
cDNA cyclic Deoxyribonucleic Acid
CPT Ceftaroline
Cls2 Cardiolipin Synthase 2
CFU Colony Forming Units
DSSA Daptomycin Susceptible Staphylococcus aureus

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DNSA Daptomycin Non-Susceptible Staphylococcus aureus
DAP Daptomycin IE
DNA Deoxyribonucleic Acid
EDTA Ethylenediaminetetraacetic acid
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FEM Factors Essential for Methicillin Resistance
HA Hospital Acquired
hVISA Heterogeneous Vancomycin Intermediate Staphylococcus aureus
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LPG Lysyl Phosphatidyl Glycerol

LTA Lipo Teichoic Acid
MIC Minimum Inhibitory Concentration
MprF Multiple Peptide Resistance Factor
MRSA Methicillin Resistant Staphylococcus aureus
NCBI National Center for Biotechnology Institute
OD Optical Density
PBPs Penicillin Binding Proteins
PVL Panton Valentine Leukocidin
PSMs Phenol Soluble Modulins

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PLs Phospholipids
PG Phosphatidyl Glycerol
PCR Polymerase Chain Reaction
qRT-PCR Quantitative Real Time Polymerase Chain Reaction
RNA Ribonucleic Acid
SCC Staphylococcal Chromosome Cassette
VraSR Vancomycin Resistance Associated Sensor Regulator
WTA Wall Teichoic Acid

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 LIST OF FIGURES
Page No.
Figure 1 Time kill assay using DSSA and DNSA strains 31

Figure 2 Expression of mprF gene in DSSA and DNSA strains 33

Figure 3 Expression of dltA gene in DSSA and DNSA strains 34

Figure 4 Expression of dltB gene in DSSA and DNSA strains 35

Figure 5 Expression of dltC gene in DSSA and DNSA strains 36

Figure 6 Expression of dltD gene in DSSA and DNSA strains 37

Figure 7 Expression of vraS gene in DSSA and DNSA strains 38

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Figure 8 Expression of femA gene in DSSA and DNSA strains 39

Figure 9 Expression of femB gene in DSSA and DNSA strains

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Figure 10 Expression of ltaS gene in DSSA and DNSA strains 41

Figure 11 Expression of walK gene in DSSA and DNSA strains 43

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Figure 12 Expression of atl gene in DSSA and DNSA strains 44

Figure 13 Expression of lytM gene in DSSA and DNSA strains 45

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Figure 14 Expression of Pbp2a gene in DSSA and DNSA strains 47

Figure 15 Expression of Pbp2 gene in DSSA and DNSA strains 48

Figure 16 Expression of Pbp1 gene in DSSA and DNSA strains 49

Figure 17 Expression of Pbp4 gene in DSSA and DNSA strains 50

Figure 18 Genomic organization and single nucleotide polymorphisms 53

(SNPs) observed in the mprF gene of DNSA strain and its
comparison with Mu50 and DSSA mprF gene sequences

Figure 19 Sequence alignment of the agrC gene of DNSA, DSSA 54

and Mu50 strains

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Figure 20 Sequence alignment of the rplV gene of DNSA, DSSA and 56
Mu50 strains

Figure 21 Sequence alignment of the rplC gene of DNSA, DSSA and 57

Mu50 strains

Figure 22 Sequence alignment of the vicR (walkR) gene of DNSA 58

and DSSA strains

Figure 23 Sequence alignment of the clpP gene of DNSA, DSSA and 60

Mu50 strains

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 LIST OF TABLES
Page No.
Table 1 MICs used for DSSA and DNSA strains in this study 20

Table 2 List of genes analyzed for gene expression studies 23

Table 3 List of genes analyzed for genetic mutation studies 27

Table 4 Genetic mutations in mprF gene of DNSA strain as 52

compared to standard Mu50 and DSSA strains

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1.0 Introduction

Methicillin resistant Staphylococcus aureus (MRSA) is a major health care concern

worldwide and is responsible for a variety of nosocomial infections including skin and

soft tissue infections, bacteremia, endocarditis, and toxic shock syndrome. Antibiotic

resistance in MRSA has posed a major threat in recent years resulting in treatment

failures. This loss of methicillin resistance has most often been due to the excision of the

staphylococcal cassette chromosome mec element (SCCmec) that carries mecA, the gene

encoding penicillin-binding protein 2A (PBP2a). Daptomycin is often used to treat

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MRSA infections but recent reports indicate an increasing resistance to this antimicrobial

agent. A combination therapy involving the use of an anti-staphylococcal -lactam

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antibiotic plus daptomycin has been explored to circumvent this problem. However, a

better understanding of the molecular mechanisms underlying the daptomycin non-

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susceptibility is required in order to develop alternate combination therapies.

Studies have shown that mutations in genes involved in regulation of

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transcription, cell wall biosynthesis, cell wall charge, autolysis and oxidative stress along

with their altered expression are principally responsible for induction of daptomycin

resistance (1). These observations form the basis of our hypothesis that identification of

gene expression profiles and genotypic alterations resulting in antibiotic resistance

would serve as important markers for identification of daptomycin non-susceptible

strains. We aim to investigate the gene expression profiles and putative genetic

determinants corresponding to the daptomycin non-susceptible (DNSA) and susceptible

(DSSA) Staphylococcus aureus phenotypes using an isogenic pair of strains in response

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to daptomycin alone or a combination antibiotic therapy. This hypothesis will be

addressed by two specific aims:

1.1 Specific Aims

Specific Aim 1: Investigate the alterations in gene expression profile of DNSA strains

of S. aureus exposed to daptomycin and ceftaroline alone or a combination

antibiotic therapy. Our hypothesis is that daptomycin non-susceptibility results from

alterations in gene expression profile in the DNSA strain as a result of exposure to

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daptomycin. The major objective of this specific aim is to identify genetic markers based
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on the expression profile of the genes that have occurred in the DNSA/DSSA strains as a

result of exposure to single or a combinatorial therapy. Earlier studies have identified

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D592 strain of S. aureus as a daptomycin susceptible strain, whereas D712 is a

daptomycin non-susceptible daughter strain of D592 (Nicasio et al, unpublished data).

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These two isogenic strains have been used in the present study. Particularly, the genes

involved in cell membrane charge (mprF, dltABCD), autolysis (atl, lytM), cell wall

synthesis (femA, femB, vraSR, ltaS, walK), and penicillin binding proteins (pbp2, pbp2a,

pbp4, pbp1) in untreated DSSA and DNSA strains or following treatment with

daptomycin and ceftaroline alone or in combinations were examined.

Specific Aim 2: Investigate the genetic alterations in DNSA strain of S. aureus

exposed to daptomycin and ceftaroline alone or a combination antibiotic therapy.

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Emergence of bacterial resistance under in vitro culture conditions occurs by spontaneous

mutations, serial passages in the presence of low dose antibiotics, or chemical

mutagenesis. Since daptomycin acts on the bacterial cell wall, it is expected that this

antibiotic will have a slow rate of inheriting resistance in contrast to antibiotics which

primarily act by targeting ribosomal DNA, and therefore acquire mutational resistance at

a much faster rate. Emergence of resistance with daptomycin against S. aureus has been

determined by spontaneous mutations. Our hypothesis is that daptomycin non-

susceptibility results from genetic alterations in the DNSA strain as a result of exposure

to daptomycin. Therefore, in Specific aim 2, we further investigated if genetic mutations

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have occurred in the DNSA strain and if these mutations can be reversed following
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ceftaroline + daptomycin combination treatment.
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2.0 Background and Significance

Staphylococcus aureus is a gram-positive cocci arranged in grape like clusters. S.

aureus causes major soft and skin tissue infections, osteomyelitis, joint infections,

bacteremia, endocarditis and toxic shock syndrome (2;3). Staphylococcal cell wall is

made up of four major components including peptidoglycan, teichoic acids (TAs),

lipoteichoic acids (LTAs) and cell surface proteins. Peptidoglycan layer is a major

component of cell wall made up of sugars and amino acids. The sugar component

consists of alternating residues of -(1, 4) linked N-acetylglucosamine and N-

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acetylmuramic acid. S. aureus contain two types of TAs which consists of wall teichoic

acid (WTA) and LTA. TAs plays a major role in growth, biofilm production, adhesion
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and virulence of gram-positive bacteria (4). WTAs are covalently linked to the

peptidoglycan layer and D- alanine residues. Addition of Alanines to WTAs causes an

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increase in positive charge and repulsion of cationic antimicrobials causing antibiotic

resistance (5). LTA plays a major role in cell division and its loss results in cell death
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(5;6).

2.1 Methicillin Resistant S. aureus (MRSA)

Methicillin was used initially to treat Staphylococcus infections but resistance

developed to methicillin and the first MRSA strain was isolated in 1961. MRSA is now a

general term used to denote antimicrobial resistance to a variety of antibiotics. MRSA

infections surpassed HIV as a cause of death in the USA in 2005 with a mortality rate of

6.3%. MRSA resistance is primarily developed due to the acquisition of Staphylococcal

chromosome cassette mec (SCCmec) carrying the mecA gene which encodes for

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penicillin binding protein 2a (PBP2a) responsible for altered binding to beta-lactam

antibiotics that results in decreased susceptibility to this class of antibiotics (7-9). The

beta-lactam antibiotics bind to the innate penicillin binding proteins and the alteration of

PBPs to PBP2a results in reduced binding. The mecA gene is under the control of two

regulatory genes mecI and mecR1. In the presence of -lactam antibiotics, mecR1 is

activated and increases expression of mecR1, mecI, mecR2 and mecA. In susceptible

strains, mecI causes the inhibition of mecA thereby resulting in decreased resistance.

However, in resistant strains mecR2 destabilizes mecI dimers, decreasing their ability to

bind to mecA promoter resulting in sustained induction of mecA transcription (10).

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MRSA is classified into two types: Community Acquired MRSA (CA-MRSA) and
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Hospital Acquired (HA-MRSA) (2). CA-MRSA emerged in early 1990s and was

characterized by presence of panton- valentine leukocidin toxin (PVL). CA-MRSA

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strains have SCCmec types IV or V which are smaller and more mobile than HA-MRSA

containing the SCCmec types I-III. The smaller version of SCCmec IV and V confer less
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resistance and explains why CA-MRSA is more susceptible to antibiotics compared to

HA-MRSA. However, CA-MRSA is more virulent compared to HA-MRSA due to the

presence of several virulence factors including toxins, adhesins and enzymes (3;11-13).

We have used an isogenic MRSA strain pair in the present study. D592 is daptomycin

susceptible and heterogeneously vancomycin intermediate S. aureus strain (hVISA) and

D712 is daptomycin non-susceptible and vancomycin intermediate strain (VISA) (14).

2.2 MRSA Virulence factors

a. Panton-Valentine Leukocidin (PVL)

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 PVL is an exotoxin encoded by two genes, lukF-PV and lukS-PV responsible for

leukocyte lysis, inflammation and lysis (2). PVL expression in the CA-MRSA is

associated with serious soft tissue infections as compared to the non-PVL strains (12;13).

b. Alpha toxin

Alpha toxin lyses macrophages and lymphocytes and alters platelet morphology

(15). This altered morphology is associated with severe thrombotic events. The amount of

alpha toxin influences the number of lesions.

c. Phenol-soluble Modulins (PSMs)

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PSMs recruit, activate, and lyse neutrophils. They are encoded by two different
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genes annotated as psma operon and psm-mec. The PSMs are more commonly associated

with CA-MRSA strains (16).

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d. Arginine catabolic mobile element (ACME)

ACME plays an important role in growth, transmission and pathogenesis of CA-

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MRSA (9).

e. Biofilms

S. aureus can form biofilms on many host tissues and implanted medical devices

resulting in a variety of chronic infections. Biofilms provide a mechanical barrier to

prevent the insertion of antimicrobials. S. aureus biofilm is embedded within a

glycocalyx primarily composed of polysaccharide intercellular antigen (PIA). Previous

reports suggest that the non-susceptible strains produce proteinaceous biofilm while the

susceptible strains produce polysaccharide biofilm (17).

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2.3 Resistance to beta-lactam antibiotics

The mechanism of action of beta-lactam antibiotics is by disruption of

peptidoglycan layer by causing acylation of transpeptidases. Beta-lactam resistance

occurs via two mechanisms: First, via production of penicillinase, mediated by blaZ

which cleaves the lactam rings and secondly through mecA that confers broader

resistance to both penicillin and beta-lactam collectively defined as methicillin resistance

(7).

MRSA resistance is primarily developed by acquisition of Staphylococcal

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chromosome cassette mec (SCCmec) carrying the mecA gene which encodes 78kDa

PBP2a responsible for altered binding to beta-lactam antibiotics leading to decreased

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susceptibility (8). PBP2a is responsible for resistance to penicillin, methicillin, oxacillin,

most cephalosporin and all other beta-lactam antibiotics. In methicillin susceptible S.

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aureus strain (MSSA), beta-lactam antibiotics bind to the native PBPs that are present on

the S. aureus cell wall which causes disruption of peptidoglycan synthesis. However,
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modification of PBP to PBP2a results in altered binding of the antibiotics and cause

peptidoglycan synthesis to take place normally making the bacteria resistant to the

antibiotic exposure (18).

2.4 Daptomycin

Because of increased reports of methicillin resistance (2;18;19), daptomycin is

now generally used to treat the MRSA infections. Daptomycin is a cyclic cationic

lipopeptide. It was approved by FDA in 2003 for treatment of skin infections caused by

gram- positive pathogens and for treatment of bacteremia and right-sided endocarditis

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 caused by S. aureus and MRSA strains in

2006 (21;22). Mechanism of action of

daptomycin occurs via two steps. The

native daptomycin molecule is anionic in

nature. Calcium binding to daptomycin is

a crucial step for daptomycin activity.

Calcium binding causes oligomerization

and micelle formation allowing

daptomycin to interact with the acidic

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and neutral membranes. Micelles are
Adapted from: Robbel and Marahiel
Adapted from.

(2010)(20)
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daptomycin to bacterial cell membranes.

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The second step allows daptomycin insertion into the bilayer, promoted by a lipid tail.

This causes depolarization by potassium efflux. This is followed by diminishing cell

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membrane negative potential and resulting in inhibition of RNA and DNA synthesis and

eventually causing death (21;23;24). However, recent reports suggest that S. aureus

strains have developed resistance to daptomycin as well (25).

2.5 Factors leading to daptomycin non-susceptibility

a. Surface Charge

The proposed mechanism of action of daptomycin requires a lesser positive

charge for daptomycin to act and bind to the bacterial cell membrane. Hence, a lesser

positive charge is a prerequisite for daptomycin to act. An increased surface positive