1.1 (blue-green). P. aeruginosa isolates can produce several colony

Introduction to Pseudomonas aeruginosa

Pseudomonas are ubiquitous
obligate aerobic Gram-Negative Bacilli
(GNB) that commonly inhabit a wide variety of nutritionally minimalistic environments
including soil, water, plants etc. These bacteria
are generally motile, with polar flagella and do not ferment carbohydrates, do
not fix nitrogen and are not photosynthetic. Among these Pseudomonas aeruginosa
(P. aeruginosa) is the most important species
affecting man and is responsible for some serious debilitating and life-threatening infections. This bacterium was first isolated in pure culture
by Gessard (1882) from wounds that discharged blue-green pus.

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1.1.1 Physiology and morphology of Pseudomonas aeruginosa

P. aeruginosa
is a physiologically versatile bacterium,
living in moist environments and commonly found in soil and water. Equipped
with large metabolic pathways P.
aeruginosa can utilize more than eighty organic compounds as a source of carbon and energy and can grow at temperatures
as high as 42?C. This gives it an
exceptional ability to colonize ecological niches where nutrients are limited.
In the hospital environment, this
bacterium is found in moist areas such as sinks and respiratory equipment and in aqueous solutions including
disinfectants, soaps, eye drops, etc.1 It is a highly adaptable organism with a large genome spanning
over 6 million base pairs, encoding 5567 genes.2 It comprises of a
relatively invariable core genome, that encodes metabolic and resistance
factors and an accessory genome that is highly variable and harbors genes
encoding virulence factors, antibiotic resistance and specific catabolic
pathways that allow its persistence in diverse environments.3 Typical biochemical characteristics
of this bacterium are positive oxidase
test, growth at 420C, arginine and gelatin hydrolysis and reduction
of nitrate. It produces a variety of pigments including pyoverdin (yellow-green
and fluorescent), pyorubin
(reddish-brown) and pyocyanin (blue-green). P. aeruginosa isolates can produce
several colony morphotypes. Large, smooth colony morphotype with flat edges and
an elevated center (‘fried egg’
appearance) is commonly isolated from clinical samples. Small colony morphotype
with small, rough and convex appearance is commonly obtained from environmental
sources.4 The mucoid morphotype that is associated with alginate
slime is often obtained from urinary tract and respiratory tract secretions.5


Infections caused by Pseudomonas

P. aeruginosa
is the third most common bacterium causing around 9–10% of nosocomial
infections.6 A major reason for its success as hospital pathogen is
its high rate of intrinsic resistance to antimicrobials, including antibiotics
and disinfectants.7  It is
also a major cause of chronic lung infections in individuals with cystic
fibrosis. Occasionally, P. aeruginosa can colonize human body sites,
with a preference for moist areas, such as the perineum, axilla, ear, nasal
mucosa, throat, and intestine. Higher
rates of colonization are reported following hospitalization, treatment with broad-spectrum antibiotics, disruption of the
physical barriers (skin or mucous membrane), in the presence of indwelling invasive
devices, and/or when there is an underlying dysfunction of the immune defense mechanisms.
Therefore, P. aeruginosa is mostly a nosocomial pathogen associated with
respiratory tract infections including ventilator-associated pneumonia (VAP), urinary
tract infections (UTIs), burn wound infections, soft tissue infections,
bacteraemia, bone and joint infections and a variety of systemic infections,
particularly in immunosuppressed patients
or patients with severe burns or malignancies.6 Some of the community-acquired infections caused by P.
aeruginosa are: urinary tract infections, otitis externa, folliculitis
acquired in swimming pools and keratitis due to wearing contact lenses. The
mucoid phenotype of P. aeruginosa chronically colonizes and infects
patients with cystic fibrosis-causing
damage to the lung tissue and compromising
the pulmonary function.


1.1.3 Virulence factors
of Pseudomonas

P. aeruginosa possesses
an arsenal of both cell-associated and
secreted virulence factors. Some of the important cell associated virulence
factors that enable its survival in diverse environmental conditions and help
in establishing infections are flagella,
pili, alginate/biofilm and lipopolysaccharide. The prominent extracellular or secreted
virulence factors are proteases,
hemolysins, cytotoxin, pyocyanin,
siderophores, exotoxin A, exoenzyme S, exoenzyme U, etc.  Production of several of these virulence
factors is coordinated by a cell density regulating mechanism termed as Quorum
Sensing (QS).8 P. aeruginosa possesses a single polar
flagellum, which plays an important role in motility, colonization, and biofilm
formation. Pili allow the bacteria to adhere to host cell surfaces, to involve in
biofilm formation and also mediate motility. Type III secretion system (TTSS) directly
injects exotoxins into the host cell’s cytoplasm and clinical isolates
expressing this system are associated with a worse clinical outcome than the
non-expressers. Quorum sensing is a cell to cell communication system involving
signaling molecules called autoinducers
and is responsible for controlling biofilm formation. Pyoverdins are
siderophores that compete with host proteins for iron chelation. Pyocyanin reacts with oxygen to form oxygen free
radicals, causing tissue damage and inhibits both lymphocyte proliferation and mucociliary
function. Alginate or slime is an extracellular polysaccharide, which has antiphagocytic
activity and resists killing by opsonization.9


1.1.4 Antibiotic resistance
in Pseudomonas aeruginosa

Antibiotic-resistant isolates of P. aeruginosa are often isolated from
cases of hospital-acquired infections. Mechanisms
of antibiotic resistance seen in this bacterium can be broadly categorized as
intrinsic, acquired or adaptive with an overlap between categories. Intrinsic resistance is due
to the failure of the antibiotic to accumulate in the cell, whereas acquired
and adaptive resistance results from the changes in the antibiotic target sites
or enzymatic inactivation of the drug.  P. aeruginosa is intrinsically
resistant to several antibiotics due to its poor outer membrane permeability and active efflux
of antibiotics. This along with the
restricted uptake through its outer membrane and coupled with secondary
resistance mediated by beta-lactamases makes it a difficult pathogen to treat.10
Chromosomal mutations
within its genome can lead to changes in the regulation
of resistance genes. It can also acquire resistance genes from other bacteria via
plasmids, transposons, and bacteriophages.11
Acquired resistance genes predominantly confer
resistance to ?-lactams
and aminoglycosides whereas chromosomal mutation
often leads to fluoroquinolone
resistance. Multidrug resistant P.
aeruginosa (MDR-PA) is a rapidly emerging challenge in clinical
practice. An isolate of P. aeruginosa is defined as MDR-PA when it shows
in vitro resistance to at least 3
classes of antibiotics such as
anti-pseudomonal cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones. MDR is a pervasive
clinical problem often associated with significant morbidity, mortality and
increased economic burden which stems from the inappropriate empiric therapy.10
For a while now carbapenems were considered as the drug of choice for
treatment of MDR infections. However in the recent past carbapenem resistance
is widely prevalent and is often mediated by metallo-beta-lactamase
(MBL) enzymes that hydrolyze carbapenems. These enzymes are encoded by genes
like bla
IMP and bla VIM carried either on the
bacterial chromosome or plasmid. Therefore there is a need to reconsider
therapeutic strategies for the effective and early appropriate empiric treatment
of infections caused by MDR isolates. Inappropriate initial empiric therapy is
a major contributor to therapeutic failure which in turn translates into high
mortality rates. Combination antimicrobial therapy with synergistic antibiotics
should be considered for empiric treatment in severely ill patients, which then
should be de-escalated to a suitable mono-therapy guided by the bacterial
antibiogram pattern.12  


1.1.5 Correlation between
antibiotic resistance and virulence of Pseudomonas aeruginosa

With growing incidence of MDR-PA,
the ever-persisting question is about the
virulence potential of these isolates. Conventional
‘antibiotic wisdom’ from long suggested that antibiotic resistance always came at
the ‘cost of fitness’ which eventually decreases the chances of survival for MDR
bacteria, thereby allowing the susceptible species to overtake resistant
species. This was the dogma in infectious diseases for long.13 Unfortunately,
the once predominant dogma that described antibiotic resistance and fitness as
an inverse relationship has not been able to sustain in the face of ever-increasing reports of antibiotic-resistant bacteria, hinting at the
possibility of compensating co-mutations.14 The regulation of virulence
and antibiotic resistance genes is a very complex mechanism which was thought
to occur as separate events, however, it is now more evident that the genetic
regulation of both is intertwined and connected. It is now clear that the
regulation of virulence genes can influence the expression of antibiotic
resistance genes and vice versa.15 At this point in time,
there is no published data from our country
comparing the virulence determinants of MDR-PA and antibiotic sensitive
isolates. Therefore this study was planned to compare the virulence potential
of MDR-PA with that of antibiotic sensitive strains
so that it can serve as a guide for future studies on newer therapeutic
strategies tackling both, antibiotic resistance and virulence in P. aeruginosa simultaneously.


1.2 Aims and objectives

1.2.1Aim of the study  


To compare and correlate the in vitro expression
of virulence factors among antibiotic sensitive and multidrug-resistant clinical isolates of P. aeruginosa in a
tertiary care hospital.


1.2.2 Objectives of the


To isolate and identify P. aeruginosa from various clinical
To study the antibiotic sensitivity of these isolates to commonly
used anti-pseudomonal agents
To phenotypically detect the production
of extended-spectrum beta-lactamase,
metallo beta-lactamase, and Amp C beta-lactamase
among cephalosporin resistant isolates of P. aeruginosa.
To detect the presence of the
following virulence factors among antibiotic sensitive and MDR isolates of
P. aeruginosa: serum bactericidal resistance, production of mannose
sensitive and resistant pili, swimming motility, twitching motility, hemolysin, phospholipase C and slime
Comparison and correlation of virulence factor production among
antibiotic sensitive and MDR isolates of P.aeruginosa.
To identify the presence of bla IMP and bla VIM genes encoding for metallo beta-lactamases in MDR P.aeruginosa.  
To study
the in vitro effect of combination antibiotics on isolates of MDR P.aeruginosa




1.3 Social relevance


P. aeruginosa is a significant cause of hospital-acquired infections because of its ability
to survive in low nutrient environments, inherent resistance to antibiotics and
its ability to form biofilms, which makes it a difficult pathogen to eradicate.
The persistence of this bacterium in the environment can be related to its
ability to form biofilms that also increase its resistance to antibiotics and
disinfectants. The large genome of this bacterium contributes to its adaptability and metabolic versatility. Currently, carbapenems are the antibiotics of
choice for treatment of severe P. aeruginosa infections. However in the
recent past resistance to this class of antibiotics is increasing worldwide and
very often carbapenem resistance mediated
by enzymes such as metallo-?-lactamases (MBL) are plasmid mediated and have a potential for rapid
dissemination. Therefore early and rapid detection of carbapenemase production
is necessary to initiate effective antibiotic treatment and also to put in
place infection control measures to prevent their dissemination in hospital
settings. With the rapid spread of multidrug-resistant
P. aeruginosa and with no newer antipseudomonal
antibiotics in the pipeline we are left with a very limited treatment options.
These options include the highly toxic
reserve antibiotics such as colistin and polymyxin B which need to be used
judiciously in patients with other underlying co-morbidities like compromised
renal function. Thus there is a pressing need
to know the local antibiotic sensitivity pattern of P. aeruginosa isolates which can help identify the appropriate agents
for initial empiric antibiotic therapy. Studying the virulence factors in the
context of antibiotic resistance will also help in designing newer therapeutic strategies to tackle MDR-PA. Therefore this
research proposal was designed to study the local antibiogram of P.
aeruginosa which will help in designing the in-house hospital antibiotic
policy. It will also compare and co-relate the virulence factors produced by
antibiotic resistant and sensitive
isolates which will help in identifying the pathogenic potential of multidrug-resistant strains. This study will
also pave the way for future research on newer modalities of treatment for
highly virulent MDR-PA.      


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