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RESEARCH THESIS

Susceptibility pattern and multi drug resistance of Pseudomonas aeruginosa infection in

burn patients at burn center

INTRODUCTION:

Basics of Pseudomonas aeruginosa:

Pseudomonas aeruginosa is a common gram negative, non-fermentative, rod shaped,

encapsulated bacterium that belongs to the Family Pseudomonadaceae, Class
Gammaproteobacteria and Order Pesudomonadales. The word Pseudomonas is derived from a
Greek word called “pseudēs” which means “false” and Monas is a Latin word which means “a
single unit”. The species name “aeruginosa” is derived from Latin which means “copper rust”
and it refers to the blue-green laboratory pigment of the cultures of species. This pigment is a
combination of two metabolites; the pyocyanin (blue) and the pyoverdine (green) that impart
blue-green color to the cultures (Kominos et al., 1972).

It is capable of causing diseases in the animals, humans as well as the plants. It is of great
medical importance as it is a multidrug resistant microorganism. It is also important due to its
omnipresence and it’s extremely advanced intrinsic methods and mechanisms of antibiotic
resistance (Bodey et al., 1983). It is highly associated with some serious medical illnesses,
nosocomial infections especially the infections in burn patients, patients on ventilator
(pneumonia) and many septic disorders (Khan et al., 2015, Haque et al., 2018). P. aeruginosa is
accountable to be the cause of approximately 10 to 20% of hospital acquired infectious diseases
like bacteremia, septic wounds leading to septic shock, hospital acquired urinary tract infections
(Streeter et al., 2016 and Orlov et al., 1979). P. aeruginosa is also the major contributor
pathogen for causing severe respiratory tract infections (Bassetti et al., 2018).

P. aeruginosa is considered an opportunistic organism to the extent that it is capable of causing

serious diseases during the existing medical conditions like Cystic Fibrosis and burns (Morello
et al., 2011 and Zhapouni et al., 2009). It is generally seen to affect the immunocompromised
patients at lot more as compared to the immunocompetent patients (Fergie et al., 1994). It is the
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causative agent behind the hot tub or Jacuzzi folliculitis in immunocompetent individuals (Yu et
al., 2007).

P. aeruginosa is citrate, oxidase and catalase positive. It means that the microorganism is
capable to convert citrate to pyruvate which enters the metabolic cycles of the organism and
produces energy. Catalase positive means that the microorganism is capable to produce oxygen
bubbles when it is exposed to hydrogen peroxide. Oxidase positive bacteria is able to produce
cytochrome c oxidase, which is an enzyme used in the electron transport chain and bacteria uses
the available oxygen in the oxygen as the end stage electron acceptor during the process of
respiration (Liu et al., 1952). It is usually found in water, soil, skin and human made environment.
It stays in normal as well as in low oxygen atmosphere as it is an obligate respire and it can use
using aerobic respiration and it can respire anaerobically on electron acceptors like nitrate (Arai
et al., 2011). In the absence of both, it is capable of fermenting arginine and pyruvate by
phosphorylation. P. aeruginosa uses iron as a source of food to survive. It is important to know
that high levels of iron are toxic to the bacteria and to overcome the toxic effects, it uses the
siderophores which helps in binding and the transport of iron.

In extreme cases when P. aeruginosa colonizes the vital organs like lungs, kidneys and the
urinary tract in general, the situation can be lethal. It resides well in the humid places and usually
the catheters, needles and other equipment are contaminated with it. It efficiently decomposes
the hydrocarbons and it has been used to break down oil from the oil spills in seas. According to
the studies P. aeruginosa is less virulent than the Staphylococcus aureus and the Streptococcus
pyrogens. However, it is capable of making extensive colonies and biofilms which are a dynamic
structure and that bestows many advantages to the other members (other bacteria/ fungi) such as
adhesion, nutritional sources, protection, antimicrobial resistance and cell transmission (Mah et
al., 2003 and Ma et al., 2009).
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Pink to red P. aeruginosa rods (Gram stained) Microscopic image of P. aeruginosa

(Todar’s online textbook of bacteriology, (researchgate.net)
K. Todar)

Looking at the genetics, we find that the genome of P. aeruginosa consists of large circular
chromosomes which carry up to 6000 open reading frames (ORF). Depending on the strains, it
sometimes has plasmids of different sizes. P. aeruginosa population forms three lineages which
are characterized by the PAO1, PA14 and PA7 genomes (Stover et al., 2000 and Mathee et al.,
2008).
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While P .
aeruginosa 
is considered
an

opportunistic pathogen, several clones have become even specialized pathogens especially in
burn patients as well as in the Liverpool epidemic strain (United Kingdom) and the AUST-02
strain (Australia) (Winstanely et al., 2009).

As an opportunistic and hospital acquired pathogen in the patients with existing diseases and low
immunity, it infects the urinary tract, open wounds, burned areas, air passage way and the blood.
It adheres, disrupts or modulates the pathways of the host cells and attacks the extracellular
matrix. It forms biofilms in order to protect itself and to cause antimicrobial resistance (Kipnis et
al., 2006).

Pathogenesis of Pseudomonas aeruginosa.

(microbiwiki.kenyon.edu)

Campylobacter jejuni and Pseudomonas aeruginosa biofilm.

(Journals.plos.org)

Only a small fraction antimicrobial agents work against P. aeruginosa and they include Penicillins,
Cephalosporins, Carbapenams, Aminoglycosides and Fluoroquinolones (Morita et al., 2014). The
extensive and frequent use of antibiotics has led to the development of antimicrobial resistance and
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is one of the leading of the failure of the antipseudomonal therapy (Paramythiotou et al., 2004,
Yayan et al., 2015 and Morita et al., 2014).

P. aeruginosa phagocytosed by a neutrophil (Gram stained)

(www.wikipedia.org)

Basics of Multi drug resistance (MDR):

Multidrug resistance (MDR) is defined as the insensitivity/ resistance of a specific

microorganism to the administered antimicrobials
which are not related structurally and have unique
molecular targets (Gerlach et al., 1986). These
antimicrobials were efficacious enough in the
past to the specific microorganism. During the
past few decades, the incidence of bacterial and
other microbial infections has increased drastically. As
a result of the continuous use of antimicrobial agents
in treating infections, many organisms and their strains have developed resistance against the
agents. As per the WHO, these agents can easily counteract the antimicrobial attack which
results into an inefficient and ineffective treatment. This leads to the spread of infection in worse
ways. It is important to note that the development of MDR is a natural phenomenon. However,
due to the rise in the number of diseases that makes the patients immunocompromised (AIDS,
Diabetes Mellitus, Autoimmunity, burns) and organ transplants, the patients get more prone to
nosocomial infections that further contribute to the outspread of MDR. WHO has listed increased
rates of MDR in organisms like Pseudomonas aeruginosa, Escherichia coli, Klebsiella
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pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis,

Neisseria gonorrhea, Salmonella and Shigella species against the Cephalosporin,
Fluoroquinolones, Carpapenam, Methicillin, Rifampicin, Isoniazid and other Antibacterial drugs.
If we talk about antifungals, we already have limited drugs available. Fungi species like
Candida, Trichosporon beigelti, neoformans and Aspergillus have developed MDR. Same is the
case with viral diseases as many viruses like Herpes simplex virus (HSV), Cytomegalovirus
(CMV), Hepatitis C virus (HCV) and Human immunodeficiency virus (HIV) are multi drug
resistant especially in transplant and chemotherapy patients. Parasitic multidrug resistance too is
observed now a day in parasites like Plasmodium, Toxoplasma gondii and Leishmania against
drugs like Chloroquine, Artemisinin and Amphotericin B (Nikaido et al., 2009).

Bone infected by P. aeruginosa in Osteomyelitis patient.

(Almay.com)

Antimicrobial drugs have been widely used for

a lot of time period now in almost all parts of the
World and surveillance in the different parts of
World has shown many organisms which are not
resistant to drugs and it is an alarming situation.
Along with high levels of MDR, they have raised
mortality and morbidity rates and now they are
known as the “super bugs” (Ghafur et al., 2013). They are a serious threat over the World to the
public health. It provoked no or reduced therapeutic outcomes and has led to the spread of
dangerous pathogens especially in burn, transplant and chemotherapy patients. TB is difficult to
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treat now a day due to the highly drug resistant strains (XDR-TB). Pneumonia is untreatable
many times due to extended spectrum β-lactamases (ESBL) mediated methods and mechanisms
induced by the causative agents leading to MDR. HIV therapy drug resistance has led to the
failure antiretroviral therapy. Such situations have incurred poor therapeutic outcomes and high
healthcare costs for the manufacture of the novel drugs for treatment (Funkenga et al., 2016).
However, there is a market difference in the resistance profile of individuals and rapid and
expanded worldwide trade and tourism has led to the potential, rise and spread of MDR.

Low therapeutic efficacy

Raised morbidity High healthcare

and mortality MDR expenses

Low and poor quality Raised chances of

of life diseases in
immunocompromised
patients
Therapeutic threat

Administration of proper doses for proper time duration still leads to poor outcomes. This failure
is due to MDR as well as poor immune response, poor drug bioavailability and raised drug
metabolism. We can classify MDR as:

 Primary resistance: Occurs when organism has never experienced particular drug.
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 Secondary resistance/ acquired resistance (MDR/ XDR): Resistance arouse after

exposure.
 Clinical based resistance: Related to drug concentrations.

(researchgate.net)

The mechanisms of microbial resistance can be understood as follows:

 Chromosomal mutations/ exchange of extrachromosomal DNA.

 Overexpression of drug target enzymes.

 Inactivation/ enzymatic degradation of amide/ ester linkage by hydrolysis.

 Drug efflux pumps

 Genetic overexpression of genes encoding for ATP binding cassette (ABC) transporter
membrane proteins (Fairlamb et al., 2016).
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Mechanism of multidrug resistance

(Jyoti T. et al., 2014)

MDR in Pseudomonas aeruginosa:

Overuse of antibiotics without prescription has accelerated the antibiotic resistance worldwide
(Martens et al., 2017). MDR in P. aeruginosa causes 40% to 60% nosocomial also called as
hospital acquired infections in different regions of the world. Infections due to P. aeruginosa are
not easy to treat especially in burned patients and it can lead to death in such patients (Toelra et
al., 2018 and Hsueh et al., 1998). MDR P. aeruginosa uses a variety of mechanisms to carry out
antibiotic resistance and some mechanisms are listed above as well. Some of these mechanisms
include over expression of efflux pumps, modification of antibiotic target, deactivation of
enzymes and biofilm formation (Pachori et al., 2019). Presence of beta lactamase enzymes is the
most common method of resistance in P. aeruginosa (Senda et al., 1996 and Vahdani et al.,
2012). Many factors like burn, age, traumas, high bacterial virulence, resistant strains, enzyme,
production of toxins as well as the movement of pathogens are the elements of hostile infection.
Superficial microbial contamination of the wound progresses and aggregates the infections in
burn patients. The level of microbial wound contamination has a direct association with the
danger of sepsis (Fitzwater et al., 2003 and Rafla et al., 2011). The increasing antibiotic resistant
strains of P. aeruginosa are an emerging global concern as well as for patients, medical
practitioners, healthcare providers and researchers in Pakistan for. With this concern, the purpose
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of this research is to evaluate the current status of antibiotic resistance of P. aeruginosa isolated
from burn patients admitted at burn center to find susceptibility pattern and MDR profile of this
pathogen.