Arita, Sean Catherine L.

BSMT 3-2
CLINICAL BACTERIOLOGY
PSEUDOMONAS AERUGINOSA INFECTION
Pseudomonas is a type of bacteria that is found commonly in the environment, like in soil
and water. Of the many different types of Pseudomonas, the one that most often causes infections
in humans is called Pseudomonas aeruginosa, which can cause infections in the blood, lungs
(pneumonia), or other parts of the body after surgery. Pseudomonas aeruginosa is a Gram-negative
bacterium, aerobic bacilli measuring 0.5 to 0.8 μm by 1.5 to 3.0 μm. Motility is by a single polar
flagellum. Species are distinguished by biochemical and DNA hybridization tests. Antisera to
lipopolysaccharide and outer membrane proteins show cross-reactivity among serovars.

This microorganism is one of the most frequent and severe causes of hospital-acquired
infections, particularly affecting immunocompromised (especially neutropenic) and intensive care
unit (ICU) patients. The majority of P. aeruginosa strains are resistant to most antibiotics currently
in use. Due to a range of mechanisms for adaptation, survival and resistance to multiple classes of
antibiotics, infections by P. aeruginosa strains can be life-threatening and are emerging as a global
public health threat.

FIGURE 1 – DIVERSE SITES OF INFECTION P. AERUGINOSA

EPIDEMIOLOGY OF PSEUDOMONAS AERUGINOSA
Pseudomonas aeruginosa is primarily a nosocomial pathogen. According to the CDC, the
overall incidence of P. aeruginosa infections in US Hospitals averages about 0.4% (4 per 1000
discharges), and the bacterium is the fourth most commonly-isolated nosocomial pathogen
accounting for 10.1% of all hospital-acquired infections. Within the hospital, P.aeruginosa finds
numerous reservoirs: disinfectants, respiratory equipment, food, sinks, taps, and mops. This
organism is often reintroduced into the hospital environment on fruits, plants, vegetables, as well
by visitors and patients transferred from other facilities.
RESERVOIR OF PSEUDOMONAS AERUGINOSA
The reservoir of an infectious agent is the habitat in which the agent normally lives, grows,
and multiplies. For Pseudomonas Aeruginosa, they live in the environment such as soil, water, and
other moist locations. It can be spread to people in healthcare settings when they are exposed to
water or soil that is contaminated with these bacteria. Resistant strains of the bacterial can also
spread in healthcare settings from one person to another through contaminated hands, equipment
(respiratory equipment, sinks, taps, and mops), humidifiers, endoscopes and endoscopes washers,
water baths and hydrotherapy pools and bathing basins. Spread occurs from patient to patient on
the hands of hospital personnel, b direct patient contacts with contaminated reservoirs, and by the
ingestion of contaminated foods and water.
P. aeruginosa was found in high concentrations (10-2500 CFU/duodenoscope); also, with
regard to the distal end, the antibiogram revealed that 60% of the samples positive for P. aeruginosa
contained strains resistant to multiple antibiotics (including carbapenems). Aeruginosa can be
transferred by a number of routes, including patient-to-patient and environmental contamination.
Due to its adaptable nature and high surviving ability, it can survive on dry inanimate surfaces in
a hospital environment from 6 hours to 6 months.
PORTAL OF EXIT FOR PSEUDOMONAS AERUGINOSA INFECTION
The main portals of exit of microbes from a patient are the mouth and airways, urethra,
anus, damaged skin (where blood and pus can emerge), and intact skin. P. aeruginosa is spread
through improper hygiene, such as from the unclean hands of healthcare workers, or via
contaminated medical equipment that wasn’t fully sterilized. Pseudomonas can easily grow in
humidifiers and types of medical equipment – catheters, for instance – that aren’t properly cleaned.
If health care workers don’t wash their hands well, they can also transfer the bacteria from an
infected patient to you. That is how the Pseudomonas Aeruginosa leave the host’s body to infect
another body.
MODE OF TRANSMISSION
There are mainly three transmission routes: (1) direct and indirect contact transmission,
(2) droplet transmission, and (3) airborne transmission. For Pseudomonas Aeruginosa, it is
commonly spread through direct contact in contaminated soil or water. In addition, P. aeruginosa
is spread through improper hygiene, such as from the unclean hands of health care workers or via
contaminated medical equipment that wasn’t fully sterilized.
PORTAL OF ENTRY FOR PSEUDOMONAS AERUGINOSA INFECTION
Portal of Entry is the opening where an infectious disease enters the host’s body. You can
get pseudomonas in many different ways. For instance, it can grow on fruits and vegetables that
you might eat, so you could get sick from eating contaminated food. It also thrives in moist areas
like pools, hot tubs, bathrooms, kitchens, and sinks.
SUSCEPTIBLE HOST OF PSEUDOMONAS AERUGINOSA INFECTION
In the hospital setting, many patients are especially susceptible for contracting infections.
P. aeruginosa rarely causes infection in the normal host, but is an efficient opportunistic pathogen
causing serious infections in patients who are mechanically ventilated, individuals who are
immunocompromised, and patients with malignancies or HIV infection. Among these risk
groups, the most vulnerable hosts are neutropenic and patients who are mechanically ventilated.
In addition, P. aeruginosa is the most prevalent chronic infection contributing to the pathogenesis
of cystic fibrosis. Because of the ubiquitous nature of P. aeruginosa and its ability to develop
resistance to antibiotics, it continues to be problematic from a treatment perspective. The
pathogenicity of P. aeruginosa is largely caused by multiple bacterial virulence factors and genetic
flexibility enabling it to survive in varied environments.
This bacterium is of particular concern to individuals with cystic fibrosis who are highly
susceptible to pseudomonal lung infections. Pseudomonas aeruginosa is also of grace concern to
cancer and burn patients as well as those people who are immunocompromised. The case fatality
rate for individuals infected with Pseudomonas aeruginosa approaches 50%. Moreover, infections
caused by Pseudomonas species include endocarditis, pneumonia, and infections of the urinary
tract, central nervous system, wounds, and musculoskeletal system.
IMMUNITY
Innate Immune Response to P.aeruginosa Infection
Innate immunity fights infections from the moment of first contact and is
composed of germline-encoded, non-clonal cellular and humoral mechanisms. These
mechanisms enable nonspecific defense against pathogens without former interactions
with infectious microbial invaders. The main components of the innate immune response
engaged in response to P. aeruginosa biofilm include neutrophils, macrophages,
dendritic cells, NK cells, and the complement system. The most solid demonstration of a
role of innate immune responses to bacterial biofilm has been obtained by introducing
human neutrophils and macrophages to P. aeruginosa biofilms devoid of planktonic
bacteria.
The observed response comprises neutrophil accumulation, respiratory burst, penetration,
phagocytosis, production of cytokines and eradication of the biofilm bacteria. In addition, P.
aeruginosa cultures with increased bacterial aggregation induced stronger respiratory burst by
neutrophils and cytokine release by macrophages. Likewise, early sampling of mouse lungs
challenged with P. aeruginosa biofilms has shown that the innate immune response involves
intense accumulation of activated neutrophils in the airways. Early accumulation of neutrophils at
the site of P. aeruginosa biofilm infection is also evident from experimentally infected chronic
wounds in mice.
Innate Immune Response in Cystic Fibrosis (CF) Patients with Chronic P. aeruginosa Lung
Infection
The innate immune response has gained particular attention in patients with CF and
chronic P. aeruginosa lung infection, due to the association between accumulation of neutrophils
in endobronchial secretions and reduced functionality of the lungs. he recruited endobronchial
neutrophils display inflammatory activity as indicated by continuing respiratory burst and
generation of nitric oxide. Accordingly, destruction of the lung tissue has been correlated with
oxidative and proteolytic lesions of endobronchial neutrophil activity. Chronic lung infections in
CF patients are associated with defective apical ion transport due to mutations in the gene encoding
the cystic fibrosis transmembrane conductance regulator. Infected CF lungs are dominated by P.
aeruginosa growing as endobronchial biofilms surrounded by numerous neutrophils and scarce
planktonic bacteria, which are subject to phagocytosis by neutrophils.
The neutrophil response in infected endobronchial secretions in CF resembles the response
in experimental in vitro and in vivo biofilms, where high numbers of neutrophils accumulate close
to the biofilm and depletion of molecular oxygen (O2) is accelerated. This is caused by the
reduction of O2 to superoxide (O2−) during the neutrophils’ active respiratory burst. Thus, the
response of neutrophils to biofilms during chronic lung infection in CF may contribute
considerably to the O2-depletion in infected CF lungs. Furthermore, as active neutrophils primarily
rely on ATP generated by anaerobic glycolysis, the high intake of glucose by neutrophils in CF
lungs as well as the enhanced level of L-lactate in sputum from CF patients with chronic P.
aeruginosa lung infection, is in agreement with a high activity of neutrophils during biofilm
infection in CF lungs. The neutrophil response to planktonic P. aeruginosa likewise includes
stimulation of the respiratory burst suggesting that neutrophil activation may also include a
response to planktonic P. aeruginosa in infected CF lungs.
Knowledge of the immune responses and bacterial defense mechanisms under conditions
of biofilm infections (i.e., pneumonia, cystic fibrosis, and chronic wounds) is important as it
constitutes an important part of the pathology of biofilm infections. We would gain a considerable
amount of knowledge to provide important treatment tools against these kinds of diseases. We may
eventually be able to damping harmful immune system activities, or to activate parts of the immune
system that can eradicate biofilm infections without causing detrimental collateral damage.
Moreover, we may be able to manipulate the bacteria and down-regulate or even eliminate the
components of biofilms that are responsible for the recalcitrance towards immune system
activities.
PATHOGENESIS
Pseudomonas aeruginosa produces many factors that may contribute to its virulence.
Almost all strains of P. aeruginosa are hemolytic on blood agar plates, and several different
hemolysins have been described. A heat-stable hemolytic glycolipid consisting of two molecules
each of L-rhamnose and 1-β-hydroxydecenoic acid has been purified. Although this hemolytic
glycolipid is not very toxic to animas (5 mg injected intraperitoneally is required to kill a mouse),
it is toxic to alveolar macrophages. Furthermore, P. aeruginosa strains isolated from respiratory
tract infections produce more hemolysin than do environmental strains, suggesting that this
glycolipid hemolysin may play a role in P. aeruginosa pulmonary infections.
Several heat-labile protein hemolysins also have been described. One of these hemolysins
may be identical to phospholipase C, which is produced by approximately 70 percent of all clinical
strains of P aeruginosa. Phospholipase C, which hydrolyzes lecithin, is of unknown toxicity, and
its role in P aeruginosa infections also remains unknown. Some strains of P aeruginosa produce
a thermolabile protein (leukocidin), which lyses leukocytes from many species including humans
but is nonhemolytic. This leukocidin (also called cytotoxin) damages lymphocytes and various
tissue culture cells and is very toxic to mice (minimum lethal dose is 1 μg). Despite its toxicity,
the role of leukocidin remains unknown.
Some strains of P aeruginosa produce large amounts of extracellular polysaccharide.
These mucoid strains usually are isolated only from patients with cystic fibrosis. The role of these
polysaccharides in the pathogenesis of P aeruginosa chronic lung infections is unknown, but they
may impede phagocytosis and impair diffusion of antibiotics and thus facilitate colonization and
persistence. Interestingly, mucoid strains are frequently deficient in production of elastase, toxin
A, and flagella, and their LPS lacks long polysaccharide side chains.
Most strains of P aeruginosa also produce one or more pigments, the most common being
pyocyanin (a phenazine pigment) and fluorescein. These pigments are nontoxic in animals.
Pyocyanin, however, retards the growth of some other bacteria and thus may facilitate colonization
by P aeruginosa. One or more of these pigments appear to function in iron acquisition by P
aeruginosa. Additional work is needed to clarify the role of these pigments in P aeruginosa
infections.
Approximately 90% of P aeruginosa strains produce extracellular protease. Three separate
proteases have been purified that differ in pH optimum, isoelectric point, and substrate specificity.
Although all are capable of digesting casein, one of them, protease II, also digests elastin. When
injected into the skin of animals, purified P aeruginosa proteases induce formation of hemorrhagic
lesions, which become necrotic within 24 hours. These proteases also cause rapid tissue destruction
when injected into the cornea of animal eyes or into rabbit lungs; they also probably contribute to
the tissue destruction that accompanies P aeruginosa eye or lung infections and may aid bacteria
in tissue invasion. Their effects, however, appear to be localized, and they are not highly toxic to
animals (LD50 = approximately 200 μg/mouse).
TOXIN A
Toxin A, the most toxic known extracellular protein of P aeruginosa, is produced by 90%
of all strains. The median lethal dose of pure toxin A is about 0.2 μg/mouse. Its toxicity has been
attributed to its ability to inhibit protein synthesis in susceptible cells. It achieves this by catalyzing
the transfer of the ADP-ribosyl moiety of nicotinamide adenine dinucleotide (NAD) onto
elongation factor 2 (EF-2) according to the following reaction:
 The resultant ADP-ribosyl-EF-2 complex is inactive in protein synthesis. This intracellular
mechanism of action of toxin A is identical to that of diphtheria toxin fragment A. Also like
diphtheria toxin, Pseudomonas toxin A is released by P aeruginosa as a proenzyme. Toxin A is
toxic to animals and cultured cells, but the proenzyme has little or no enzymatic activity.
EXOENYME S
A second ADP-ribosyltransferase, exoenzyme S, has been described. Exoenzyme S
catalyzes the transfer of ADP-ribose onto a number of GTP-binding proteins, including the product
of the proto-oncogene c-H-ras (p2lC-H-ras); however, it does not modify elongation factor 2.
Exoenzyme S is produced by about 90% of clinical isolates of P aeruginosa. Transposon-induced
S-deficient mutants are less virulent in several animal models than is their S-producing parental
strain; thus, exoenzyme S may be involved in the pathogenesis of some P aeruginosa infections.

FIGURE 2 – COMPARISON OF THE STRUCTURE AND FUNCTION OF TOXIN

A AND ITS FRAGMENTS

LABORATORY DIAGNOSIS
Diagnosis of P aeruginosa depends on its isolation and laboratory identification. It grows
well on most laboratory media and commonly is isolated on blood agar plates or eosin-
methylthionine blue agar. It is identified on the basis of its Gram morphology, inability to ferment
lactose, a positive oxidase reaction, its fruity odor, and its ability to grow at 42° C. Fluorescence
under ultraviolet radiation helps in early identification of P aeruginosa colonies and also is useful
in suggesting its presence in wounds. Other pseudomonads are identified by specific laboratory
tests.
Pseudomonas aeruginosa and other Pseudomonas organisms are aerobic, non-fermentative,
non-enterobacterial gram-negative bacilli. Obtain 2 sets of blood cultures (i.e, aerobic and
anaerobic bottles) from different sites before starting empiric antibiotics. The following laboratory
results are helpful to confirm a pseudomonal infection:
 1. CBC counts revealing leukocytosis with a left shift and bandemia, which indicates possible
presence of toxic granulations or vacuoles.
2. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels, which may be
elevated in infection.
3. Metabolic profile revealing any electrolyte abnormalities, degree of dehydration, and
worsening renal function.

TREATMENT OF PSEUDOMONAS AERUGINOSA

Pseudomonas aeruginosa is frequently resistant to many commonly used antibiotics.
Although many strains are susceptible to gentamicin, tobramycin, colistin, and amikacin, resistant
forms have developed. The combination of gentamicin and carbenicillin is frequently used to treat
severe Pseudomonas infections. Several types of vaccines are being tested, but none is currently
available for general use.
The spread of P aeruginosa can best be controlled by observing proper isolation procedures,
aseptic technique, and careful cleaning and monitoring of respirators, catheters, and other
instruments. Topical therapy of burn wounds with antibacterial agents such as mafenide or silver
sulfadiazine, coupled with surgical debridement, has dramatically reduced the incidence of P
aeruginosa sepsis in burn patients.
Pseudomonas aeruginosa is frequently resistant to many commonly used antibiotics. Although
many strains are susceptible to gentamicin, tobramycin, colistin, and amikacin, resistant forms
have developed, making susceptibility testing essential. The combination of gentamicin and
carbenicillin is frequently used to treat severe Pseudomonas infections, especially in patients with
leukopenia. Several types of vaccines are being tested, but none is currently available for general
use.
STRUCTURE, CLASSIFICATION, AND ANTIGENIC TYPES
Pseudomonas aeruginosa is a Gram-negative rod measuring 0.5 to 0.8 μm by 1.5 to 3.0 μm.
Almost all strains are motile by means of a single polar flagellum, and some strains have two or
three flagella. The flagella yield heat-labile antigens (H antigen). The significance of antibody
directed against these antigens, aside from its value in serologic classification, is unknown.
Clinical isolates usually have pili, which may be antiphagocytic and probably aids in bacterial
attachment, thereby promoting colonization.
The cell envelope of P aeruginosa, which is similar to that of other Gram-negative bacteria,
consists of three layers: the inner or cytoplasmic membrane, the peptidoglycan layer, and the outer
membrane. The outer membrane is composed of phospholipid, protein, and lipopolysaccharide
(LPS). The LPS of P aeruginosa is less toxic than that of other Gram-negative rods. The LPS of
most strains of P aeruginosa contains heptose, 2-keto-3-deoxyoctonic acid, and hydroxy fatty
acids, in addition to side-chain and core polysaccharides. Recent evidence suggests that the LPS
of a large percentage of strains isolated from patients with cystic fibrosis may have little or no
polysaccharide side chain (O antigen), and that this finding correlates with the polyagglutinability
of these strains with typing sera.
Pseudomonas aeruginosa is a non-fermentative aerobe that derives its energy from oxidation
rather than fermentation of carbohydrates. Although able to use more than 75 different organic
compounds, it can grow on media supplying only acetate for carbon and ammonium sulfate for
nitrogen. Furthermore, although an aerobe, it can grow anaerobically, using nitrate as an electron
acceptor. This organism grows well at 25° C to 37° C, but can grow slowly or at least survive at
higher and lower temperatures. Indeed, the ability to grow at 42° C distinguishes it from many
other Pseudomonas species. In addition to its nutritional versatility, P aeruginosa resists high
concentrations of salt, dyes, weak antiseptics, and many commonly used antibiotics. These
properties help explain its ubiquitous nature and contribute to its preeminence as a cause of
nosocomial infections.

FIGURE 3 – STRUCTURE AND PATHOGENIC MECHANISMS OF P.AERUGINOSA

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