JOURNAL

CLUB
PRESENTER : Dr. RESHMA V.P

MODERATOR : Dr. AMIT R. UGARGOL

Phenotypic & Genotypic Analysis of Biofilm
production by Pseudomonas aeruginosa
from infection and colonization samples
Rodrigo Lira Rodrigues, Jailton Lobo da Costa Lima, Kêsia
Xisto da Fonseca Ribeiro de Sena and Maria Amélia Vieira
Maciel
 Journal of Brazilian Society of Tropical Medicine
 Vol:53
 2020
 INTRODUCTION
 Pseudomonas aeruginosa- opportunistic pathogen associated
with health care related infections

 Immunosuppressed individuals, ICUs

 2nd & 3rd degree burns
 Patients on mechanical ventilation
 Biomaterials – catheters, prostheses, contact lenses
 UTI
 Corneal infections
 INTRODUCTION (contd.,)
 Increase the rate of mortality & morbidity

 Potentially multidrug resistant

 BIOFILM production (80%):

 Virulence & bacterial resistance
 Persistence of infection
 Ineffective action of antimicrobials
 Escape from phagocytes
 INTRODUCTION (contd.,)
 QUORUM SENSING (QS)
 Complex biochemical communication mechanism
 Microbes secrete quorum sensing molecules to communicate
number and types of cells among the members of biofilm
 At high concentrations -> transcription of specific genes ->
biofilm production

 QS enables P.aeruginosa to respond to the self inducting molecules

called acyl-homoserine lactones (AHLs) that target Las and Rhl
genes
 INTRODUCTION (contd.,)

Genes Signaling Receptors

• lasI molecules • LasR
• rhlI
• OdDHL • RhlR
• pqsA
• BHL • PqsR
• PQS
 INTRODUCTION (contd.,)
 Studyaimed at analyzing P.aeruginosa isolates from colonized
& infected patients,

 to verify biofilm production phenotypically

 to search for QS genes (lasI, lasR, rhlI,rhlR)
 to undertake an analysis of the clonal profile of the isolates
 METHODS
Bacterial Isolates & their sources
 Patients
admitted to a public hospital in Recife, Brazil
 2018-2019

 Total 31 P.aeruginosa isolates

 21- infection isolates
 10- colonization isolates

 Stored at (-20ºC)
 Frozen isolates reactivated in BHI medium, incubated for 24 h
at 37ºC
 Then seeded in cetrimide agar and incubated for 24 h at 37ºC
 METHODS (contd.,)
Biofilm production test
P.aeruginosa isolates grown in BHI broth for 24h at 37ºC

200µL of bacterial suspension applied to 96 well polystyrene plates in triplicate

for micro titration; incubated for 24 h at 37ºC

Bacterial suspensions removed & each well washed with 250 µL of saline

Fixation using 200 µL of methanol for 15 min

Plates dried at room temperature and stained with 200 µL of crystal violet for
5 min

Plates washed under running water & dried at room temperature

Absorbance at A570 measured using ELISA plate reader and samples

classified as described by Stepanovic
 METHODS (contd.,)
Biofilm production test
 The optical density for each isolate (ODi) was obtained by averaging
the three wells

 This value was compared with the optical density of the negative
control (ODc).

 The isolates were classified into four categories based on the average
of the ODi obtained relative to the ODc.

 The classification criteria for each category included

 cells that were not adherent (ODi ≤ ODc)
 weakly adherent (+) (ODc < ODi ≤ 2×ODc)
 moderately adherent (++) (2×ODc < ODi ≤ 4×ODc)
 strongly adherent (4×ODc < ODi)
METHODS (contd.,)
Detection of genes related to biofilm production
• Total DNA extraction performed using Brazol kit
• DNA quantified via spectrophotometry

• Genes associated with QS (lasI, lasR, rhII,rhlR) amplified

via PCR
• 30 cycles of denaturation at 94ºC for 1 min, annealing at
52ºC for 1min, extension step at 72ºC for 1.5min

• Blue green stained PCR products were run on a 2 % agarose

gel using electrophoresis
• Visualized under UV light
METHODS (contd.,)
Detection of genes related to biofilm production (contd.,)
METHODS (contd.,)
Molecular typing of the isolates
 The 31 isolates were analyzed via molecular typing by using the
enterobacterial repetitive intergenic consensus-based PCR
(ERIC-PCR) technique to determine the clonal profile of the
strains.
 The PCR was performed in a total volume of 25 μL per tube
containing
 100 ng of DNA
 10 pmol of primers (ERIC-1 [5'-
ATGTAAGCTCCTGGGGATTCAC-3']; ERIC-2
[5'AAGTAAGTGACTGGGGTGAGG-3'])
 1x buffer
 200 μM deoxyribonucleotide triphosphate
 1.5mM of MgCl2
1 U of DNA Taq Polymerase
METHODS (contd.,)
Molecular typing of the isolates (contd.,)
 The amplification parameters for ERIC-PCR were
 Denaturation at 95ºC for 3 min
 30 cycles at 92ºC for 1 min
 Annealing at 36ºC for 1 min
 Extension step at 72ºC for 8 min
 Final extension at 72ºC for 16 min.
 The PCR products were stained with Blue Green
 Analyzed by agarose gel electrophoresis using a 1.5 % agarose gel.
 The DNA bands were visualized under UV light and
photodocumented for clonal analysis
RESULTS
Origin of isolates
 Infection isolates:
 Blood (33.3%)
 Tracheal secretion & urine samples (23.8%)

 Colonization isolates : rectal swab culture (90% )

RESULTS (contd.,)
Phenotypic analysis of biofilm production
 58.1 % (18/31) were biofilm producers
II (10)

II(8)
CI (7)

CI(3)
II(2)
II (1)
 RESULTS (contd.,)
Quorum sensing gene analysis

 All31 isolates had the four QS genes studied (lasI, lasR, rhlI,
and rhlR)
 RESULTS (contd.,)
Clonal profiles of isolates
 26 distinct genetic profiles were identified, that showed high genetic
variability between the isolates

I
I
CI
DISCUSSION
 Infection isolates – obtained from blood, tracheal secretions,
urine – similar to other studies

 Quantitative phenotypic test (gold std) to detect biofilm

production- other studies show a higher percentage (73.7% -
98.6%)

 Adhesion profile of biofilm : predominance of weakly adherent

isolates
DISCUSSION(contd.,)
 Detection of the presence of the QS genes associated with the
phenotypic analysis of biofilm production can help in the evaluation of
their regulation.

 Genotypic analysis of 31 isolates showed a 100 % detection rate for all

four investigated genes.

 41.9 % (13∕31) of the isolates did not produce biofilms in the

quantitative analysis.

 This may occur due to the non-expression of these genes or because of

the presence of mutations in the genes that regulate the QS system
 mutations at position 53 of the LasR protein, which is close to the
binding region for its autoinducer, N-(3-oxododecanoyl) homoserine
lactone (OdDHL)
DISCUSSION(contd.,)
 Isolates lacking the lasR and lasI genes were unable to form a biofilm,
and the results indicate that these genes play an important role in QS
and the formation of biofilms.

 Isolates with mutations in the genes associated with the QS system had
a lower degree of virulence

 26 distinct genetic profiles of the isolates were obtained and similar

results seen in other studies

 Antibiotic therapy promotes the artificial selection of bacteria for

greater drug resistance
DISCUSSION(contd.,)
 The analysis of genetic diversity showed the occurrence of a clone
composed of four isolates, one of them II and three CI.

 Dissemination between patients plays an important role in the

acquisition of P. aeruginosa colonization on the skin and mucous
membranes, and this process precedes the infection that occurs later.

 Prior rectal colonization by P. aeruginosa is a key factor for the

development of infection.

 Health professionals may directly or indirectly be involved in the

microorganism spread chain
DISCUSSION(contd.,)
 The manifold and diverse mechanisms employed by P. aeruginosa
to survive antibiotic treatment while growing in a biofilm represent
an important therapeutic challenge

 The acquisition of possible resistance genes is also an important

factor

 This was not analyzed in the present study.

 Therefore, the emphasis on surveillance culture is important in the

implementation of the infection control program
CONCLUSIONS
 Described the biofilm production in infection and colonization isolates of
P. aeruginosa from patients admitted to a hospital.

 Detected the presence of high genetic diversity between the isolates,

including a clone composed of among infection and colonization isolates,
indicating that dissemination had occurred in the hospital environment.

 The results indicate the importance of the detection of biofilms in P.

aeruginosa in both colonization and infection isolates, especially from
complex environments such as ICUs.

 The strategy outlined in this study can be used for monitoring and studying
strains that can cause infections in hospitalized patients, though the
formation of biofilms remains an important therapeutic challenge .
Thank
you……