GENETICS OF ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS SPECIES · genetics of antibiotic resistance in...

GENETICS OF ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS SPECIES

A SCHOLARLY PAPER PRESENTED TO

THE DEPARTMENT OF NATURAL SCIENCES IN CANDIDACY FOR THE DEGREE OF

MASTER OF SCIENCE

By ERICA L. CONNER

NORTHWEST MISSOURI STATE UNIVERSITY MARYVILLE, MISSOURI

APRIL, 2016

Genetics of Antibiotic Resistance 2

Running Head: GENETICS OF ANTIBIOTIC RESISTANCE

Genetics of Antibiotic Resistance

In Staphylococcus Species

Erica L. Conner

Northwest Missouri State University

SCHOLARLY PAPER APPROVED

Scholarly Advisor Date

Dean of Graduate School Date


I. Introduction

A. General Problem of Antibiotic Resistance to Medicine

Antibiotics are chemical substances used to prevent and treat infections caused by

microorganisms, such as bacteria, parasites and fungi. Antibiotic medications play a role

by suppressing or inhibiting the growth of microorganisms (Sanders et al. 2011). There

are more than 150 antibiotics currently available, but only 125 are effectively used for

treatment (Zuchora-Walske 2014). Antibiotics have benefitted society (Fabbretti et al.

2011) by providing treatments for tuberculosis, gonorrhea, methicillin-resistant

Staphylococcus aureus (MRSA), bronchitis, urinary infections, and many others (Tindall

et al. 2013). Yet, antibiotic resistance has become a worldwide issue that has expanded

and persisted among all nations on earth. New strains of antibiotic-resistant microbes

migrate easily, and they often create a “worldwide threat” to society (Levy 2002).

Developing countries have been impacted greatly due to malnutrition, poor living

conditions, and lack of medical sources (Alanis 2005). Over-the-counter antibiotics can

be obtained more easily in developing countries than in developed nations where their

access is more restricted (Alanis 2005). Resistant strains evolved during times of

environment instability, such as war, and migrations introduced new strains in

communities. Some antibiotics are no longer effective or efficient to treat infections

(Baquero and Campos 2003). The biggest challenge is when the most clinically

significant pathogens become resistant to most widely effective drugs (Lee et al. 2013).

One of the settings in which antibiotic resistance is most concerning is a hospital. In

the United States (US), numerical data for antibiotic resistance have been collected from


hospitalized patients since 2002 (Collins 2008). Yearly, more than 35 million US patients

require hospital care due to infection or disease (Wenzel and Edmond 2001).

Additionally, exposure to microorganisms in intensive care units presents a 60% risk of

acquiring nosocomial infections. In other words, these infections that are caused by

pathogens that acquired resistance to antibiotics (Haddadin et al. 2002). Nearly 2 million

patients develop hospital-acquired infections during treatment, 55% of which involve

antibiotic-resistant bacteria (Stone 2009).

Some bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), have

become resistant to antibiotics and spread throughout hospitals, clinical settings, and

other areas. Nowadays, there are only a few effective drugs that can control MRSA

(David and Daum 2010). In the U.S., National Nosocomial Infections Surveillance (NNIS)

stated that approximately 60% of S. aureus infections derived from intensive care units

were methicillin resistant, and the number of infections rose from 29% in 2005 to 49% in

2009 (Sydnor and Perl 2011). Vancomycin resistance in Enterococcus faecium

infections increased from 9,829 infections in 2000 to roughly 22,000 infections in 2006

(Sydnor and Perl 2011). With restricted options to cure infections, healthcare providers

propose to increase medical fees and introduce patients to medications with potentially

harmful? side effects (David and Daum 2010). Without effective antimicrobial agents,

any individual with an infection is approximately 70% more likely to die (Frieden et al.

2013).

Researchers have seen a consistent and rapid increase in antibiotic resistance,

especially in recent years. Sydnor (2011) stated that hospital epidemics can be

minimized if accurate surveillance of nosocomial infections are installed, best practices


are implemented to prevent and treat them, and health care personnel are trained to

avoid transmission of infectious microbes from patient to patient (Sydnor et al. 2011). As

of 2011, the Netherlands was reported to have the lowest antibiotic consumption rate in

Europe, and this country believes antibiotic resistance can be prevented by reducing the

dosage of antibiotics prescribed to a patient (Vandenbroucke - Grauls 2014).

Community environments have also shown signs of bacterial infections that are

resistant to antibiotics. Vancomycin resistance in Enterococcus faecium infections have

shown an increase percentage of 124% between 2000 to 2006 (Sydnor and Perl 2011).

For instance, Mycobacterium tuberculosis is a community-acquired pathogen

responsible for tuberculosis (TB), which infects 30% of the human population and

accounts for nearly 2 million deaths annually (Jensen et al. 2005). Until 2002,

streptomycin was routinely used to treat patients with tuberculosis (Gillespie 2002). Drug

resistance developed because M. tuberculosis was continuously exposed to a single

drug. Gillespie studies (2002) showed that a combination of isoniazid, pyrazinamide,

rifampin, and ethambutol drugs were able to control and prevent tuberculosis from

expanding (Gillespie 2002). As of 2014, a combination of multiple drugs diminished the

number of spontaneous mutations and increased the treatment rate by 48% (Fonseca et

al. 2015). Hence, antibiotic-resistant pathogens have become a major concern not only

in the healthcare facility, but also in community settings (Tomasz 1994).

B. Mortality Rate

Mortality and morbidity rates are escalating due to the prevalence of antibiotic

resistance strains in intensive care units (Hanberger et al. 2014). Non-resistant strains

are nonpathogenic, while antibiotic resistant strains are pathogenic, leading to an


increase death rate (Hanberger et al. 2014). In the US, about 2 million people

developed bacterial infections from 2005 through 2008 that were resistant to more than

one antibiotic, resulting in an estimated 99,000 deaths (Kallen et al. 2010). In 2011, the

EU had nearly 2 million people with nosocomial infections that lead to 200,000 deaths. In

the EU, housing of vulnerable patients within the same area increases the probability of

resistant strains to pass from one person to another (Guggenbichler et al. 2011).

Based on Table 1, multi-drug resistance (MDR) M. tuberculosis (TB) is one of the

three organisms that causes the highest mortality in humans. Highly diverse populations

and poor sanitation leads to development of multidrug resistance, which contributes to

95% of mortality rates in low- and middle-income states (Drobniewski et al. 2015). As of

2007 in India, MDR TB was twice as common in TB patients living with HIV versus in TB

patients without HIV because patients were showing spontaneously resistant mutants in

reserve drugs, such as ofloxacin (Isaakidis et al. 2011).

Despite the fact that MDR TB is curable, its treatment depends upon extensive

chemotherapy that is exceptionally costly for low-income nations. Efforts to prevent the

spread of the infection highly depend on the socioeconomic status of the country (Olson

et al. 2012). Implementation of new programs, effective screening tests, and efficient

therapy options assist reducing the mortality rates in US patients. Maintaining the low

mortality of TB in the U.S. will demand continued prevention and control efforts,

specifically fast diagnosis, guaranteed accomplishment of treatment, and efficient and

complete reporting (Geiter 2000).

Vancomycin-resistant Enterococcus (VRE) infections are also emerging as

serious health risks. VRE is the fourth most common antibiotic-resistant pathogen with


the highest expected annual causes in the US and the third most common with the

highest annual deaths. Several drugs have been used effectively against VRE, but its

mortality rate (Table 1) still spiked (Cho et al. 2013). A patient who contracts VRE is

considered immunosuppressed because he/she has been previously exposed to the

organism during a major surgery or other medical procedures, which have been treated

with multiple antibiotics (Collins 2008). These infections pose a 10% risk of death in

patients with graft transplant, but almost 70% in those with endocarditis, tumors, and

liver transplants (Kapur et al. 2000). Nonetheless, some authorities are more skeptical

and tend to admit that patients might have other medical conditions that could lead to the

increase of mortality and not necessarily attribute all mortalities to VRE infections (Cho

et al. 2013). A potential reason for high mortality rates among VRE infections is because

VRE can potentially transfer genetic traits to S. aureus, another organism with high

fatality rates (Cetinkaya et al. 2000).

According to Table 1, drug-resistant Campylobacter jejuni and Neisseria

gonorrhoeae infections are the most common cases of antibiotic resistant infections,

followed by MRSA which continues to be the number one antibiotic resistant pathogens

with highest mortality rate in the US. Carbapenem-resistant Enterobacteriaceae (CRE)

infections are relatively more predominant than MDR Acinobacter infections by showing

100 more annual deaths in the US (Gupta et al. 2011). Vancomycin-resistant

Staphylococcus aureus (VRSA) infections have shown no mortality rate in the US in

comparison with drug-resistant Candida. Drug-resistant Candida infections contribute to

the leading cause of invasive candidiasis due to the delays on finding appropriate and

efficient antifungal therapeutic agents (Pfaller and Diekema 2007).


Antibiotic- Resistant pathogens Expected annual cases of

antibiotic resistant

infections in the US

Expected annual

deaths in the US

Vancomycin-resistant

Staphylococcus aureus (VRSA)

N/A 0

Drug-resistant Mycobacterium

tuberculosis

9,582 1,631

Drug-resistant Candida 3,400 220

Multidrug-resistant Acinetobacter 7,300 500

Carbapenem-resistant

Enterobacteriaceae

9,000 600

Vancomycin-resistant Enterococcus 20,000 1,300

Methicillin-resistant Staphylococcus

aureus (MRSA)

80,461 11,285

Drug-resistant Neisseria gonorrhoeae 246,000 N/A

Drug- resistant Campylobacter Jejuni

(Gillespie et al. 2006)

310,000 120


Table 1 - Comparison of annual antibiotic resistant infections with annual deaths in U.S patients due to several types of bacteria (most data condensed from text in (Frieden 2013)).

C. Health Care Costs

Antibiotic resistance is a financial burden on the healthcare system. Physicians

are obligated to treat individual patients with the most effective drugs for an infection.

Health care institutions believe that hospital expenses are a form of reimbursing

physicians for their service to society (Roberts et al. 2009). Patients, on the other hand,

demand better healthcare service and better quality of life for them and for those around

them (Roberts et al. 2009).

Antibiotics for bacterial infections are costly, and sometimes hospitalization is

required until the infection is cleared, further raising costs (Roberts et al. 2009). In the

US, Ventola’s (2015) statistics showed that antibiotic-resistant infections incur roughly

$20 billion in annual healthcare costs and estimated that patients with antibiotic-resistant

infections cost from $19,000 to $29,000. According to a study by Dellit (2007), VRE

infections required up to 17 days of hospital care with an average cost of $27,000.

Antibiotic-resistant Pseudomonas aeruginosa infections incurred hospital fees of

$54,081 per patient, compared to $22,116 for those infected with antibiotic-susceptible

strains (Slama 2008).

S. aureus infections are expensive to treat, and MRSA infections are more costly

than those that are methicillin-sensitive (Singh et al. 2006). Inpatient treatment, which

includes overall/basic hospitalization costs, antibacterial drugs, preliminary exams, and

imaging will total close to $35,000 for patients with resistant strains (Filice 2010).


Assuming that 15% of the US patients become infected annually, costs for only MRSA

infections would total approximately $45 million (Filice 2010).

Currently, several elements have played a vital role in the cost of controlling

infection. The cost to create a new antibiotic is recently calculated at $1 billion (Slama

2008). Not only are novel antibiotics needed, but surveillance within each hospital must

be augmented (Slama 2008). Hospital administration plays a vital role on controlling

resistant pathogens, as well as enforcing rules to limit resistant strains from spreading.

Hospitals or medical centers can only initiate surveillance programs or policy changes to

improve infection control if there is financial support. Without financial support, resistant

pathogens will continue to develop and spread, and the quality of health care worldwide

will be drastically affected (Slama 2008).


II. Methods Used by Bacteria to Acquire Antibiotic Resistance

A. Basis for Antibiotic Resistance

Numerous mechanisms (illustrated in Figure 1) are used by bacteria to avoid

antibiotic treatment, such as reducing drug uptake, actively pumping antibiotics outside

of the cell, enzymatic modification of antibiotics, altering antibiotics’ target sites,

overproduction of antibiotic’s target, and metabolic bypass of the antibiotic’s target

(Wilcox 2004).

Figure 1 - Different antibiotic-resistance strategies (Wilcox 2004).


Decreased drug intake is an antibiotic-resistance mechanism used by bacteria

(Lee et al. 2013). Aminoglycosides are one of the main antimicrobial classes that are

addressed by reduced drug uptake (Lee et al. 2013). Resistance to aminoglycosides,

which block protein synthesis, can be acquired by modifying the number and structure of

the porin channels that allow passage of the antibiotic into the cell (Bonomo and Szabo

2006). Bacteria tend to be resistant to the number of aminoglycosides molecules present

in the cytoplasm (Ramirez and Tolmasky 2010). As a result, aminoglycoside uptake will

decrease, and they cannot enter the cytoplasm, bind to ribosomes and inhibit protein

synthesis (Ramirez and Tolmasky 2010).

Actively pumping drugs outside of the cell is accomplished by efflux pumps, as

demonstrated in Figure 1 (Webber and Piddock 2003). Efflux pumps are energy-

dependent (active transport) mechanisms that move antibiotics and other elements

outside of the cell (Ramirez and Tolmasky 2010). Pumps can be specific to a single

antibiotic or function with multiple targets (Nikaido 2009). Pumps that are functional

against multiple antibiotics are called multidrug efflux pumps and can lead to multidrug

resistance (Nikaido 2009). Genetic elements encoding efflux pumps may be expressed

from chromosomes and plasmids (Weinstein 2005). As an example, quinolones are a

class of antibiotics responsible for inhibiting DNA gyrase and type IV topoisomerase -

two enzymes important for bacterial DNA replication (Collin et al. 2011). Efflux pumps

can remove quinolones from the cell before their concentration is sufficient to inhibit

DNA metabolism (Drlica et al. 2009). Often times, this mechanism is employed with both

Gram-positive (e.g. Staphylococcus sp.) and Gram-negative (e.g. Pseudomonas

aeruginosa) bacteria (Drlica et al. 2008).


Enzymatic modification of antibiotics utilizes enzymes that are capable of altering

or destroying the antibiotic’s structure before it reaches its target (Figure 1). Enzymes

are selective due to their different specificities for their target (Mahon et al. 2014). A

common example of enzymatic modification of an antibiotic is found with aminoglycoside

resistance. Enzymes in aminoglycoside-resistant bacteria catalyze the modification at

different hydroxyl and ammonium groups of the 2-deoxystreptamine nucleus (nucleus

linked to various sugars through glycosidic linkages) by the addition of an acetyl, a

nucleotidyl, or phosphate group by acetyltransferases, nucleotidetransferases, or

phosphotransferases, respectively (Ramirez and Tolmasky 2010). Addition of these

functional groups (illustrated on Figure 2) to an antibiotic blocks their interaction with

ribosomes, leaving the bacterium drug resistant (Ramirez and Tolmasky 2010).

Figure 2 –Aminoglycoside Resistance: Mechanism of Action (Jia et al. 2013)


Bacteria may also alter target sites of antibiotics. Isolates of Mycobacterium

tuberculosis have become resistant to ciprofloxacin due to mutations of gyrA and gyrB

that combine to form DNA gyrase (Aubry et al. 2006). DNA gyrase has a crucial function

in DNA replication, where it nicks DNA, introduces negative supercoils, then re-joins

DNA (Collin et al. 2011). Quinolones, such as ciprofloxacin bind to GyrA and interfere

with its ability to cut DNA strands and reconnect them (Collin et al. 2011). However,

some strains of Mycobacterium tuberculosis produce mutant proteins that have changed

their physical structure, blocking ciprofloxacin from binding and inhibiting its function

(Collin et al. 2011).

Another example of altering antibiotics’ target sites to gain resistance can be

found with enzymes responsible for peptidoglycan synthesis. Peptidoglycan synthesis

during cell growth requires the activity of penicillin binding proteins (PBPs) that break

and reform peptide interbridges between peptidoglycan polymers (Kohanski et al. 2010).

PBPs can be targeted by β-lactam antibiotics (e.g. penicillin and methicillin) as an

effective means for halting cell growth (Kohanski et al. 2010). Upon structural

modification of the PBPs through mutations, PBPs have low affinity for these antibiotics,

making them useless (Kohanski et al. 2010).

Resistance to carbapenems can occur when bacteria produce carbapenem-

hydrolyzing enzymes (carbapenemases) that fall into two categories (serine

carbapenemases and metallo-β-lactamases) based on the reactive site of the enzymes

(Bush 2010). Serine carbapenemases also known as class A carbapenemases, undergo

a substitution that changes the active site (Queenan and Bush 2007). On the other hand,


metallo--β-lactamases (class B carbapenemases) cleave the beta-lactam ring of the

antibiotic and disrupt its activity (Queenan and Bush 2007).

Overproduction of an enzyme that is targeted by an antibiotic is a fourth

resistance mechanism employed by antibiotic-resistant bacteria (Nikaido 2009). Enzyme

overproduction can be used in response to sulfonamides, which mimic p-aminobenzoic

acid (PABA), a substrate of dihydropterate synthetase (Behera 2010). If PABA

conversion to folic acid by dihydropterate synthetase (DHPS) is blocked by

sulfonamides, biosynthesis of purines and several amino acids is blocked (Behera

2010). A cell can create an overabundance of PABA, diluting the antibiotic’s

concentration against its target molecule (Behera 2010). Therefore, a smaller fraction of

dihydropterate synthetase molecules will bind PABA, instead of the sulfonamide

antibiotic, preserving some enzyme function (Behera 2010).

A gene’s promoter site (sequence that initiates a gene’s transcription into

mRNA) can be altered to increase production of an antibiotic’s substrate (Nikaido 2009).

For example, trimethoprim functions by obstructing a different step in folic acid

production (Vilcheze and William 2012). Trimethoprim binds to an enzyme known as,

dihydrofolate reductase, which suppresses the reduction of dihydrofolic acid to

tetrahydrofolic acid (Vilcheze and William 2012). Trimethoprim resistance results from

overproduction of the enzyme dihydrofolate reductase (DHFR). If the promoter is altered,

more DHFR gene will be transcribed and translated into above-normal levels of DHFR

(Vilcheze and William 2012). This excess of DHFR enzyme compensates for those

enzyme molecules inhibited by trimethoprim and retains net activity in the cell (Vilcheze

and William 2012).


B. Transfer of Antibiotic Resistance

Genetic diversity in bacterial populations is a result of mutations, vertical gene

transfer, and horizontal gene transfer (Bennett 2008). Specific mechanisms that allow

the horizontal transfer of DNA material between bacteria are transformation,

transduction and conjugation (Bennett 2008).

1. Vertical gene transfer

The evolution of pathogenic bacteria is associated with innumerous influencing

factors dictated by their population genetics. Vertical gene transfer (VGT) or vertical

evolution is a mechanism in which genetic material is transmitted from a mother cell to

daughter cells during cell division (Vogan and Higgs 2011). VGT and mutation are the

mechanisms responsible for Darwin’s principles of evolution and natural selection and

the basic principles of Mendel’s work: spontaneous mutations on the bacterial

chromosome confer resistance to a member of the bacterial population (Gogarten et al.

2002). Mutations are considered rare, but bacterial growth rates are so rapid that

resistance can become dominant in a population very quickly (Davies and Davies 2010).

2. Horizontal gene transfer

Horizontal gene transfer (HGT) is defined as the transfer of genetic material

between individual bacteria of the same or different species (Aminov 2011). HGT relies


on three fundamental mechanisms in terms of genetic exchange: transduction,

transformation or conjugation (Aminov 2011).

3. Transduction

Transduction is the passage of genes from one bacterium to another via bacteria-

specific viruses called bacteriophages (Huddleston 2014). Bacteriophages replicate by

infecting bacterial cells and using them as a host to produce more virus particles. Upon

multiplication, virus particles accumulate, burst the cell and are released into the

surrounding environment to begin another round of infection (Huddleston 2014).

Bacteriophages are generally very host-specific, often infecting only small numbers of

strains within a species and rarely infecting more than one species. Therefore,

transduction is an HGT method that is viable for only closely related species.

Transduction occurs within the lytic cycle, which could be delayed due to a

lysogenic cycle. During a lysogenic cycle, phage DNA is combined into the

bacterial chromosome (known as a prophage) and remains dormant for some

length of time (Blackstock 2014). Usually, due to environmental cues (UV

damage to the cell, starvation, etc.), the phage genome will undergo excision

from the bacterial chromosome and begin the lytic cycle (Russell et al. 2013).

Defective excision steps play a crucial role because assembling phage heads

can accidentally and randomly package host DNA, instead of its own DNA

(Koneman 2006). Subsequent infection of host cells by these phage particles

would transfer DNA from the previous host to the new host. This also means that


this phage particle is defective for replication, and the next host cell will not die as

a result of infection. Therefore, transduction can be associated with antibiotic

resistance because antibiotic resistance genes are just as likely to be

accidentally packaged and transduced as any other gene (Koneman 2006).

4. Conjugation

Plasmid-mediated conjugation takes place in Bacillus subtilis, Streptococcus

lactis, and Enterococcus faecalis but is not present as frequently in Gram-positive as

Gram-negative bacteria (Mayer 2010). Plasmids can carry genes needed for formation

of a sex pilus, and, therefore, conjugation. Overall, conjugation is the most common

mechanism of horizontal gene transfer in bacteria (Huddleston 2014). Conjugation, as

the term indicates, is a physical linkage between two bacteria by direct cell-to-cell

contact. A sex pilus (tube-like appendage) is required for bacterial conjugation in Gram-

negative bacteria (Bennett 2008). Conjugation takes place when DNA is transferred

directly from a donor to a recipient bacterium through the pilus (Parija 2009). Transferred

genetic material can contain not only the genes required to make pili, but other

segments of the donor chromosome, as well. If antibiotic-resistance genes are among

those transferred, conjugation will lead to the spread of antibiotic resistance (Griffiths et

al. 2000). Not all bacteria are capable of producing sex pili, but conjugation can exist

between bacteria of different species (Griffiths et al. 2000).

Gram-positive cells are not known to produce sex pili, but conjugation is still

possible. In Gram-positive bacteria sticky surface molecules are formed in order to bring

the two bacteria together in what is known as a mating bridge (Grohmann et al. 2003).

This bridge allows cells to aggregate, and DNA can then be transferred from the donor

to the recipient cells.


5. Transformation

Transformation is a natural process of HGT, during which genetic material in the

environment is transferred across the cytoplasmic membrane, leading to integration of

foreign DNA within the host chromosome (Huddleston 2014). The ability to acquire

naked, foreign DNA is referred to as natural competence (Chen and Dubnau 2004).

During the natural transformation process, foreign DNA links to a DNA translocase

protein made by the host and enters the host cell (Domingues et al. 2012). Only one

single-stranded DNA molecule is retained by the host cell, while the other is degraded by

nucleases. The translocated, single-stranded DNA may then be integrated into the

bacterial chromosome by a RecA-mediated transformation, if the foreign DNA was linear

(Domingues et al. 2012). However, plasmid DNA (small, closed-circular DNA; discussed

later) may be maintained as a plasmid in the host’s cytoplasm. Acquired plasmid or

chromosomal DNA will continue to replicate and be expressed along with the host’s

original DNA (Bennett 2008). If the foreign DNA contained genes that encode antibiotic

resistance, the “transformed” cell is now resistant and can transfer this gene to its

daughter cells (Giedraitiene et al. 2011).

6. Transposons

Transposons are also important sources of antibiotic resistance acquired via

horizontal gene transfer (Huddleston 2014). Transposons are mobile DNA elements that

integrate into the chromosomal or plasmid DNA of prokaryotes (Lodish 2000). In other

words, transposons have the ability to move a short piece of DNA from one location to

another within a cell. Inverted repeats (IR) are short, palindromic sequences of


nucleotides that read in reverse direction and flank the gene(s) within the transposon to

facilitate recombination into the host DNA (Bousios et al. 2010). Between the inverted

repeats is the gene for transposase, whose protein product detects the ends of the

insertion sequences (Bousios et al. 2010). Transposases do not usually move

transposons to specific sequences in the chromosome; therefore it moves (hops) the

transposon into random positions (Alberts et al. 2002). In some transposons, the original

transposon leaves a copy of itself at the progenitor site while replicating itself in other

locations in a process called, replicative transposition (Skipper et al. 2013). Gene

sequences disrupted by transposition are no longer capable of forming their intended

products and are “knocked out” (Hickman et al. 2011)

The simplest transposable elements are known as insertion sequences, which

carry only the transposase genes necessary to insert into and excise itself from a

molecule of DNA (Hickman et al. 2011). However, standard transposons typically

contain inverted repeats, a transposase gene and unrelated genes encoding metal-

antibiotic resistance markers, proteins needed for causing disease, or simply metabolic

genes (Juhas et al. 2009). Insertion sequences can also lead to formation of composite

transposons (Wagner 2006). Composite transposons are normally a pair of insertion

sequence elements that flank a sequence of DNA composed of one or more genes,

often coding for antibiotic and/or metal resistance (Wagner 2006). The terminal insertion

sequence is able to transpose by itself, which can be seen in transposon Tn5, Tn9 and

Tn10 families (Wagner 2006). Also, the Tn3 family of transposases contains genes for a

transposase, β-lactamase and resolvase (enzyme used for excising the transposon from

the host DNA) in which all are located in between the inverted terminal repeats (Mayers


2009). β-lactamase is a resistance gene used for the β-lactam antibiotics, including

penicillin and its derivate (Mayers 2009).

7. Bacterial plasmids

Plasmids are independently-replicating, double-stranded DNA molecules that are

separate from chromosomal DNA in prokaryotic cells (Murray et al. 2012). Plasmids are

generally up to approximately 10 kilobases in size and are often considered mini-

chromosomes (Bennett 2008). Plasmids can be described as having narrow- or broad-

host range (Gelder et al. 2008). Narrow-host range (NHR) plasmids are the most

common plasmids in nature. A limited number of NHR plasmids have been developed in

specific hosts to be second chromosomes due to their stability in a particular strain and

an inability to replicate in other strains or species. This stability allows NHR plasmid size

to successfully increase by acquiring genes from the host’s chromosome due to

transposition and recombination events (Gelder et al. 2008). Broad-host-range (BHR)

plasmids can be transferred to and replicate in relatively diverse and distant bacterial

species than can NHR plasmids (Gelder et al. 2008). Hence, NHR plasmids seemed to

play a role as genetic storage for the same group of species, while BHR act as an active

gene transporter between diverse species (Gelder et al. 2008). Occasionally, plasmids

possess characteristics that provide some selective advantage to the host bacterium,

such as virulence elements, adherence and antibiotic resistance genes (Mahon et al.

2014). The expansion of plasmids is definitely something that the public health sector

wants to avoid (Gelder et al. 2008). With better technology, researchers will be able to

analyze the epidemiology of the plasmids.


C. Development of Antibiotic Resistance

Antibiotics function either by altering the bacterial structures or by interfering with

fundamental physiological mechanisms of the bacteria (e.g. growth, DNA replication,

gene expression, etc.) (Kohanski et al. 2010). Bacteria may acquire or be innately

resistant to an antibiotic because they have no sites where molecules can enter the cell,

because efflux pumps expel the antibiotic, or the cell is capable of producing enzymes

that interfere with and destroy the antibiotics (Fernandez and Handock 2012). There are

numerous reasons that can lead to the survival of the bacteria, regardless which drug is

used to prevent infections from developing (Davies and Davies 2010).

The use and misuse of antibiotics is another factor that allows the expansion of

resistant organisms. To make matters worse, clinicians prescribe antibiotics that perhaps

are unnecessary, and patients increase the problem by requesting drugs for every

infection (Weckx 2012). Diseases for which antibiotics are prescribed, like sinus

infections, are expanding, especially at day-care settings (Oxford et al. 2013). With an

increase in day-care environments, infections will surge; therefore, more antibiotics will

be used, leading to an increased spread of drug resistance. Viral infections (e.g.

influenza or cold) are often treated with unnecessary drugs at patients’ demands,

increasing chances of creating resistant bacterial strains that can evolve. Even when

drugs are prescribed correctly, patients fail to use them appropriately. Therefore, the

weak organisms are killed, but the strongest will persist and propagate within the

population (Weckx 2012).

Even when antibiotics are used correctly, problems can arise. Travel increases

the probability of transferring drug resistance from one country to another at jet speed.

Hospitalized patients often times are being medicated with several drugs, which create a


favorable situation for multi-resistant strains to develop and thrive. Hospitalized patients

often have compromised immune systems, which increases their vulnerability to many

infections, in particular those who are antibiotic-resistant (Oxford et al. 2013).

To conclude, a variety of factors such as population growth, usage and misuse of

antibiotics have all contributed to development of antibiotic resistance. Hopefully, the

evolution of resistance can be stopped eventually, if society begins to change its

behavior and attitude towards infectious diseases. Precautions should be taken serious

for not only the patients’ benefit, but for the good of all humankind (Davies and Davies

2010).


III. The Genus Staphylococcus

A. Natural Habitat and Host Range

Worldwide, Staphylococcus species are notorious human pathogens and

are the focus of comprehensive studies among researchers. More than 40

Staphylococcus species cause diseases known to be clinically and economically

significant (Lindsay 2008). Public health has become threatened by antibiotic-

resistance strains that pass among humans, and it is thought that species or their

habitats might correlate with type of infection (Lindsay 2008). There is a wide host

range among species that belong to the genus Staphylococcus. For instance, S.

aureus can be isolated from horses, rabbits, humans, and ruminants and cause

skin infections, septic arthritis, and others (Lindsay 2008).Overall, the pathogens

among the staphylococcus species can be colonize among man and other

mammals. Depending upon the host, infections can range from a mild skin

infections to a more severe type of syndrome. Table 2 shows the host range and

infections caused by several species of staphylococci.


Table 2: Hosts of Staphylococcus species according to the type of infections.

Referen

ce

Pathogen Host Associated Disease

Mahon et

al. 2011

S. aureus Humans

and

Animals

Skin and wound

infections, scalded skin

syndrome, toxic shock

syndrome, toxic

epidermal necrolysis,

and food poisoning

Raz et al. 2005 S. saprophyticus Humans Cystitis an urinary tract

infections

Lindsay

2008

S. delphini Horse

and

cows

Pyoderma

Lindsay

2008

S. hyicus Pig Exudative dermatitis

Lindsay

2008

S. sciuri Pig Greasy Pig syndrome

Vela et

al. 2012

S. xylosus Gerbils

and

birds

Nasal dermatitis in

gerbils, avian

staphylococcosis and

Pyelonephritis

Tibra et

al. 2009

S. gallinarum

Chickens Endophthalmitis

Hoffman

et al.

2007

S. auricularis Humans

(infants)

Opportunistic

infections

Bocher

et al.

2009

S. lugdunesis Humans Endocarditis,

septicemia, meningitis,

skin and soft tissue

infections

Wang et

al. 2013

S. intermedius Canines Pyoderma and skin

tissue infections in

dogs

Lindsay

2008

S. epidermidis Cows

and

goats

Mastitis


B. How are Staphylococcus Infections Acquired?

An individual is exposed to diverse strains of microorganisms daily

without causing any infections. Human microbiota are usually nonpathogenic, as

long as they remain within their normal habitat (Reid et al. 2011). When normal

microbiota are displaced, infections can occur. Human skin is one of the

favorable sites for pathogens to establish a direct interaction and potentially

cause infections (Ki and Rotstein, 2008). Infections associated with staphylococci

can range from minor skin problems to life-threatening systemic infections (Ki and

Rotstein, 2008). A variety of conditions may be associated with staphylococcal

infections. Therefore treatment depends on the site and severity of the illness (Ki

and Rotstein, 2008).

1. Bacteremia

Bacteremia, the presence of bacteria in the blood, is one of the main routes

taken to destabilize the immune system of an individual (Delost 2014). Bacteremia

cases begin showing signs of fever, chills and hyperventilation but at late stages

can travel through the internal organs (heart, brain and lungs), catheters and

implanted devices, such as pacemakers (Delost 2014). Bacteria enter the

bloodstream through small cuts and wounds, following by the adhesion to inner

membranes of the tissues, which subsequently triggers a cascade of infections

(Cabell et al. 2003).

2. Food Poisoning Food poisoning is derived from ingesting contaminated products.

Symptoms such as nausea, vomiting, diarrhea, dehydration, and low blood


pressure can present (Argudin et al. 2010). Often times, negligent food workers

contract staphylococcal infections and continue to process food without further

precautions. Once S. aureus is present inside of the human body, it can trigger

infections (Argudin et al. 2010). S. aureus is capable of forming a broad variety of

toxins, such as staphylococcal enterotoxins (SEs) responsible for creating potent

biological effects, such as damage of gastrointestinal tract (Dinges et al. 2000).

Usually SEs are active in low amounts and are capable of resisting heat and

cannot be eliminated by cooking. Acid-resistant SEs can withstand the pH of the

digestive tract (Loir et al. 2003).

3. Skin

Staphylococcal infections are normally skin infections, such as boils,

impetigo, and staphylococcal scalded skin syndrome (SSSS) (Proft 2013). Usually,

bacteria invade superficial layers of the skin via open wounds or cuts. Boils, for

instance, are skin infections created by pockets of pus that developed in a hair

follicle or oil gland (Murray et al. 2008). The skin over the infected site normally

shows some redness and swelling (Murray et al. 2008). In this case, the boil

breaks open and pus drains and causes other infections. Boils generally develop

under the arms or around the groin (Ibler and Kromann 2014). SSSS forms blisters

due to the production of toxins that disrupts the epidermis, leaving the affected

area exposed to harmful pathogens. Clinically, SSSS is seen to spread throughout

large portions of the body (Bukowski et al. 2010). A defined area of infection

created by SSSS is commonly diagnosed as impetigo (Bukowski et al. 2010).


C. Risk Factors for Infections

1. Recent Hospitalization

Regardless of intense efforts implemented by infection control programs,

bacteria remain persistent in hospital settings where they can infect susceptible

people, primarily those with weakened immune systems (Sydnor and Perl 2011).

Infections can occur before, during, or after surgery if certain precautions are not

followed (Sydnor and Perl 2011). In a hospital setting, staphylococci infect critically

ill patients more than any other organism because of the way they spread.

Generally staphylococci, in particular S. aureus, diffuse by skin-to-skin contact

between doctors, nurses, or visitors (Barnes 2010). Normally, someone who

contracts a staphylococcal infection, such as MRSA, can stay hospitalized for up to

10 days (Barnes 2010). Therefore, precautions such as maintaining proper

personal protective equipment to protect patients and physicians must be followed

(Barnes 2010). However, what makes staphylococci significant is that several

species have become resistant to antibiotics. Methicillin-resistant S. aureus

(MRSA) is a prime example of antibiotic resistance to methicillin and penicillin

(Nikaido 2009). With limited treatments, it becomes extremely difficult to control the

spread of infection. The best way to minimize the problem is to maintain good

hygiene between health care workers and extending these protocols to visitors and

patients (Barnes 2010).

2. Residing in a Long-Term-Care Facility

MRSA is 20% more common in long-term-care facilities (LTCFs) than it is

in hospitals (Mazur and Gudiol 2009). Urinary tract infections are the number one


risk found among elderly that reside in the LTCFs, followed by respiratory and

gastrointestinal infections (Bonono 2000). As a whole, these diseases comprise

94% of the infections in a LTCF due to the lack of training among staff and proper

care of residents (Engelhart et al. 2005). Some individuals admitted to a care

facility are likely to carry MRSA and have the ability to spread it to other patients

(Bonono 2000). As the population ages and technology advances to provide better

care for patients, both the number and proportion of people residing in LTCFs will

increase (Bonono 2000). Hospital-acquired infections differ from those contracted

in community settings because it is an older population of patients with a broad

range of disabilities (Mathei et al. 2007). Some characteristics of a nursing-home

setting promote the growth of infectious diseases. Residents tend to be clustered

in confined areas and daily activities usually involve groups (Mathei et al 2007).

Also, healthcare workers might have limited training in how to control infectious

diseases. Understaffing is also a common problem in LTCFs, and caregivers are

often too occupied to worry about infectious control (Mathei et al. 2007).

3. Indwelling devices

As opportunistic pathogens, staphylococci can migrate from medical

equipment to an individual’s internal organs (Otto 2008). Dialysis, catheters,

pacemakers and prosthetic devices are examples of invasive, indwelling devises

that are used routinely in hospital facility (Guggenbichler et al. 2011). As an

example, S. epidermidis can directly and indirectly cause infections, despite its role

as a constituent of human skin’s normal microbiota. Patients with weakened and

debilitated immune systems are more vulnerable to S. epidermidis infections when

introduced to the organism via the implantation of medical devices (Otto 2008).


According to Baddour, in 2003 roughly 53% and 20% of the patients became

infected with S. aureus and coagulase-negative staphylococci (CoNS),

respectively, as a result of prosthetic vascular graft. Indwelling devices side-step

the normal defense mechanism of the epidermal surface and provide a habitat in

which pathogens can proliferate protected from the patient’s immune defenses

(Mehta et al. 2014). Therefore, proper maintenance and cleaning of these devices

could reduce the risk of staphylococcus infections (Mehta et al. 2014).

D. Physiology of How Staphylococci Cause Disease

1. Human Pathogens

Staphylococci are known to be opportunistic pathogens, meaning that they

take advantage of a wound or weakened immune system within a host. Infections

are predominantly found in hair follicles, sutures, and even along the respiratory

tract (Mahon et al. 2011). Upon entering skin or mucous membranes,

staphylococci are able to produce and secrete several components that allow them

to survive in the host and cause damage to host tissues (Coates et al. 2014).

Pathogenicity of staphylococci can be attributed to various virulence factors,

including surface proteins (Protein A), exfoliatin toxins, and biofilm formation

(Crossley et al. 2009).

Staphylococcus aureus has on its cell wall a surface protein that is referred

to as Protein A, which is capable of attaching to a fragment of the antibody

Immunoglobulin G (Mahon et al. 2011). IgG is a protein synthesized by B cells that

protects the internal tissues and binds to foreign substances upon entering the


body (Janeway et al. 2001). Once bound by IgG, foreign bodies are typically

engulfed and destroyed by phagocytic white blood cells. However, Protein A of S.

aureus binds in the wrong orientation to the IgG surface (Tattlybaeva et al. 2013).

As a result, white blood cells show decreased ability to ingest and kill them, and

potential for diseases increases (Tattlybaeva et al. 2013).

Exfoliatin Toxins (ETs), also referred to as epidermolytic toxins, are known

to cause SSSS (affecting large parts of the skin) and can have implications in

bullous impetigo (affecting limited areas of the skin) (Bukowski et al. 2010). Serine

proteases are enzymes present in ETs responsible for cleaving desmosomal

cadherins on the extracellular layer of the skin (Bukowski et al. 2010).

Desmosomes are cell junctions composed of cadherins linked to catenins that

interact to form strong mechanical attachments between basement membrane and

epithelial cells (Garrod and Chidgey 2007). Disruption of desmosomal cadherins

causes cell attachments to weaken and, as a result, clinical manifestation of SSSS

ensues (Garrod and Chidgey 2007). During early stages of SSSS, fever, fatigue

and malnutrition are often followed by erythematous rash (large fluid blisters).

Bullous impetigo refers to large liquid filled blisters, specifically present at the site

of infection (Bukowski et al. 2010). Diagnosis involves PCR or randomly amplified

polymorphic DNA analysis (Bukowski et al. 2010). Strains producing ETs are

strictly treated intravenously with antibiotics. ET-producing strains contain the eta

gene, which encodes for exfoliative toxin A (Magni 2010), is only positive in 10% of

MRSA cases (Bukowski et al. 2010). Hence, a vast range of antibiotics can still be

effective, according to the patient’s needs (Bukowski et al. 2010).


Biofilms formed by staphylococci are responsible for the microorganisms’

adherence to each other in an aqueous matrix (Schommer et al. 2011). Adherence

takes place due to a secreted matrix, referred to as extracellular polymeric

substance (EPS). EPS can be made of extracellular components such as DNA,

proteins, and polysaccharides, although polysaccharides and proteins are most

common and abundant (Flemming et al. 2007). Biofilms on living or nonliving

surfaces show increased resistance to antibiotics and disinfectants. Infections in

cystic fibrosis patients and indwelling medical devices become problematic when

biofilms are involved (Joo and Otto 2012).

Development of bacterial pathogenicity depends greatly upon the formation

of the biofilm and the interaction between bacteria and the host. Biofilms are

capable of hiding bacteria from antimicrobial agents and affect the host immune

system during and after the infection (Joo and Otto 2012). Diagnoses of biofilms

can be accomplished by performing microscopy and culture studies (Hassan et al.

2011). Current treatment involving a combination of a StaphVAX (polysaccharide

conjugate vaccine) and vancomycin usually shows effectively reduced signs of

infection. However, further treatments must be studied to provide a better and

efficient approach (Brady et al. 2011).

2. Non-Human Pathogens

As referred to in Table 2, several species of Staphylococcus are animal

pathogens or promote infections that impact humans and animals. Pathogenicity of

these species is also due to a variety of opportunistic infections including skin

lesions, pyoderma, and mastitis.


S. hyicus is found predominantly associated with pigs, particularly along

nasal and vaginal mucosa (Dworkin et al. 2006). S. hyicus causes exudative

epidermatitis, more commonly referred to as Greasy Pig Disease (Casanova et al.

2011). Greasy Pig Disease, as the name suggests, causes greasy, peeling skin

due to the production of an ET that forms blisters that disrupts spinosum

epithelium cells. The spinosum epithelium becomes fragile and ruptures, allowing

entry of pathogens (Fudaba et al. 2005). To control the spread of infections, this

disease can be treated using a combination of procaine penicillin G and novobiocin

for a period of 5 days (Park et al. 2013).

S. intermedius is naturally present among skin microbiota of all canines,

and no pathogenicity is known for humans (Tanner et al. 2000). S. intermedius

directly affects canine skin flora but indirectly impacts their central nervous system

(Koneman 2006). Infections that occur at the central nervous system level are

poorly understood. The brain barrier is an extremely complex membrane capable

of preventing the entry of invasive pathogens. Toxins and other organisms may

enter through the skull and the selective membranes, but that rarely occurs

because of the brain membrane isolation (Durdik et al. 2009). Development of

pyoderma occurs when S. intermedius invades the superficial layer of the skin and

hair follicles without formation of a scab (Futagawa-Saito et al. 2006). Without a

scab or coating, the epidermis becomes too fragile, superficial scale is produced,

and debris will accumulate in and around hair follicles, leading to hair loss

(Zachary and McGavin 2013). Infections normally respond well to cefotaxime

therapy (Durdik et al. 2009).


S. lentus is primarily found in animals that are used as a source of aliment,

such as cattle, poulty, and goats (Schwendener and Perreten 2012). Rarely, S.

lentus can transmit infections to humans, but most commonly this organism can

inflame the breast tissues of sheep and goats, leading to pain, redness and

soreness (Schwendener and Perreten 2012). Once the breast is infected, milk

becomes very cloudy due to the presence of clots. Normal milk contains

predominately epithelial and white blood cells (Rupp et al. 2008). If mammary

gland infection occurs, the number of neutrophils will increase in the bloodstream

(Rupp et al. 2008). Hence, by performing a somatic cell count it is possible to

determine that cows and/or goats with high cell counts will have greater risk to

contract clinical mastitis in comparison to those with lower somatic cell counts

(Rupp et al. 2008). Treatment should be focused on culture results obtained from

milk samples (Pieterse and Todorov 2010). Dry-off (non-lactating) period is an

efficient method to cure mastitis (Pieterse and Todorov 2010). Antibacterial agents

may be utilized during the non-lactating period in the infected animal. Once the

dry-off period is completed, prophylactic antibiotics can be administered to prevent

subsequent infections (Pieterse and Todorov 2010).


E. Diagnosis

1. Selective Testing for Identification of Staphylococcus species Staphylococci testing is accomplish by doing several and selective

laboratory examinations. The schema for identification of Staphylococcus species

relies primarily on microscopic confirmation of a Gram-positive coccus, followed by

a catalase-positive test (Mahon et al. 2011). Catalase-positive cultures then

undergo coagulase testing. Coagulase-positive results are seen mainly in S.

aureus, whereas coagulase-negative cultures (CoNS) are seen mostly in other

staphylococcus species, such in S. epidermidis (Foster 1996). CoNS can then be

examined for oxidase/bacitracin susceptibility. Cultures resistant to

oxidase/bacitracin will proceed with novobiocin susceptibility test (Mahon et al.

2011). Most CoNS are novobiocin-sensitive, whereas S. saprophyticus is known to

be novobiocin-resistant (Mahon et al. 2011).

2. Salt Tolerance and Mannitol Fermentation

Many media are utilized for identification of staphylococci, but certain

media are more useful than others (Tille 2013), such as Mannitol Salt Agar (MSA),

Blood Agar (BA), and Muller-Hinton Agar (MHA). MSA is a selective and

differential media with 7.5% of NaCl concentration that favors the growth of Gram-

positive bacteria. Any isolates forming colonies on MSA are considered salt-

tolerant. MSA also differentiates mannitol fermentation to acids by changing color

from red to yellow. Non-fermenters of mannitol leave the medium red (Mahon et al.

2011). Staphylococcus aureus ferments mannitol and the medium turns into


yellow color while other non-pathogenic staphylococci will not ferment mannitol

and the media will remain red in color (Mahon et al. 2011).

3. Catalase Catalase is an enzyme that catalyzes the conversion of hydrogen peroxide

into water and oxygen. Detection of catalase is a key feature for differentiation of

staphylococci from streptococci. Bacteria are characterized as catalase positive

when bubbles are formed when cells contact hydrogen peroxide. Staphylococcus

species are catalase-positive, whereas species of Streptococcus are catalase-

negative (Mahon et al. 2011).

4. Coagulase Reaction

Staphylococcus and Yersinia are two of the few genera that can produce

coagulase (Lackie 2010). Coagulase is an enzyme that converts fibrinogen to

fibrin, which forms clots that encapsulate cells. Coagulase occurs when clumping

factor binds to the cell walls of bacteria that react with fibrinogen. Then fibrinogen

dissolves, allowing cells to clump together (Kateete et al. 2010). Coagulase

differentiates staphylococcal isolates, and it can be classified as CoNS or

coagulase-positive staphylococcus (CPS) (Foster 1996). CoNS isolates are the

most frequent in medical microbiology laboratory because they survive in dead or

decomposing matter, but CPS isolates are considered to be the most pathogenic

(Foster 1996).

5. Clumping Factor

Clumping factors are heat-stable proteins that reside in the cell wall of S.

aureus (Tille 2013). These proteins cause clumping of fibrinogen or fibrin.


Disruption of the bacterial cell wall allows the release of clumping factor, which can

then act upon the fibrinogen in the plasma, allowing blood cells to aggregate (Tille

2013). Despite the fact that clumping factor and coagulase might have similarities,

they prove to be distinct from each other. Coagulase is an enzyme created by

many bacteria that distinguishes S. aureus from CoNS species (Becker et al.

2014). The confusion occurs because clumping factor is a portion of coagulase

that is bound to the bacterial cell wall and links directly to fibrinogen. Clumping

factor and coagulase act independently from each other (Josefsson et al. 2001).

6. Hemolysins Hemolysins are one of many features utilized to differentiate staphylococcal

species from other organisms. Hemolysins are microbial proteins that can lyse red

blood cells (RBCs) by disrupting their cell membrane. Many hemolysins are pore-

forming toxins which induce lysis of RBCs by creating pores in their cytoplasmic

membrane (Thompson et al. 2011). This can be visually seen by performing a

blood agar test (Nayak et al. 2013).

Blood Agar (BA) is a nutritive medium composed of tryptic soy agar

amended with 5-10% of sheep, rabbit, or horse blood and is used to grow a variety

of bacteria. The identification of each species would depend on the hemolytic

activity (Mahon et al. 2011). If RBCs are completely destroyed by a hemolysin and

a clear zone forms around the colonies. This is referred to as β-hemolysis. In the

agar, partial destruction shows a greenish color underneath the colony because

bacteria contain hydrogen peroxide that converts hemoglobin (red) into

methemoglobin (green). Non-hemolytic or γ-hemolysis is the result of no


destruction of RBCs and therefore, the agar does not undergo any changes

(Engelkirk et al. 2011).

S. haemolyticus, S. lugdunensis and S. aureus can produce hemolytic

zones around the colonies (Mahon et al. 2011). Others such as, S. epidermidis are

referred to be non-hemolytic (Mahon et al. 2011). However, S. epidermidis shows

partial destruction of hemoglobin in the RBCs, referred to as α-hemolysis

(Engelkirk et al. 2011).

7. Production of Deoxyribonucleases Testing for enzymes which degrade DNA, deoxyribonucleases (DNAses), is

essential to the identification of potentially pathogenic bacteria (Kateete et al. 2010).

Testing involves streaking the organisms on an agar medium that also contains DNA

and a dye which changes color in the presence of the degraded DNA (Tille 2013).

Several staphylococcal species are able to produce this enzyme, such as S. aureus, S.

epidermidis, S. intermedius (Pammi et al. 2013), and S. hyicus (Bhatia and Zahoor

2007).

8. Thermostable Nuclease (TNase)

TNase is an enzyme that is produced only by a few species of

Staphylococcus (Enoch et al. 2008), including S. aureus, S. hyicus and S.

intermedius. TNase testing is performed by placing blood broth from the blood

culture in heated environment (60 °C) and then transfering the suspension to an

agar medium for up to 4 hours at room temperature. If the DNA has been

degraded a clearing zone will form, demonstrating the TNase activity (Enoch et al.

2008).


9. Sensitivity to Novobiocin and Methicillin

Mueller-Hinton Agar (MH) with 2% NaCl makes the agar selective to

staphylococcus species (Mahon et al. 2011). MH contains acid digest of Casein,

beef extract, starch and agar, specifically to select pathogenic Neisseria species

(Mahon et al. 2011). The translucent appearance of the agar helps determine the

susceptibility to antimicrobial agents. The Kirby-Bauer method continues to be a

very common method to measure bacterial resistance. If bacteria growth is

inhibited in the presence of a certain antibiotic, a zone of inhibition (no visible

growth) is formed (Mahon et al. 2011).

Novobiocin is an antimicrobial agent discovered in Streptomyces niveus

that disrupts the normal ATP formation and its activity (Leboffe and Pierce 2012).

Most CoNS can be distinguished by using the novobiocin test. Novobiocin

sensitivity is tested by placing a 5-μg disk containing the antibiotic onto Muelher-

Hinton agar (MH) for 24 hours at room temperature (Leboffe and Pierce 2012). A

novobiocin-susceptible isolate forms a zone of inhibition greater than 16 mm in

diameter around the disk. If the organism is resistant to novobiocin, no zone would

be formed. S. saprophyticus is often used as a positive control because it is

resistant to novobiocin (Leboffe and Pierce 2012).

The Kirby-Bauer test differentiates from novobiocin testing by using a

choice of antibiotics that should be directly applied into the MH and then classified

as being Resistant (R), Intermediate (I) or Susceptible (S) according to the

organism in the study.


S. aureus is said to be resistant to methicillin upon determining that no

visible growth occurred in the agar (Mahon et al. 2011). Based on Clinical

Laboratory and Standards Institute guidelines, S. aureus is resistance to methicillin

when the minimal inhibitory concentration (MIC) is > 4 mg/L and susceptible when

the MIC is ≤ 4 mg/L (Brown et al. 2005).

F. Treatment

Staphylococci are considered to be the most pathogenic organisms in

healthcare facilities, as well as in society (Mahon et al. 2011). Without prevention,

they can impact the health of an individual at any age. The choice of an

antimicrobial agent should follow certain criteria, such as drug sensitivity, site of

infection and disease condition. The two main types of treatment used are surgery

or antibiotic chemotherapy (Leekha et al. 2011). In the majority of cases, patients

that require surgical treatment also need antibiotics. However, each individual

requires personalized treatment based upon their medical condition (Leekha et al.

2011).

Some CoNS, such as S. epidermidis, are known to cause UTIs and serious

inflammation but have become resistant to drugs such as methicillin, gentamicin,

ampicillin, and erythromycin (Villari et al. 2000). Currently, S. epidermidis is treated

by using vancomycin or rifampin (Villari et al. 2000). Nearly 20% of UTIs are

derived from S. saprophyticus, which are novobiocin resistance, and are treatable

with multiple antibiotics (Raz et al. 2005). S. haemolyticus is considered to be the

most antibiotic-resistant organism among the CoNS family (Barros et al. 2012).

Strains have become resistant to multiple drugs such as penicillins, ampicillin,


gentamicin, and ciprofloxacin (Barros et al. 2012). Vancomycin was reported to

treat patients that were infected with S. haemolyticus (Biavasco et al. 2000).

CoNS tend to create problems when it comes to animal infections, as well

(Mahon et al. 2011). Pigs typically contract S. hyicus, which causes exudative

epidermidis (Casanova et al. 2011). This disease is highly contagious to other pigs

and potentially to humans through skin exfoliation (Casanova et al. 2011). No

satisfactory treatment has yet been determined (Park et al. 2013). However, the

use of topical antibiotics (procaine penicillin G and novobiocin) appeared to be the

most efficient way to prevent infection from spreading (Park et al. 2013).


IV. Pathogenesis of Staphylococcus aureus

A. Mode of Pathogenicity

Staphylococcus aureus is a well-known bacterium for its commensal role in animal

health. It is commensal because it can harmlessly colonize a host’s nasal cavities,

armpits, vagina, or pharynx (Jenkins et al. 2015). Commensal bacteria protect the

superficial layer of our body from colonization of potentially pathogenic bacteria. For

instance, S. aureus produces bacteriocins (toxins created by bacteria that target other

bacteria) that impede pathogenic Staphylococcus epidermidis from increasing in number

(Chiller et al. 2001).

A versatile, pathogenic bacterium is formed when an organism successfully

overcomes the normal host defense protection and causes disease (Chiller et al. 2001).

S. aureus causes superficial lesions, such as boils or furuncles, systemic infections

(endocarditis and osteomyelitis), food poisoning, and toxic shock syndrome (Jenkins et

al. 2015). Infections may result from a combination of actions between multiple genetic

factors or single virulence factors (Jenkins et al. 2015). Infections commonly result from

wounds that allow staphylococci to access neighboring tissues and the bloodstream.

Infections can remain localized or become systemic, depending upon the interaction

between virulence factors and host-defense mechanisms (Anderson et al. 2012).

1. Adherence to Host Tissue

S. aureus is found primarily in the nares, and nearly 20% of the human

population is considered to be carriers for this pathogen, which increases risk of

contracting an infection (Naber 2009). Cells are capable of binding to host cells via

extracellular-matrix elements with the support of microbial-surface-components


recognizing adhesive-matrix molecules (MSCRAMM), which are a collection of adhesive

molecules (Clarke and Foster 2006). MSCRAMM adhere to fibrinogen (protein that

assists with the formation of blood clots) via different binding sites (Cognasse et al.

2015). Consequently, MSCRAMM function during early stages of infections. Due to the

broad range of MSCRAMM types, a variety of S. aureus strains may initiate an array of

diseases (Bien et al. 2011). For instance, coagulase is a type of adhesive molecules in

the MSCRAMM which links to prothrombin to produce staphylothrombin (Cognasse et al.

2015). Staphylothrombin converts fibrinogen into fibrin, leading to platelet aggregation

and allowing adherence of S. aureus to platelets (Vanassche et al. 2012). Fibrin clots

protect cells from phagocytosis, and, as a result, they will persist and cause staph

infections (Cheng et al. 2010). All clinical isolates of S. aureus are known to be

coagulase-positive, making it a classical phenotypic marker for identifying this bacterium

(Cheng et al. 2010).

2. Invasion of Host Tissues

The invasion of S. aureus into host tissues activates production of a wide variety

of extracellular proteins, each with a distinct role (Bien et al. 2011). Alpha-toxin, also

referred to as alpha hemolysin, is one of several pore forming toxins and a critical

virulence factor for S. aureus against platelets, erythrocytes, and monocytes (Berube

and Wardenburg 2013). S. aureus produces alpha-hemolysin as a single molecule to

facilitate the linkage with eukaryotic cell membranes (Frank et al. 2012). On target

membranes, its subunits oligomerize to create heptameric rings with a central pore

(Berube and Wardenburg 2013). The central pore formed on the target membrane can

now permit leakage of simple ions (e.g. Ca2+) that would result in apoptosis (Berube and

Wardenburg 2013).


Beta-toxin is composed of sphingomyelinase, a fatty-acid enzyme responsible for

catalyzing sphingomyelin into ceramide and phosphorylcholine (Kurek et al. 2013).

Sphingomyelinase is found in animal cell membranes capable of targeting membranes

rich in this lipid (Kurek et al. 2013). Beta-toxin lyses erythrocytes by crossing the lipid

membrane and promoting the release of their contents (Fournier and Philpott 2005).

Normally, beta-toxin is expressed by S. aureus strains in non-human infections to a

greater extent than in human isolates.

Delta-toxin, despite its small size, is produced by 97% of strains of S. aureus

(Dinges et al. 2000). It is also speculated that nearly 60% of coagulase-negative

staphylococci (CoNS) form delta-toxin, especially S. epidermidis (Noble 2004). Delta-

toxin is said to be lytic to a variety of cells by the disintegration of their membranes.

However, its pathogenicity remains unknown (Turnidge et al. 2002).

Gamma-toxin and Panton-Valentine-Leukocidin (PVL) are additional toxins

produced by S. aureus. Each individual toxin creates secreted proteins, called S and F

subunits.

S subunit stands for slow-eluting proteins during purification on an ion exchange

column responsible for identifying the cell specificity of the cytolysin (Sabour and

Griffiths 2010). S class is created as a single molecule that ended up linking tightly to

other S molecules, forming a multi-complex structure. This complex structure can then

attach and produce tiny pores on the membranes of the neutrophils (Sabour and Griffiths

2010). F subunit refers to fast-eluting proteins during purification on an ion-exchange

column. F subunit attaches primarily to red blood cells and then to S subunits to

perforate their membranes (Fluit and Schmitz 2003). The difference between the


Gamma toxin and PVL toxin is that gamma toxin binds strongly to red blood cells, while

PVL binds to white blood cells, specifically neutrophils (Nilsson et al. 1999). Overall, S

and F subunits act together in the destruction of membranes on both toxins (Fluit and

Schmitz 2003). Gamma-toxin is present in 99% of S. aureus strains whereas only 2%-

3% of strains produce PVL toxins (Lydyard et al. 2009).

3. Avoidance of Host Defenses

S. aureus can interfere with innate and adaptive immune responses in numerous

ways (Liu 2009). Capsular polysaccharide (CP) is composed of a variety of

polysaccharides present on the surface of S. aureus, and the majority of strains express

CPs in vivo or in vitro (Turnidge et al. 2002). In the presence of CPs, phagocytosis is

inhibited by preventing cell-surface structure identification by receptors on the surface of

a phagocyte. Therefore, CPs prevent complement activation and prevent S. aureus from

being recognized by the host’s immune system (Mahon et al. 2014). As a result, the

host defense mechanism no longer fights against the infection (Moroni et al. 2011).

Superantigens belong to a class of antigens that initiate a non-specific T-cell

response to release cytokines, causing severe inflammation (Proft and Fraser 2003).

Enterotoxins belong to the family of superantigens that stimulate T-cell production due to

the interaction of the major histocompatibility complex class II molecules on antigen

presenting cells and T cell receptors (Proft and Fraser 2003). Among numerous

enterotoxins, staphylococcal superantigens A, B, and C contain superantigenic features.

Enterotoxins have been associated with autoimmune diseases, such as systemic lupus

erythematosus, and rheumatic arthritis, and are involved in abnormal immunologic

stages, such as psoriasis and atopic dermatitis (Turnidge et al. 2002). S. aureus avoids

the immune system because it expresses different types of superantigens that corrupt


the normal humoral immune response, leading and immunosuppression (Foster 2005).

Superantigens help S. aureus avoid the host immune system by stimulating a large

amount of T cells, which secrete large amount of interleukin-2. Interleukin- 2 is a protein

that, in large amounts, can induce vomiting. Overstimulation by superantigens is not

always beneficial to the host and can lead to shock and death (Freeman-Cook et al.

2006).

Toxic shock syndrome toxin is caused by a superantigen known to be produced by

Staphylococcus aureus (Mahon et al. 2014). These toxins are not processed by APCs,

but instead TSS toxins attach directly to the outside portion of an MHC II antigen on

APCs and to the outside portion of the T cell receptor of the T helper cells.

Superantigens are non-specific, so they can bind without antigen specificity and

stimulate many helper T cells. Excessive amounts of interleukin- 2 produced by T cells

will enter the bloodstream, instead of only acting locally as they normally do.

Overproduction of interleukin-2 circulating in the bloodstream initially induces vomiting

and nausea, but left untreated can become fatal (Freeman-Cook et al. 2006).


A. Modes of Antibiotic Resistance

The spread of antibiotic resistance and the occurrence of multiple drug resistant

pathogenic bacteria have negatively impacted the medical field due to the lack of

effective therapeutic treatment for bacterial infections, such as S. aureus (Lowry 2003).

S. aureus can develop resistance to antibiotics by altering the antibiotic target, trapping

the antibiotic, or through efflux pumps (Pantosti et al. 2007).

1. Alteration of the Antibiotic Target

S. aureus tolerates the cytotoxic activity of β-lactam antibiotics by modifying the

normal penicillin-binding proteins, such as PBP2a (Stapleton and Taylor 2002). PBP2a

modification is best seen in methicillin-resistant S. aureus (MRSA) due to the mecA gene

that is responsible for encoding PBP2a. MRSA forms PBP2a as a fifth PBP, in addition

to four PBPs found in all S. aureus strains (Zapun et al. 2008). β-lactams have a

reduced affinity for PBP2a, and enzyme function will not be disturbed even if it is in the

presence of methicillin or any other β-lactam (Drawz and Bonomo 2010).

Vancomycin-resistant strains can also modify antibiotic targets D-Alanyl-D-

Lactate of peptidoglycan precursors (Kwun et al. 2013). According to Figure 3, D-Ala-D-

Lac production requires VanS, a membrane detector of vancomycin that regulates the

levels of phosphorylation of VanR. VanR is a transcriptional inducer of the van operon

composed of membrane bound histidine kinase and cytoplasmic response regulator

vanHAX (Depardieu et al. 2007). VanH is a dehydrogenase that converts pyruvate to D-

Lac, while VanA acts as a ligase which assemblies the formation of an ester bond

between D-Ala and D-Lac precursors (Kwun et al. 2013). The resistance takes place

because vancomycin does not interact with D-Ala-D-Lac in peptidoglycan (Courvalin


2006). VanX is an enzyme composed of two peptides that cleaves the normal D-Ala-D-

Ala peptidoglycan. Lastly, VanY is a D,D-carboxypeptidase that cleaves the terminal D-

Ala precursors that are formed if D-Ala-D-Ala is not broken by VanX (Cetinkaya et al.

2000). Once D-Ala-D-Ala is substituted for the D-Ala-D-Lac in peptidoglycan production,

S. aureus strains are resistant to vancomycin (Cetinkaya et al. 2000).

Figure 3- Mechanism of action of vancomycin resistance in S. aureus (Hughes 2003).

2. Exclusion of the Antibiotic

Daptomycin is an antibiotic created by Streptomyces roseosporus, a soil

actinomycete, that is used to treat systemic infections with Gram-positive bacteria

(Steenbergen et al. 2005). Daptomycin is composed of a 13-amino-acid-lipopeptide

chain that is trapped by cytoplasmic membranes, which leads to membrane perforation,

K+ efflux (Steenbergen et al. 2005), and apoptosis of the cell (Straus and Hancock


2006). It is believed that resistance to daptomycin arises due to a modification of

bacterial cell walls and might be correlated to vancomycin resistance in vancomycin-

intermediate S. aureus (VISA) strains (Camargo et al. 2008). VISA strains show

peptidoglycan layers that are joined together to prevent vancomycin and daptomycin

access to the cell membrane (Yu et al. 2012). Daptomycin is a large molecule and is

unable to cross through the porin channels of modified walls (Kohanski et al. 2010).

3. Efflux Pumps

Efflux pumps are mechanisms that transport elements, like antibiotics, from the

interior to the exterior of the cell (Webber and Piddock 2003). Some efflux pumps are

specific to one antibiotic while others can accommodate a variety of antibiotics

(multidrug) and be responsible for a multitude of bacterial resistance (Nikaido 2009). S.

aureus is known to have 30 efflux pumps for quinolones (Costa et al. 2013). NorA is an

efflux pump for fluoroquinolones in S. aureus (Costa et al. 2013). norA is a chromosomal

gene that encodes for three alleles that differ by up to 10% in their nucleotide sequences

(Costa et al. 2013). According to the literature, flouroquinolone resistance has increased

with the overexpression of norA gene. The resistance to fluoroquinolones can be due to

mutations in the promoter region of the norA gene through the action of regulatory

proteins (Costa et al. 2003). Thus, if mutations take place at the norA promoter region,

transcription level is affected and consequently, overexpression of the gene associated

with a mutation will occur. As a result, the efflux pump suffered a misfolding due to the

inability of the regulator protein to bind to the promoter and act against S. aureus (Couto

et al. 2008).


4. Mode of mecA gene

SCCmec stands for staphylococcal cassette chromosome mec, which is part of a

family of mobile genetic elements that encode genomic islands with a methicillin-

resistance gene known as mecA (Katayama et al. 2000). SCCmec elements that do not

have mecA gene have cassette chromosome recombinase (ccr) gene instead (Ito 2009).

ccr genes code for recombinases that allow deletions or insertions to take place in the

SCCmec chromosome in order to obtain the correct orientation for transcription (Zong

2013). mec complex carries copies of plasmids with resistant genes while ccr complex is

responsible for the mobility of SCCmec. Five types of ccr and four classes of mec

complex (mecA being the most important) work together to form SCCmec cassette

(Katayama et al. 2003). All methicillin-resistant S. aureus (MRSA) strains harbor at least

one SCCmec element in their chromosome, some of which include a mecA gene that

encodes PBP2a (Lowry 2003). PBP2a is a protein which binds to peptidoglycan and

disrupts the peptide bond in cross-linkages of peptidoglycan chains. Penicillin-binding

proteins have a natural affinity for penicillin, methicillin, and other β-lactam/β-lactam-like

antibiotics (Drawz and Bonomo 2010). When PBPs are bound to one of these

antibiotics, they no longer catalyze cross-linkage of peptidoglycan in the cell wall. This

weakens the cell walls, and cells are more likely to lyse (Drawz and Bonomo 2010).

However, PBP2a shows a decreased affinity to these drugs, conferring antibiotic

resistance (Stapleton and Taylor 2002).


MRSA strains have the ability to activate mecA expression in the presence of β-

lactam antibiotics and repress mecA expression in their absence (Stapleton and Taylor

2002). In reference to Figure 4, if methicillin, penicillin or any other β-lactam is not

present, mecA expression is repressed by MecI, which binds to the promoter of mecA

and inhibits transcription (Stapleton and Taylor 2002). In the presence of β-lactams,

expression of mecA is activated by MecR1, a cell surface protein whose role is to detect

β-lactams. Upon detection, the MecR1 inhibitor is disrupted by deoxyribozymes which

reduce mecRI and PBP2a mRNAs expression, and thus facilitate expression of mecA

(Meng et al. 2009).


Figure 4- Mechanism of action of mecA gene in the presence and absence of beta-

lactam antibiotics (Arede et al. 2012).


5. Specific Treatment for Staphylococcus aureus

S. aureus infections are the most alarming of all staphylococci. In the US, nearly

60% of infections with S. aureus are caused by methicillin-resistant strains, and this

bacterium is thought to be the most difficult to cure (Sakoulas and Moellering 2008). As it

was previously stated, strains can cause food poisoning, toxic shock syndrome, MRSA,

MSSA, and skin infections (Turnidge et al. 2002). Despite the fact that these diseases

are serious, they are worsened because they are also resistant to a number of

antibiotics. Treatment options are limited, complex, and expensive (Sakoulas and

Moellering 2008). Without effective antibiotics, it becomes nearly impossible to treat

such complicated infections (Davies and Davies 2010). Forrest and Tamura (2010)

believe that a combination of antibiotics can improve the efficacy of the treatment

against MRSA versus the use of a single monotherapy.

1. Methicillin-Resistant Staphylococcus aureus (MRSA)

To effectively reduce and stop MRSA development, it is imperative to test clinical

isolates against antibiotics using disk-diffusion (Kirby-Bauer) tests on agar plates (Skov

et al. 2006). Kirby-Bauer tests determine an isolate’s sensitivity to a certain antibiotic. In

the presence of an efficient antibiotic, a zone of inhibition will appear on agar plates

(Skov et al. 2006). Vancomycin, for example, is considered an efficient antibiotic to treat

MRSA infections (Appelbaum 2007). On the other hand, methicillin disks show no zone

of inhibition, indicating that bacteria are uninhibited (Skov et al. 2006). Antibiotic therapy

should be continually administered for 72 hours to MRSA patients until multiple sets of

blood cultures are obtained, even if the symptoms have improved. Early stoppage of

therapy can allow MRSA to survive and develop further antibiotic resistance (Hughes


2008). Overall, each patient may require a personalized approach based on the type and

severity of infection and status of the individual (Hughes 2008).

For patients with severe MRSA infections (more prone to death), a combination

of two or more drugs, like vancomycin, linezolid, and sulfamethoxazole-trimethoprim is

considered to be the most efficient treatment (Deresinski 2009). Minor skin infections

may respond well to mupirocin. Mupirocin is utilized mainly as a topical agent that

specifically and irreversibly binds to isoleucyl tRNA synthetase, thus preventing protein

formation (Gisby and Bryant 2000). The effectiveness of mupirocin has been observed in

surgical and bronchopulmonary infections (Watanabe et al. 2001).

For patients with endocarditis, teicoplanin is known to be the most efficient

treatment. The recommended target is more than 20 mg/L of plasma reached by

teicoplanin prior to the next dose administered, which is refered to as trough

concentration (Ueda et al. 2014). Long-term use of teicoplanin does not lead to

resistance, but isolates can become resistance to vancomycin (Cetinkaya et al. 2000).

Alternative drugs are available for patients with less severe infections. Normally, a

combination of oral therapy with two active agents, such as rifampin or fluoroquinolones,

can be prescribed (Leekha et al. 2011). Oral therapeutic antimicrobial agents are

discouraged for long-term use because of risk for occurrence of resistance and adverse

effects of the antibiotics (Turnidge et al. 2002).

2. Methicillim-Susceptible Staphylococcus aureus (MSSA)

In the 1990s, methicillin-susceptible S. aureus (MSSA) strains were studied and

known from infections in previously healthy patients. (David et al. 2011). Currently,

MSSA infections are treated with penicillinase-resistant penicillins, such as dicloxacillin


and oxacillin in oral or injectable form (Rayner and Munckhof 2005). A wide range of

researchers have adopted these drugs because penicillinase-resistant penicillins destroy

bacteria and show a reduced incidence of adverse reactions (Fair and Tor 2014).

Studies have shown that outpatients with serious staphylococcal infections have

presented satisfactory outcomes (Turnidge et al. 2002).

Cephalosporins have been a useful alternative to penicillinase-resistant

penicillins due to their stability against staphylococcal β-lactamase. Patients with a

history of mild allergies or intolerance to penicillins have been able to use

cephalosporins as a treatment (Turnidge et al. 2002). Nonetheless, cephalosporins are

not considered a safe choice for patients who suffer from severe allergies, such as

angioedema or anaphylaxis. This can be due to partial cross-allergenicity between

penicillins and cephalosporins which can create severe hypersensitivity (Rayner and

Munckhoff 2005). In these cases, clindamycin or vancomycin are known to be most

effective treatments (Rayner and Munckhof 2005).

Currently, β-lactams combined with β-lactam inhibitors, such as amoxicillin/

clavulanic acid, or ampicillin/sulbactam are effective against Gram-negative anaerobes.

This combination of drugs gives a broader spectrum, a considerable advantage when

staphylococci are present in mixed infections with Gram-negative bacteria, including

anaerobes (Turnidge et al. 2002).


V. Conclusion

Antibiotics developed to answer the demands of the global society. Antibiotics

became one of the resources used to treat and prevent diseases. During World War II,

penicillin saved millions of soldiers and ultimately changed the course of history (Ventola

2015). Until 1950s, antibiotics were responding well to the demands of society and

controlled nearly all major bacterial infections during WWII (Ventola 2015). Bacteria

began to survive in the presence of the most common antibiotics used in the medicine

era. Today, several studies have shown and prove that inappropriate and unnecessary

use of antibiotics plays a very crucial role in the expansion of antibiotic resistance

(McEachran et al. 2015).

Bacteria are capable of passing genetic material from one bacterium to another,

leading to the appearance of mutations. Mutations when developed can confer

resistance to antibiotics. Despite warnings from Ventola and other researchers,

antimicrobial agents continue to be misused and overprescribed all over the world

(Ventola 2015). In the U.S., data collected in 2010 states that up to 50% of antibiotics

where inappropriately prescribed to the patients (Ventola 2015). Zdziarski study shows

that unnecessary drug prescription has led to foodborne and waterborne infections,

gastrointestinal complications, antibiotic oversensitivity, ecosystem modification, and

disturbances in human and animal evolution (Zdziarski et al. 2003).

Further studies should investigate how to minimize antibiotic resistance by

controlling the over-prescription of drugs. Not every disease requires antibiotics. In the

UK and Thailand, self-medication is easily available over the counter, and without


evaluation people continue to abuse antibiotics (Pechere 2001). However, the

Netherlands is one of the northern European countries with the lowest incidence of

antibiotic resistance because laws have been implemented prohibiting over the counter

drugs. For instances, physicians are the only ones allowed to prescribe drugs that are

used to treat the infections. If a patient has common flu or cold, antibiotics cannot be

prescribed (Rossignoli and Calvenna 2007). In livestock settings, the European Union

committee implemented drug prescription forms in order to control the usage of

antibiotics in food animals and farms. A threshold limit determined by the European

committee is controlled to ensure that the usage of antibiotics are been met in farm

animals (Bartlett et al. 2013). Also, educating the general public of the risks associated

with misuse of drugs can facilitate and control the bacterial spread (Grigoryan et al.

2010). By following hygiene guidelines and effective disinfection procedures in the

community and in the hospital, the Netherlands believes that it can play a vital role in

minimizing the risks of antibiotic resistance (Deurenberg et al. 2006).

In the US, staphylococcal infections have imposed severe health conditions and

economic burden in over 2 million people every year (Stone et al. 2005). In 2005, after

hand hygiene compliance was implemented, successful results arose in the US hospitals

for MRSA but have not yet been seen in community settings (Carlet et al. 2012). On the

other hand, Netherlands communities found that MRSA skin abscesses less than 5 cm

in size are effectively treated with an incision and drainage instead of using antibiotics.

Scandinavia has followed similar measures to control antibiotic resistance and has the

lowest MRSA prevalence of all European countries, at only 0.6% (Deurenberg et al.

2006). Even though in the US laws have been implemented, guidelines have been

established and educational programs have been campaigned, society still has


preconceived habits concerning antimicrobial agents and their side effects. The scientific

community would highly benefit if further research could be done on how to effectively

educate society, pharmacists, physicians, nurses, and parents of every nation (Carlet et

al. 2012). National and international committees should together propose effective

strategies to address antibiotic resistance among humans and animals, in particular the

interaction between livestock feed and the environment (McEachran et al. 2015).


VI. Acknowledgement

Through the years there have been many blockades and detours in the journey.

Without the help of my family, friends and colleagues, I would have never been able to

achieve my goal of obtaining a master’s degree.

First and foremost I give thanks to my loving and supportive family. My parents

and my brother for always believing in me, for their unconditional support and

understanding. My parents thought me to be persistent, dedicated and extremely hard

worker and never give up when detours appear in the way. After living such an

adventure life at young age, I learnt how to survive and how to succeed, and my

commitment leads me to where I am today. I am truly blessed and grateful for what my

parents have thought me, in order to reach my goals.

Second, I want to share my deepest gratitude to my husband. I truly appreciate

his understanding during many late evenings spent studying, reading, and writing that

consumed my time over this past few years.

For this scholarly paper, I would like to thank my advisor: Dr. James Campbell,

for his time and interest toward my project. I am very appreciative of the support and

guidance given to me by Dr. James Campbell. His guidance and support were critical to

the success of this paper. I am forever grateful for all his patience and time committed

to reviewing and commenting on the numerous drafts that I presented.


VII. References

Alanis A. 2005. Resistance to Antibiotics: Are we in the post-antibiotic Era?

Archives of Medical Research 36(6): 697-705.

Alberts B, Johnson A, Lewis J. 2002. Molecular Biology of the Cell. 4th Edition.

New York: Garland Science.

Aminov R. 2011. Horizontal gene exchange in environmental microbiota. Front

Microbiol 2: 158.

Anderson A, Miller A, Donald R, Scully I, Nanra J, Cooper D, Jansen K. 2012.

Development of a multicomponent Staphylococcus aureus vaccine designed to counter

multiple bacterial virulence factors. Hum Vaccin Immunother 8(11): 1585-1594.

Appelbaum P. 2007. Microbiology of Antibiotic Resistance in Staphylococcus

aureus. Clin Infect Dis 45(supplement 3): S165-S170.

Arede P, Milheiriço C, Lencastre H, Oliveira D. 2012. The Anti-Repressor MecR2

Promotes the Proteolysis of the mecA Repressor and Enables Optimal Expression of β-

lactam Resistance in MRSA. PLoS Pathog 8(7): e1002816.

Argudin M, Mendoza M, Rodicio M. 2010. Food Poisoning and Staphylococcus

aureus enterotoxins. Toxins (Basel) 2(7): 1751–1773

Aubry A, Veziri N, Cambau E, Truffot-Pernot C, Jarlier V, Fisher M. 2006. Novel

Gyrase Muttions in Quinolone-Resistant and Hypersusceptible Clinical Isolates of

Mycobacterium tuberculosis: Functional Analysis of Mutant Enzymes. Antimicrob Agents

Chemother 50(1): 104-112.

Baddour L, Bettmann M, Bolger A, Epstein A, Ferrieri P, Gerber M, Gewitz M,

Jacobs A, Levison M, Newburger J, Pallasch T, Wilson W. Baltimore R, Falace D,


Shulman S, Tani R, Taubert K. 2003. Nonvalvular Cardiovascular Device-Related

Infections. Circulation 108: 2015-2031.

Baquero F, Campos J. 2003. The tragedy of the commons in antimicrobial

chemotherapy. Revista Espanola de Quimioterapia. 16(1):11-3.

Barnes B. 2010. Negligence, medical malpractice, vicarious liability, or patient

responsibility: Who should pay when a patient contracts MRSA from a Healthcare

facilty? Indiana Health Law Review 7: 335.

Barros E, Ceotto H, Bastos M, Santos K, Giambiagi-deMarval M. 2012.

Staphylococcus haemolyticus as an Important Hospital Pathogen and Carrier of

Methicillin Resistance Genes. J Clin Microbiol 50(1): 166-168.

Bartlett J, Gilbert D, Spellberg B. 2013. Seven ways to preserve the miracle of

anitbiotics. Clin Infect Dis 56(10):1445-1450.

Becker K, Heilmann C, Peters G. 2014. Coagulase-Negative Staphylococci. Clin

Microbiol Rev 27(4): 870-926.

Behera D. 2010. Textbook of Pulmonary Medicine. Jaypee Brothers Publishers

1: 269.

Bennett P. 2008. Plasmid encoded antibiotic resistance: acquisition and transfer

of antibiotic resistance genes in bacteria. Br J Pharmacol 153(1): S347-S357.

Berube B, Wardenburg J. 2013. Staphylococcus aureus α-Toxin: Nearly a

Century of Intrigue. Toxins 5: 1140-1166.

Bhatia A, Zahoor S. 2007. Staphylococcus aureus Enterotoxins: A Review.

Journal of Clinical and Diagnostic Research 3:188-197.


Biavasco F, Vignaroli C, Lazzarini R, Varaldo P. 2000. Glycopeptide

Susceptibility profiles of Staphylococcus haemolyticus Bloodstream isolates. Antimicrob

Agents Chemother 44(11): 3122.

Bien J, Sokolova O, Bozko P. 2011. Characterization of virulence factors of

Staphylococcus aureus: Novel function of known virulence factors that are implicated in

activation of airway epithelial proinflammatory response. Journal of Pathogens doi:

10.4061/2011/601905.

Blackstock J. 2014. Guide to Biochemistry. Butterworth-Heinemann. Pp 234.

Blondal K, Rahu K, Altraja A, Viiklepp P, Rhau M. 2013. Overall and cause-

specific mortality among patients with tuberculosis and multidrug resistant tuberculosis.

Int J Tuber Lung Dis 17(7): 961-8.

Bocher S, Tonning B, Skov R, Prag J. 2009. Staphylococcus lugdunensis, a

common cause of skin and soft tissue infections in the community. J Clin Microbiol 47(4):

946-950

Bonomo R, Szabo D. 2006. Mechanism of multidrug resistance in Acinetobacter

species and Pseudomonas aeruginosa. Clin Infect Dis 43:S49-S56.

Bonono R. 2000. Multiple Antibiotic-resistant Bacteria in Long-term-care Facilities:

An emerging problem in the practice of Infectious disease. Clin Infect Dis 31(6): 1414-

1422.

Bousios A, Darzentas N, Tsaftaris A, Pearce S. 2010. Highly conversed motifs in

non –coding regions of Sirevirus retrotransposons: the Key for their pattern of

distribuition within and across plants? BMC Genomics 11:89.


Brady R, Graeme A, O’May, Leid J, Prior M, Costerton J, Shirtliff. 2011.

Resolution of Staphylococcus aureus Biofilm Infection Using Vaccination and Antibiotic

Treatment. Infect Immun 79(4):1797-1803.

Brown D, Edwards D, Hawkey P, Morrison D, Ridgway G, Towner K, Wren M.

2005. Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-

resistant Staphylococcus aureus (MRSA). Journal of Antimicrobial Chemotherapy 56:

1000-1018.

Bukowski M, Wladyka B, Dubin G. 2010. Exfoliative Toxins of Staphylococcus

aureus. Toxins (Basel) 2(5): 1148–1165.

Bush K. 2010. Bench-to-bedside review: The role of β-lactamases in antibiotic-

resistant Gram-negative infections. Crit Care 14(3): 224.

Cabell C, Abrutyn E, Karchmer A. 2003. Bacterial endocarditis: The Disease,

treatment and prevention. Circulation 107:e185-187.

Camargo I, Neoh H, Cui L, Hiramatsu K. 2008. Serial Daptomycin Selection

Generates Daptomycin-nonsusceptible Staphylococcus aureus Strains with a

Heterogeneous Vancomycin-Intermediate Phenotype. Antimicrob Agents Chemother

52(12): 4289-4299.

Carlet J, Jarlier V, Harbarth S, Voss A, Goossens H, Pittet D. 2012. Ready for a

world without antibiotics? The Pensières Antibiotic Resistance Call to Action.

Antimicrobial Resistance and Infection Control 1:11.

Casanova C, Iselin L, Sendi P. 2011. Staphylococcus hyicus bacteremia in a

Farmer. J Clin Microbiol 49(12): 4377-4378.

Centinkaya Y, Falk P, Mayhall C. 2000. Vancomycin-Resistant Enterococci. Clin

Microbiol Rev 13(4):6866-707.


Chen I, Dubnau D. 2004. DNA uptake during bacterial transformation. Nat. Rev.

Microbiol 2 (3): 241–9.

Cheng A, McAdow M, Kim H, Bae T, Missiakas D, Schneewind O. 2010.

Contribution of Coagulase towards Staphylococcus aureus Disease and Protective

Immunity. PLOS Pathog 6(8): e1001036.

Chiller K, Selkin B, Murakawa G. 2001. Skin Microflora and Bacterial Infections of

the Skin. Journal of Investigative Dermatology Symposium Proceeding 6:170-174.

Cho S, Lee D, Choi S, Kwon J, Kim S, Choi J, Park Y, Choi J, Yoo J. 2013.

Impact of vancomycin resistance on mortality in neutropenia patients with enterococcal

bloodstream infection: a retrospective study. BMC Infect Dis 13:504.

Clarke S, Foster S. 2006. Surface adhesins of Staphylococcus aureus. Adv

Microb Physiol 51:187-224

Coates R, Moran J, Horsburgh M. 2014. Staphylococci: colonizers and pathogen

of human skin. Future Microbiol 9(1): 75-91.

Cognasse H, Damien P, Chabert A, Pozzetto B, Cognasse F, Garraud O. 2015.

Platelets and Infections- complex interactionsw with bacteria. Front Immunol 10:3389.

Cole S. 2014. Who will develop new antibacterial agents? Phil Trans R Soc

369(1645): 1471-2970.

Collin F, Karkare S, Maxwell A. 2011. Exploiting bacterial DNA gyrase as a drug

target: current state and perspectives. Appl Microb Biotechnol 92(3): 479-497.

Collins S. 2008. Preventing Health Care-Associated Infections. In: Hughes G.

Patient Safety and Quality: An Evidence-Based Handbook for Nurses. Agency for

Healthcare Research and Quality (US): 41.


Costa S, Viveiros M, Amaral L, Couto I. 2013. Multidrug Efflux Pumps in

Staphylococcus aureus: an Update. Open Microbiol J 7: 59-71.

Courvalin P. 2006. Vancomycin Resistance in Gram-positive cocci. Clin Infect

Dis 42(supplement 1): S25- S34.

Couto I, Costa S, Viveiros M. 2008. Efflux-mediated response of Staphylococcus

aureus exposed to ethidium bromide. J Antimicrob Chemother 62:504–13.

Crossley K, Jefferson K, Archer G, Fowler V. 2009. Staphylococci in Human

Disease. Second edition. Oxford (UK). Wiley-Blackwell.

David M, Boyle-Vavra S, Zychowski D, Daum R. 2011. Methicillin-Susceptible

Staphylococcus aureus as a Predominantly Healthcare-Associated Pathogen: A

Possible Reversal of Roles? PLoS One 6(4): e18217.

David M, Daum R. 2010. Community-Associated Methicillin-Resistant

Staphylococcus aureus: Epidemiology and Clinical Consequences of an Emerging

Epidemic. Clin Microbiol Rev 23(3): 616-687.

Davies J, Davies D. 2010. Origins and evolution of antibiotic resistance. Microbiol

Mol Biol Rev 74(3): 417-433.

Dellit T, Owens R, McGowan J, Gerding D, Weinstein R, Burke J,Huskins W,

Paterson D, Fishman N, Carpenter C, Brennan P, Billeter M, Hooton T. 2007. Infectious

Diseases Society of America and the Society for Healthcare Epidemiology of America

Guidelines for Developing an Institutional Program to Enhance Antimicrobial

Stewardship. Clin Infect Dis 44(2): 159-177.

Delost M. 2014. Introduction to Diagnostic Microbiology for the Laboratory

Sciences. Burlington: MA. Jones & Bartlett Publishers.


Depardieu F, Podglajen I, Leclercq R, Collatz E, Courvalin P. 2007. Modes and

Modulations of Antibiotic Resistance Gene Expression. Clin Microbiol Rev 20(1): 79-114.

Deresinski S. 2009. Vancomycin in Combination with Other Antibiotics for the

Treatment of Serious Methicillin-Resistant Staphylococcus aureus Infections. Clin Infect

Dis 49(7): 1072-1079.

Deurenberg R, Vink C, Kalenic S, Friedrich A, Bruggeman C, Stobberingh E.

2006. The molecular evolution of methicillin-resistant Staphylococcus aureus. Clinical

Microbiology and Infection 13(3): 222-235.

Dinges M, Orwin P, Schlievert P. 2000. Exotoxins of Staphylococcus aureus. Clin


Domingues S, Harms K, Fricke W, Johnsen P, Silva G. 2012. Natural

Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes

between Bacterial Species. PLoS Pathog 8(8): e1002837.

Drawz S, Bonomo R. 2010. Three Decades of β-Lactamase Inhibitors. Clin

Microbiol 23(1): 160-201.

Drlica K, Hiasa H, Kerns R, Malik M, Mustaev A, Zhao X. 2009. Quinolones:

Action and Resistance Updated. Curr Top Med Chem 9(11): 981-998.

Drlica K, Malik M, Kerns RJ, Zhao X. 2008. Quinolone-mediated bacterial death.

Antimicrob Agents Chemother 52:385–92.

Drobniewski F, Cooke M, Jordan J. 2015. Systematic review, meta-analysis and

economic modeling of molecular tests for antibiotic resistance in tuberculosis.

Southampton (UK): NIHR Journals library 19.34.


Durdik P, Fedor M, Jesenak M, Hamzikova J, Knotkova H, Banovcin P. 2009.

Staphylococcus intermedius – rare pathogen of acute meningitis. International Journal of

Infectious Diseases 14(3): e236-238.

Dworkin M, Falkow S, Rosenberg E, Schleifer K, Stackebrandt E. 2006. The

Prokaryotes: Volume 4 Bacteria: Firmicutes, Cyanobacteria. 3rd edition. Minneapolis

(MN). Springer.

Engelhart S, Hanses-Derendorf L, Exner M, Kramer M. 2005. Prospective

surveillance for healthcare-associated infections in German nursing home residents. J

Hosp Infect. 60(1):46

Engelkirk P. Duben-Engelkirk J, Gwendolyn R, Burton W. 2011. Burton’s

Microbiology for the Health Sciences. 9th Edition. Lippincott Williams & Wilkins:

Baltimore, MD.

Enoch D, Cooke F, Guha S, Brown N. 2008. Thermostable nuclease: a study of

clinical impact. J Antimicrob Chemother 61(3): 754-755.

Fabbretti A, Gualerzi C, Brandi L. 2011. How to cope with the quest for new

antibiotics. FEBS Lett 585(11):1673-81.

Fair R, Tor Y. 2014. Antibiotics and Bacterial Resistance in the 21st Century.

Perspect Medicin Chem 6:25-64.

Fernandez L, Handcock R. 2012. Adaptive and Mutational Resistance: Role of

Porins and Efflux Pumps in Drug Resistance. Clin Microbiol 25(4): 661-681.

Filice G, Nyman J, Lexau C, Lees C, Bockstedt L, Como-Sabetti K, Lesher L,

Lynfield R. 2010. Excess Costs and Utilization Associated with Methicillin Resistance for

Patients with Staphylococcus aureus Infection. Infection Control and Hospital

epidemiology 31:4.


Flemming H, Neu T, Wozniak D. 2007. The EPS matrix: The “House of Biofilm

Cells” J Bacteriol 189(22): 7945-7947.

Fluit C, Schmitz F. 2003. MRSA: current perspectives. Horizon Scientific Press.

Norfolk: England.

Fonseca J, Knight G, McHugh T. 2015. The complex evolution of antibiotic

resistance in Mycobacterium tuberculosis. International Journal of Infectious Diseases

32: 94-100.

Forrest G, Tamura K. 2010. Rifampin Combination Therapy for

Nonmycobacterial Infections. Clin Microbiol Rev 23(1): 14-34.

Foster T. 1996. Staphylococcus. In: Baron S. Medical Microbiology. 4th edition.

Galveston (TX). Available from: http://www.ncbi.nlm.nih.gov/books/NBK8448.

Foster T. 2005. Immune evasion by staphylococci. Nat Rev Microbiol 3: 948-958.

Fournier B, Philpott D. 2005. Recognition of Staphylococcus aureus by the Innate

Immune System. Clin Microbiol Rev 18(3): 521-540.

Frank K, Zhou T, Moreno-Vinasco L, Hollett B, Garcia J, Wardenburg J. 2012.

Host Response Signature to Staphylococcus aureus Alpha-Hemolysin Implicates

Pulmonary Th17 Response. Infect Immun 80(9): 3161-3169.

Freeman-Cook L, Freeman-Cook K, Alcamo E, Heymann D. 2006.

Staphylococcus aureus infections. Infobase Publishing: USA.

Frieden T. Antibiotic Resistance Threats in the United States. U.S Department of

Health and Human Services. CDC. 2013. 114 p.

Fudaba Y, Nishifuji K, Andresen L, Yamaguchi T, Komatsuzawa H, Amagai M,

Sugai M. 2005. Staphylococcus hyicus exfoliate toxins selectively digest porcine

desmoglein 1. Microbial Pathogenesis 39: 171-176.

http://www.ncbi.nlm.nih.gov/books/NBK8448


Futagawa-Saito K, Ba-Thein W, Sakurai N, Fukuyasu T. 2006. Prevalence of

virulence factors in Staphylococcus intermedius isolates from dogs and pigeons. BMC

Veterinary Research 2:4.

Garrod D, Chidgey M. 2007. Desmosomal structure, composition and function.

Apical Junctional Complexes Part I 1778(3): 572-587.

Geiter L. 2000. Ending Neglect: The Elimination of Tuberculosis in the United

States. Washington (DC): National Academies Press.

Gelder L, Williams J, Ponciano JM, Sota M, Top EM. 2008. Adaptive plasmid

evolution results in host-range expansion of a broad-host-range plasmid. Genetics. 178:

2179-2190.

Giedraitiene A, Vitauskiene A, Naginiene R, Pavilonis A. 2011. Antibiotic

resistance mechanisms of clinically important bacteria. Medicina (Kaunas) 47(3): 137-

46.

Gillespie I, O’Brien S, Frost J, Tam C, Tompkins D, Neal K, Syed Q, Farthing M.

2006. Investigating vomiting and/or bloody diarrhea in Campylabacter Jejuni infection. J

Med Microbiol 55(6):741-6.

Gillespie S. 2002. Evolution of Drug Resistance in Mycobacterium tuberculosis:

Clinical and Molecular Perspective. Antimicrob Agents Chemother 46(2): 267-274.

Gisby J, Bryant J. 2000. Efficacy of a New Cream Formulation of Mupirocin:

Comparison with Oral and Topical agents in Experiemental skin infections. Antimicrob

Agents Chemother 44(2): 255-260.

Gogarten P, Doolittle W, Lawrence J. 2002. Prokaryotic Evolution in Light of

Gene transfer. Mol Biol Evol 19(12): 2226-2238.


Griffiths A, Miller J, Suzuki D. 2000. An Introduction to Genetic Analysis 7th

edition. New York: W. H. Freeman.

Grigoryan L, Monnet D, Haaijer-Ruskamp F, Bonten M, Lundborg S, Verheij T.

2010. Self-Medication with Antibiotics in Europe: A case for action Curr Drug Safety 5(4):

329-332.

Grohmann E, Muth G, Espinosa M. 2003. Conjugative Plasmid Transfer in Gram-

Positive Bacteria. Microbiol Mol Rev 67(2): 277-301.

Guggenbichler J, Assadin O, Boeswald M, Kramer A. 2011. Incidence and clinical

implication of nosocomial infections associated with implantable biomaterials- catheters,

ventilator-associated pneumonia, urinary tract infections. GMS Krankenhhyg Interdiszip

6(1): Doc18.

Gupta N, Limbago B, Patel J, Kallen A. 2011. Carbapenem-Resistant

Enterobacteriaceae: Epidemiology and Prevention. Clin Infect Dis 53(1): 60-67.

Haddadin A, Fappiano S, Lipsett P. 2002. Methicillin resistant Staphylococcus

aureus (MRSA) in the intensive care unit 78: 385-392.

Hanberger H, Antonelli M, Holmborn M, Lipman J, Pickkers P, Leone M, Rello J,

Sakr Y, Walter S, Vabhems P, Vincent J. 2014. Infections, antibiotic treatment and

mortality in patients admitted to ICUs in countries considered to have high levels of

antibiotic resistance compared to those with low levels. BMC Infect Dis 22(14): 513.

Hassan A, Usman J, Kaleen F, Omair M, Khalid A, Igbal M. 2011. Evaluation of

different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis

15(4): 305-11.

Hickman A, Chandler M, Dyda F. 2011. Integrating prokaryotes and eukaryotes:

DNA transposases in light of structure. Crit Rev Biochem Mol Biol 45(1): 50-69.


Hoffman D, Brown G, Lombardo F. 2007. Early-onset sepsis with Staphylococcus

auricularis in an extremely low-birth weight infant - an uncommon pathogen. Journal of

perinatology: official journal of the California Perinatal Association 27(8): 519–20.

Huddleston J. 2014. Horizontal gene transfer in the human gastrointestinal tract:

potential spread of antibiotic resistance genes. Infect Drug Resist 7:167-176.

Hughes D. 2003. Exploiting genomics, genetics and chemistry to combat

antibiotic resistance. Nature Reviews Genetics 4: 432-441.

Ibler K, Kromann C.2014. Recurrent furunculosis – challenges and management:

a review. Clin Cosmet Invest Dermatol 7:59-64.

Isaakidis P, Cox H, Varghese B, Montaldo C, Da Silva E, Mansoor H,

Ladomirska J, Sotgiu G, Migliori G, Pontali E, Saranchuk P, Rodrigues C, Reid T. 2011.

Ambulatory multi-drug resistant tuberculosis treatment outcomes in a cohort of HIV-

infected patients in a slum setting in Mumbai, India. PLoS One 6(12):e28066.

Ito T. 2009. Classifiction of Staphylococcal Cassette Chromosome mec

(SCCmec): Guidelines for reporting Novel SCCmec elements. Antimicrob. Agents

Chemother 53(12): 4961-4967.

Janeway C, Travers P, Walport M. 2001. Immunology: The Immune System in

Health and Disease. 5th edition. New York (NY): Garland Science.

Jenkins A, Diep B, Mai T, Nhung V, Warrener P, Suzich J, Stover C, Sellman B.

2015. Differential Expression and Roles of Staphylococcus aureus Virulence

Determinants during Colonization and Disease. mBio 6(1): e022 2-14.

Jensen P,Lambert L, Iademarco M, Ridzon R. 2005. Guidelines for preventing

the transmission of Mycobacterium tuberculosis in health-care settings, 2005. MMWR

Recommend. Rep. 54:1-141.


Jia X, Zhang J, Sun W, He W, Jiang H, Chen D, Murchie A. 2013. Riboswitch

Control of Aminoglycoside Anitbiotic Resistance. Cell 152(1-2): 68-81.

Joo H, Otto M. 2012. Molecular basis of in vivo biofilm formation by bacterial

pathogens. Chem Biol 19(12): 1503-13.

Josefsson E, Hartford O, O’Brien L, Patti J, Foster T. 2001. Protection against

Experimental Staphylococcus aureus Arthritis by Vaccination with Clumping factor A, a

novel virulence determinant. J Infect Dis 184(12): 1572-1580.

Juhas M, Meer J, Gaillard M, Harding R, Hood D, Crook D. 2009. Genomic

Islands: tools of bacterial horizontal gene transfer and evolution. FEMS Microbiol Rev

33(2): 376-393.

Kallen A, Mu Y, Bulens S, Reingikd A, Petit S, Gershman K, Ray S, Harrison L,

Lynfield R, Dumyati G, Townes J, Schaffner W, Patel P, Fridkin S. 2010. Health care

associated invasive MRSA infections, 2005-2008. The Journal of the American Medical

Association, 304(6), 641-647.

Kapur D, Dorsky D, Feingold J, Bona R, Edwards R, Aslanzadeh J, Tutschaka P,

Bilgrami S. 2000. Incidence and outcome of vancomycin-resistant enterococcal

bacteremia following autologous peripheral blood stem cell transplantation. Bone

Marrow Transplantation 25(2): 147-152.

Katayama Y, Ito T, Hiramatsu K. 2000. A New Class of Genetic Element,

Staphylococcus Cassette Chromosome mec, Encodes Methicillin Resistance in

Staphylococcus aureus. Antimicrob Agents Chemother 44(6): 1549.

Katayama Y, Takeuchi F, Ito T, Ma X, Ui-Mizutani Y, Kobayashi I, Hiramatsu K.

2003. Identification in Methicillin-Susceptible Staphylococcus hominis of an active


primordial mobile genetic element for the staphylococcal cassette chromosome mec of

Methicillin-Resistant Staphylococcus aureus. J Bacteriol 185(9): 2711-2722.

Kateete D, Kimani C, Katabazi F, Okeng A, Okee M, Nanteza A, Joloba M,

Najjuka F. 2010. Identification of Staphylococcus aureus: DNase and Mannitol salt agar

improve the efficiency of the tube coagulase test. Ann Clin Microbiol Antimicrob 9: 23.

Keshavjee S, Gelmanova I, Pasechnikov A, Mishustin S.Andreew Y, Yedilbayev

A, Murlherjee J, Rich M, Nardell E, Farmer P, Kim J, Shin S. 2008. Treating Multidrug-

Resistant Tuberculosis in Tomsk, Russia. Ann N Y Acad Sci 1136:1-11.

Ki V, Rotstein C. 2008. Bacterial skin and soft tissue infections in adults: A review

of their epidemiology, pathogenesis, diagnosis, treatment and site of care. Can J Infect

Dis Med Microbiol 19(2):173-184.

Kohanski M, Dwyer D, Collins J. 2010. How antibiotics kill bacteria: from targets

to networks. Nat Rev Microbiol 8(6): 423–435.

Koneman E. 2006. Koneman’s Color Atlas and Textbook of Diagnostic

Microbiology. 6th edition. Baltimore (MD). Lippincott Williams & Wilkins.

Kurek K, Lukaszuk B, Piotrowska D, Wiesiolek P, Chabowska A, Zendzian-

Piotrowska M. 2013. Metabolism, Physiological Role, and Clinical Implications of

Sphingolipids in Gastrointestinal Tract. Biomed Res Int 908907.

Kwun M, Novotna G, Hesketh A, Hill L, Hong H. 2013. In Vivo Studies Suggest

that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug

to d-Ala-d-Ala Termini in the Peptidoglycan Cell Wall. Antimicrob Agents Chemother

57(9): 4470-4480.

Lackie J. 2010. Oxford: Dictionary of biomedicine. New York: NY. Oxford

University Press.


Leboffe M, Pierce B. 2012. Microbiology: Laboratory Theory & Application. Third

Edition. US. Morton Publishing Company.

Lee C, Cho H, Jeong B, Lee S. 2013. Strategies to Minimize Antibiotic

Resistance. Int J Environ Res Public Health 10(9): 4274-4305.

Leekha S, Terrell C, Edson R. 2011. General Principles of Antimicrobial Therapy.

Mayo Clin Proc 86(2): 156-167.

Levy S. 2002. Factors impacting on the problem of antibiotic resistance. J

Antimicrob Chemother 49(1):25-30.

Lindsay J. Staphylococcus: Moleculear Genetics. Norfolk, UK: Caister Academic

Press; 2008. 255 p.

Liu G. 2009. Molecular Pathogenesis of Staphylococcus aureus infection. Pediatr

Res 65 (5 Pt 2): 71R-77R.

Lodish H, Berk A, Zipursky S. 2000. Mobile DNA. Molecular Cell Biology. 4th

edition. New York: W. H. Freeman.

Loir Y, Baron F, Gautier M. 2003. Staphylococcus aureus and food poisoning.

Genet Mol Res 2(1): 63-76.

Lowry F. 2003. Antimicrobial resistance: the example of Staphylococcus aureus.

J Clin Invest 111(9): 1265-1273.

Lydyard P, Cole M, Holton J, Irving W, Porakishvili N, Venkatesan P, Ward K.

2009. Case Studies in Infectious Disease: Staphylococcus aureus. Garland Science:

608.

Magni M. 2010. Detection of Bacteria, Viruses, Parasites and Fungi: Bioterrotism

Prevention. Springer Science & Business Media, ISBN: 978904818543-6.


Mahon C, Donald C, Manuselis G. 2014. Textbook of Diagnostic Microbiology.

Elsevier 5(12): 270.

Mahon C, Lehman D, Manuselis G. 2011. Textbook of Diagnostic Microbiology

fifth edition. Elseier, ISBN: 9780323089890.

Mathei C, Niclaes L, Suetens C, Jans B, Buntinx F. 2007. Infections in residents of

nursing homes. Infectious disease clinics of North America 21(3): 761-772.

Mayer G. 2010. Exchange of genetic information. Microbiology and Immunology

Online 8(6).

Mayers D. 2009. Antimicrobial drug resistance: mechanism of drug resistance.

Human Press 1(8): 81.

Mazur A, Gudiol F. 2009. Methicillin- Resistant Staphylococcus aureus in long-

term-care facilities. Clin Microbiol Infect 7:26-30.

McEachran A, Blackwell B, Hanson J, Wooten K, Mayer G, Cox S, Smith P.

2005. Antibiotics, bacteria, and antibiotic resistance genes: aerial transport from cattle

feed yards via particulate matter. Environ Health Perspect 123:337–343

Mehta Y, Gupta A, Todi S, Myatra S, Samaddar D, Patil V, Bhattacharya P,

Ramasubban S. 2014. Guidelines for prevention of hospital acquired infections. Indian J

Crit Care Med 18(3): 149-163.

Meng J, Wang H, Hou Z, Fu J, Ma X, He G, Xue X, Jia M, Luo X. 2009. Novel

Anion Liposome-Encapsulated Antisense Oligonucleotide Restores Susceptibility of

Methicillin-Resistant Staphylococcus aureus and Rescues Mice from Lethal Sepsis by

targeting mecA. Antimicrob Agents Chemother 53(7): 2871-2878.

Moroni P, Pisoni G, Cremonesi P, Castiglioni B. 2011. Molecular Detection of

Human bacterial Pathogens: Staphylococcus. CPR Press 28: 309- 311.


Murray P, Rosenthal K, Pfaller M. 2008. Medical Microbiology. Philadelphia: PA.

Mosby Elsevier.

Murray P, Rosenthal K, Pfaller M. 2012. Medical Microbiology. Elsevier Health

Sciences pp 960.

Naber C. 2009. Staphylococcus aureus Bacteremia: Epidemiology,

Pathophysiology, and Management Strategies. Clin Infect Dis 48(4): S231-S237.

Nayak A, Green B, Beezhold D. 2013. Fungal Hemolysins. Med Mycol 51(1): 1-

16.

Nicasio A, Kuti J, Nicolau D. 2008. The current state of multidrug-resistant Gram-

negative bacilli in North America. Pharmacotherapy 28:235-249.

Nikaido H. 2009. Multidrug Resistance in Bacteria. Annu Rev Biochem 78:119-

146.

Nilsson I, Hartford O, Foster T, Tarkowski A. 1999. Alpha-toxin and Gamma-toxin

jointly promote Staphylococcus aureus virulence in Murine septic arthritis. Infect Immun

67(3): 1045-1049.

Noble W. 2004. The Skin Microflora and Microbial Skin Disease. Cambridge

University Press. Cambrigde, UK.

Olson N, Davidow A, Winston C, Chen M, Gazmararian J, Katz D. 2012. A

national study of socioeconomic status and tuberculosis rates by country of birth, United

States, 1996–2005. BMC Public Health 12: 365.

Otto M. 2008. Staphylococcus epidermidis – the “accidental” pathogen. Nat Rev

Microbiol 7(8): 555-567.


Oxford J, Goossens H, Schedler M, Sefton A, Sessa, Van der Velden A. 2013.

Factors influencing inappropriate antibiotic prescription in Europe. Educ Prim Care 24:

291-3.

Pammi M, Liang R, Hicks J, Mistretta T, Versalovi J. 2013. Biofilm extracellular

DNA enhances mixed species biofilms of Staphylococcus epidermidis and Candida

albicans. BMC Microbiology 13: 257.

Pantosti A, Sanchini A, Monaco M. 2007. Mechanism of antibiotic resistance in

Staphylococcus aureus. Future Microbiol 2(3): 323-34.

Papp-Wallace K, Endimiani A, Taracila M, Bonomo R. 2011. Carbapenems:

Past, Present, and Future. Antimicrob Agents Chemother 55(11): 4943.

Parija. 2009. Textbook of Microbiology & Immunology. Elsevier India. pp 580.

Park J, Friendship R, Poljak Z, Weese J, Dewey C. 2013. An investigation of

exudative epidermitis (greasy pig disease) and antimicrobial resistance patterns of

Staphylococcus hyicus and Staphylococcus aureus isolated from clinical cases. Can Vet

J 54(2): 139-144.

Pechere J. 2001. Patients Interviews and Misuse of antibiotics. Clin Infect Dis

33(3): S170-S173.

Pfalller M, Diekema D. 2007. Epidemiology of Invasive Candidiasis: a Persistent

Public Health Problem. Clin Microbiol Rev 20(1): 133-163.

Pieterse R, Todorov S. 2010. Bacteriocins- exploring alternatives to antibiotics in

mastitis treatment. Braz J Microbiol 41(3): 542-562.

Proft T, Fraser J. 2003. Bacterial Superantigens. Clin Exp Immunol 133(3): 299-

306.


Proft T. 2013. Bacterial Toxins: Genetics, Cellular Biology and Practical. Norfold:

UK. Horizon Scientific Press.

Queenan A, Bush K. 2007. Carbapenemases: the versatile β-Lactamases. Clin


Ramirez M, Tolmasky M. 2010. Aminoglycoside Modifying Enzymes. Drug Resist

Update. 13(6): 151-171.

Rayner C, Munckhof W. 2005. Antibiotics currently used in the treatment of

infections caused by Staphylococcus aureus. Intern Med J 35(2): S3-16.

Raz R, Colodner R, Kunun C. 2005. Who Are You? – Staphylococcus

saprophyticus? Clin Infect Dis 40(6): 896-898.

Reid G, Younes J, Van der Mei H, Gloor G, Knight R, Busscher H. 2011.

Microbiota restoration: natural and supplemented recovery of human microbial

communities. Nature Reviews Microbiology 9: 27-38.

Roberts R, Hota B, Ahmad I, Scott R, Foster S, Abbasi F, Schabowski S, Kampe

L, Ciavarella G, Supino M, Naples J, Cordell R, Levy S, Weisntein R. 2009. Hospital and

Societal Costs of Antimicrobial-Resistant Infections in a Chicago Teaching Hospital:

Implications for Antibiotic Stewardship. Clin Infect Dis 49(8): 1175-1184.

Rossignoli A, Clavenna A. 2007. Antibiotic prescription and prevalence rate in the

outpatient paediatric population: analysis of surveys published during 2000-2005. Eur J

Clin Pharmacol 63:1099-1106.

Rupp R, Bergonier D, Dion S, Hygoneng M, Aurel R, Robert-Graunie C, Foucras

G. 2008. Response to somatic cell count-based selection for mastitis resistance in a

divergent selection experiment in sheep. J Dairy Sci 92(3): 1203-19.


Sabour P, Griffiths M. 2010. Bacteriophages in the Control of Food- and

waterborne pathogens. American Society for Microbiology Press. Washington, DC.

Sakoulas G, Moellering R. 2008. Increasing Antibiotic Resistance among

Methicillin-Resistant Staphylococcus aureus Strains. Clin Infect Dis 46: S360-S367.

Sanders M, McKenna K, Lewis L, Quick G. 2011. Mosby’s Paramedic Textbook.

4th ed. Massachusetts: Jones & Barlett Learning.

Schommer N, Christner M, Hentschke M, Ruckdeschel K, Aepfelbacher M, Rohde

H. 2011. Staphylococcus epidermidis Uses Distinct Mechanisms of Biofilm Formation to

Interfere with Phagocytosis and Activation of Mouse Macrophage-Like Cells 774A.1.

Infect Immnun 79(6): 2267-2276.

Schwendener S, Perreten V. 2012. New MLSb resistance gene erm (43) in

Staphylococcus lentus. Antimicrob Agents Chemother 56(9): 4746-4752.

Singh A, Goering R, Simjee S, Foley S, Zervos M. 2006. The application of

molecular techniques to the study of hospital infection. Clin Microbiol Rev 19(3): 512-30.

Skipper K, Andersen P, Sharma N, Mikkelsen J. 2013. DNA transposon-based

gene vehicles – scenes from an evolutionary drive. Journal of Biomedical Science 20:92.

Skov R, Smyth R, Larsen A, Bolmstrom A, Karlsson A, Mills K, Frimodt-Moller N,

Kahlmeter G. 2006. Phenotypic Detection of Methicillin Resistance in Staphylococcus

aureus by Disk Diffusion Testing and Etest on Mueller-Hinton Agar. J Clin Microbiol

44(12): 4395-4399.

Slama T. 2008. Gram-negative antibiotic resistance: there is a price to pay.

Critical Care 12(4): S2.

Stapleton P, Taylor P. 2002. Methicillin resistance in Staphylococcus aureus. Sci

Prog 85(Pt 1): 57-72.


Steenbergen J, Alder J, Throne G, Tally F. 2005. Daptomycin: a lipopetide

antibiotic for the treatment of serious Gram-positive infections. J Antimicrob Chemother

55(3): 283-288.

Stone P, Braccia D, Larson E. 2005. Systematic review of economic analyses of

health care-associated infections. Am J Infect Control 33:501-509

Stone P. 2009. Economic burden of healthcare-associated infections: an

American perspective. Expert Rev Pharmacoecon Outcomes Res 9(5): 417-422. ,

Braccia D, Larson E. 2005. Systematic review of economic analyses of health care-

associated infections. Am J Infect Control 33:501-509.

Straus S, Hancock R. 2006. Mode of action of the new antibiotic of Gram-positive

pathogens daptomycin: Comparison with cationic antimicrobial peptides and

lipopeptides. Biochimica et Biophysica Acta 1758(9): 1215-1223.

Sydnor M, Emily R, Perl T. 2011. Hospital Epidemiology and Infection Control in

Acute-Care Settings. Clin Microbiol Rev. 24(1): 141–173.

Tanner M, Evertt C, Youvan D. 2000. Molecular Phylogenetic Evidence for

Noninvasive Zoonotic Transmission of Staphylococcus intermedius from a Canine Pet to

a Human. J Clin Microbiol 38(4): 1628-1631.

Tattlybaeva E, Nikiyan H, Vasilchenko A, Deryabin D. 2013. Atomic force

microscopy recognition of protein A on Staphylococcus aureus cell surfaces by labelling

with IgG–Au conjugates. Beilstein J Nanotechnol 4: 743-749.

Thompson J, Cronin B, Bayley H, Wallace M. 2011. Rapid assembly of a

multimeric membrane protein pore. Biophys J 101(11): 2679–83.


Tibra N, Jalali S, Reddy A, Narayanan R, Agarwal R. 2009. Traumatic

endophthalmitis caused by Staphylococcus gallinarum. Journal of Medical Microbiology

59(3): 365–366.

Tille P. 2013. Bailey & Scott’s Diagnostic Microbiology. Thirteenth edition.St.

Louis: MO. Elsevier.

Tindall W, Sedrak M, Boltri J. 2013. Patient-Centered Pharmacology.

Philadelphia: F.A Davis Company.

Tomasz A. 1994. Multiple-antibiotic resistant pathogenic Bacteria. The New

England Journal of Medicine 330(17): 1247-1251.

Turnidge J, Rao N, Chang F, Fowler V, Kellie S, Arnold S, Lee B, Tristan A.

2002. Staphylococcus aureus. Antimicrobial Therapy and vaccines 2: 631-58.

Ueda T, Takesue Y, Nakajima K, Wada Y, Tsuchida T, Takahshi Y, Ishihara M,

Kimura T, Uchino M, Ikeuchi H. 2014. High-dose regimen to achieve novel target trough

concentration in teicoplanin. J infect Chemother 20(1): 43-7.

Vanassche T, Kaushot A, Verhaehen J, Peetermans W, Van Ryn J, Schneewind

O, Hoylaerts M, Verhamme P. 2012. Fibrin formation by staphylothrombin facilitates

Staphylococcus aureus-induced platelet aggregation. Thromb Haemost 107(6): 1107-21.

Vandenbroucke-Grauls. 2014. Antimicrobial resistance in the Netherlands: a

natural experiment? Front Public Health 2(5).

Vela J, Hildebrandt K, Metcalfe A, Rempel H, Bittman S, Topp E, Diara M. 2012.

Characterization of Staphylococcus xylosus isolated from broiler chicken barn

bioaerosol. Poultry Science 91(12): 3003–3012.

Ventola C. 2015. The Antibiotic Resistance Crisis. P T 40(4): 277-283.


Vilcheze C, Williams J. 2012. The Combination of Sulfamethoxazole,

Trimethoprim, and Isoniazid or Rifampin Is Bactericidal and Prevents the Emergence of

Drug Resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 56(10):

5142-5148.

Villari T, Sarnataro, Iacuzio. 2000. Molecular Epidemiology of Staphylococcus

epidermidis in a Neonatal Intensive Care Unit over a Three-Year Period.” Journal of

Clinical Microbiology. 38(5): 1740-1746

Vogan A, Higgs P. 2011. The advantages and disadvantages of Horizontal gene

transfer and the emergence of the first species. Biol Direct 6(1): 10.1186/1745-6150-6-1.

Wagner A. 2006. Cooperation is Fleeting in the World of transposable elements.

PloS Comput Biol 2(12): e162.

Wang N, Neilan A, Klompas M. 2013. Staphylococcus Intermedius Infections:

Case Report and Literature Review. Infect Dis Rep 5(1): e3.

Watanabe H, Masaki H, Asoh N, Watanabe K, Oishi K, Kobayashi S, Sato A,

Sugita R, Nagatake T. 2001. Low Concentrations of Mupirocin in the Pharynx following

Intranasal Application May Contribute to Mupirocin Resistance in Methicillin-Resistant

Staphylococcus aureus. J Clin Microbiol 39(10): 3775-3777.

Webber M, Piddock L. 2003. The importance of efflux pumps in bacterial

antibiotic resistance. J Antimicrob Chemother 51(1): 9-11.

Weckx L. 2012. Antibiotics: from use to abuse. Braz J Ortohinolaryngol 78(2): 2-

2.

Weinstein R. 2005. Efflux Pump and Nosocomial Antibiotic Resistance: A primer

for hospital Epidemiologists. Clin Infect Dis 40(12): 1811-1817.


Wenzel R, Edmond M. 2001. The impact of hospital-acquired bloodstream

infections. Emerg Infect Dis 7(2): 174-177.

Wilcox S. 2004. Cationic Peptides: A new hope. The Science Creative Quarterly

< http://www.scq.ubc.ca/cationic-peptides-a-new-hope/>. Accessed 2014 July 20.

Wisplinghoff H. 2004. Nosocomial bloodstream infections in US hospitals:

analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect

Dis 39:309-317.

Yu R, Dale S, Yamamura D, Stankus V, Lee C. 2012. Daptomycin-

nonsusceptible, vancomycin-intermediate, methicillin-resistant Staphylococcus aureus

endocarditis, Can J Infect Dis Med Microbiol 23(2): e48.

Zachary J, McGavin M. 2013. Pathologic Basis of Veterinary Disease. Fifty

Edition. St. Louis: MO. Elsevier

Zapun A, Contrearas-Martel C, Vernet T. 2008. Penicillin-binding proteins and β-

lactam resistance. Oxford University Press 32(2): 361-385.

Zdziarski P, Simon K, Majda J. 2003. Overuse of high stability antibiotics and its

consequences in public and environmental health. Acta Microbiologica Polonica 52(1):5-

13.

Zong Z. 2013. Characterization of a complex context containing mecA but lacking

genes encoding cassette chromosome recombinases in Staphylococcus haemolyticus.

BMC Microbio 13: 64.

Zuchora-Walske C. 2014. Antibiotics. North Mankato (MN): ABDO Publishing

Company.

GENETICS OF ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS SPECIES · genetics of antibiotic resistance in...

Documents

Transcript of GENETICS OF ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS SPECIES · genetics of antibiotic resistance in...