Incidence of Pseudomonas aeruginosa with Special Reference to Drug Resistance and Biofilm Formation from Clinical Samples in Tertiary Care Hospital

Full text

Incidence of
Pseudomonas
aeruginosa
with Special
Reference to Drug Resistance
and Biofilm Formation from
Clinical Samples in Tertiary
Care Hospital
Received Date: 07 April 2016 – Accepted Date: 23 May 2016 – Published Online: 06 June 2016
Copyright © 2016
INTRODUCTION
Pseudomonas aeruginosa
(
P. aeruginosa
) is an aerobic, gram negative rod-shaped, motile
bacterium1. Moist sites in the hospital are known reservoirs of
P. areuginosa
strains. They are often multidrug resistant due to intrinsic and acquired
determinants2. It can survive with low levels of nutrients and grow in tem-
peratures ranging from 4 to 42°C3,4. It can cause urinary tract infections,
respiratory infections, dermatitis, soft tissue infections, bacteremia, bone
and joint infections and gastrointestinal infections. It is responsible for a
variety of systemic infections, particularly in patients with severe burns, bed
ulcers, and in patients suffering from cancer or AIDS5,6.
Biofilms have an enormous impact on healthcare, and are estimated to
be associated with 65% of nosocomial infections7. Biofilm is a structured
RESEARCH ARTICLE
J. D. Andhale*,
R. N. Misra,
N. R. Gandham,
K. M. Angadi,
S. V. Jadhav,
C. R. Vyawahare,
M. Pawar,
S. Hatolkar
Dr. D. Y. Patil Medical College, Hospital and
Research Centre (D. Y. Patil Vidyapeeth, Pune),
Pimpri, Pune, 411018, India

Address reprint requests to
*Mr. J. D. Andhale, [Ph. D. Scholar],
Department of Microbiology, Dr. D. Y. Patil
Medical College, Hospital and Research
Centre (D. Y. Patil Vidyapeeth, Pune), Pimpri,
Pune, 411018, India
E-mail: jd.nsk2008@gmail.com

Article citation: Andhale JD, Misra
RN, Gandham NR, Angadi KM, Jadhav
SV, Vyawahare CR et al. Incidence of
Pseudomonas aeruginosa with spe-
cial reference to drug resistance and
biofilm formation from clinical samples in
tertiary care hospital. J Pharm Biomed Sci
2016;06(06):387–391.
Available at www.jpbms.info
Statement of originality of work: The
manuscript has been read and approved by all
the authors, the requirements for authorship have
been met, and that each author believes that the
manuscript represents honest and original work.
Sources of funding: None.
Ethical approval: The study was approved by
the institutional ethics committee.
Acknowledgments: The authors would like
to extend their thanks to the management and
the head of the institute for giving opportunity
to conduct research. They are also thankful to
members of the D. Y. Patil Vidyapeeth for their
help and support. The authors acknowledge the
assistance provided by Neelam Sing (Senior
Technician) at different stages of this study.
Competing interest / Conflict of interest: The
author(s) have no competing interests for financial
support, publication of this research, patents
and royalties through this collaborative research.
All authors were equally involved in discussed
research work. There is no financial conflict with
the subject matter discussed in the study.
Disclaimer: Any views expressed in this paper
are those of the authors and do not reflect the
official policy or position of the Department of
Defense.
NLM Title J Pharm Biomed Sci
CODEN JPBSCT
2230-7885
ISSN No
ABSTRACT
Background Pseudomonas aeruginosa (P. aeruginosa ) is an aerobic, gram negative,
motile rod and possesses a variety of virulence factors. Antimicrobial resistance is an
innate feature of bacterial biofilms.
Objectives Determination of prevalence, antibiotic susceptibility and biofilm production
of P. aeruginosa isolates from clinical samples.
Materials and Methods A prospective study was carried out from the period of June
2014 to December 2014 in Microbiology Department, Dr. D. Y. Patil Medical College, Pune.
The study included a total of 300 various clinical samples received in the department of
Microbiology from different wards for routine culture and sensitivity test. The samples
were processed and isolates were identified by standard protocol. All isolates were tested
for phenotypic detection of biofilm formation and antibiotic resistance pattern.
Results Out of 300 clinical samples, 30 samples were positive for P. aeruginosa (10%).
Maximum of 19 isolates were from pus/wound swab (63.33%) followed by urine 5 (20%).
23 (76.66%) were from males and 7 (23.33%) were from females. Maximum prevalence
belonged to the age group of 41–60 years of age 14 (46.66%), followed by patients of
60–80 years of age 8 (26.66%). A total of 13 of the 30 isolates (43.33%) showed biofilm
production. 66.66% (10/15) of multiple antibiotic resistant isolates showed biofilm
production. P. aeruginosa was highly resistant to ceftazidime 50% and least resistant to
imipenem 10%.
Conclusion The results confirmed P. aeruginosa is a common pathogen isolated from
various clinical samples of patients. In this study, the antibiotic resistance was signifi-
cantly higher among biofilm-producing P. aeruginosa than non-producer. Imipenem was
found to be the most effective antimicrobial agent. Use of ceftazidime should be restricted
as it found least effective. To avoid rapid emergence of drug resistant strains, periodic
testing of biofilm formation and antibiotic sensitivity should be carried out to detect the
resistance trends. As this is a hospital-based epidemiological data, present study will
help for implementation of better patient management and infection control strategies.
KEYWORDS P. aeruginosa, biofilm, Imipenem, ceftazidime
DOI http://dx.doi.org/10.20936/jpbms/160259

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J. D. Andhale
community of bacterial cells enclosed in a self-produced
polymeric matrix adherent to an inert or living surface.
Organisms producing biofilm are far more resistant
to antimicrobial agents than non-producers8. Bacterial
biofilms cause chronic infections, because they show
increased tolerance to antibiotics and disinfectant chem-
icals as well as resisting phagocytosis and other com-
ponents of the body’s defence system. Biofilm growth
is associated with an increased level of mutations as
well as with quorum-sensing-regulated mechanisms.
Chromosomal lactamase, up-regulated efflux pumps
and mutations in antibiotic target molecules in bacteria
also contribute to the survival of biofilms. Biofilms can
be prevented by early aggressive antibiotic prophylaxis
or therapy, and they can be treated by chronic suppres-
sive therapy9. Antimicrobial resistance is an innate fea-
ture of bacterial biofilms10. The mechanisms responsible
for antimicrobial resistance in organisms producing bio-
films may be delayed penetration of the antimicrobial
agents through the biofilm matrix, altered growth rate
of biofilm organisms and other physiological changes
due to the biofilm mode of growth11. Many studies have
shown that biofilm formation is higher in MDR strains12.
Infection with multidrug resistant (MDR) strains of
P. aeruginosa
are of great concern for hospitalised patients13.
P. aeruginosa
is one of the main organisms responsi-
ble for drug-resistant nosocomial infections
. P. aeruginosa
is becoming day-by-day a very common pathogen. It
may colonise healthy humans without causing disease.
But sometimes, its potential to act as a pathogen, can
be identified without any doubt due to its isolation in
clinical samples with positive disease impact and as an
agent of nosocomial infection. Injudicious administra-
tion of broad-spectrum antibiotics, instrumentation
and intrinsic resistance of microorganisms antimicro-
bial agents contribute to make
P. aeruginosa
a nosocomial
pathogen14.
Keeping these facts in mind, this study was under-
taken to study the prevalence, detection of biofilm pro-
duction and antibiotic sensitivity pattern of
P. aeruginosa
among the clinical isolates.
MATERIALS AND METHODS
This study was conducted at the Department of
Microbiology in Dr. D. Y. Patil Medical College, Hospital
and Research Centre, Pimpri, Pune. It is a tertiary care
centre and teaching hospital. The study was conducted
from June 2014 to December 2014.
Ethical statement
The study was approved by Institutional Ethical Committee
(Dr. D. Y. Patil Vidyapeet Pune).
Approximately, 300 clinical samples were included
in this study. The samples received for routine culture
and sensitivity from various wards of both sexes with
different ages having clinical infection were included.
The samples were taken from the department of micro-
biology, Dr. D. Y. Patil medical College, Hospital and
Research Centre, Pune. They were cultured onto Mac
Conkey’s, nutrient and blood agar plates, and then
incubated at 37°C for overnight. After obtaining the
pure strains, the strains were subjected to biochemical
tests to identify the isolate.
P. aeruginosa
was identified
by colonial morphology, gram stain, a positive oxidase,
catalase, citrate, production of pyocyanin pigments and
motility test.
Antimicrobial susceptibility
Antimicrobial susceptibility was performed on Mueller–
Hinton agar by standard disc diffusion method recom-
mended by Clinical and Laboratory standards Institute.
This was done by dipping a sterile swab, carefully swab-
bing the entire surface of Mueller–Hinton agar plates. The
antibiotics used against the
P. aeruginosa
were as follows:
amikacin (30 µg), ceftazidime (30 µg), ceftazidime/
clavulanic acid (30/10 µg), ceftazidime/tazobactum
(30/10 µg), carbenicillin (100 µg), ciprofloxacin (5 µg),
gentamicin (10 µg), imipenem (10 µg) and piperacillin
(100 µg). Then, the antibiotic discs were placed on the
surface of the inoculated plates and gently pressed. The
plates were incubated at 37°C for 18–24 h. The diame-
ter of inhibition zone was measured in millimetres and
isolates were scored as sensitive or resistant by com-
paring with values recommended on standard charts15.
P. aeruginosa
ATCC27853 was used as the quality control
organism in antimicrobial susceptibility determination.
Evaluation of biofilm formation by tube method
A qualitative assessment of biofilm formation was car-
ried out as described by Christensen et al.16. A loopful of
isolates from overnight culture plates was inoculated in
10 ml of trypticase soy broth with 1% glucose in test
tubes and incubated at 37°C for 24 h. After incuba-
tion, the tubes were decanted and washed with phos-
phate-buffered saline (pH 7.3) and dried. Then, the
dried tubes were stained with crystal violet (0.1%).
Excess stain was removed, and the tubes were washed
with distilled water and dried in inverted position and
then observed for biofilm formation. Tests were per-
formed in triplicate at three different times. Biofilm
was considered positive when a visible film lined the
wall and the bottom of the tube. The amount of biofilm
formed was recorded as a A) strong, B) weak and C)
negative.
RESULTS
A total of 300 samples were collected from June 2014
to December 2014. Out of total 300 various clinical
samples investigated, 30 strains of
P. aeruginosa
were iso-
lated and identified by conventional microbiological
procedures. The rate of isolation of
P. aeruginosa
was 10%.

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Isolation of
P. aeruginosa
from clinical samples
Of these 30 strains of
P. aeruginosa
, 23 (76.66%) were
from males and 7 (23.33%) were from females (Table 1).
Most of them belonged to the age group of 41–60
years, (14, 46.66%), followed by patients of 60–80 years
of age (8, 26.66%) (Table 2).
19 (63.66%) were from Pus and wound sample,
which were the predominant source of specimens of
P. aeruginosa
, and clinical isolates followed by urine 6 (20%)
and sputum 4 (13.33%), body fluid 01 (3.33%) (Table 3).
In our study, a total of 13 of the 30 isolates (43.33%)
showed biofilm production and 17/30 (56.66%) iso-
lates were negative (Figs. 1–3).
Among 13 isolates, biofilm production was strongly
positive for 10/13 (33.33%) isolates, 10% (3/13) iso-
lates were weakly positive and 56% 17/30 of non- biofilm
producers (Table 4).
In our study, 10 out of 15 (66.66%) multiple drug
resistant
P. aeruginosa
(
MDRPA
) isolates showed biofilm
production (Table 5), out of which 9/15 (60%) showed
strong biofilm production and 1/15 (6.66%) weak.
5/15 (33.33) MDRPA were negative (Table 5).
Out of 30 isolates, 9 (30%) were sensitive to all anti-
biotics tested and 15 (50%) isolates were multi-drug
resistant. In this study,
P. aeruginosa
was highly resistant to
ceftazidime 50%, ceftazidime + clauvlinic acid 46.66%,
carbenicillin 46.66%, ceftazidime + tazobactum 40%,
Table 1 Gender-wise distribution of clinical isolates of
P. aeruginosa (n = 30).
Sr. no. Gender Number Percentage (%)
1. Male 23 76.66
2. Female 07 23.33
3. Total 30 99.99
Table 2 Prevalence of P. aeruginosa based on age.
Sr. no. Age in years Number Percentage (%)
1. 0–20 06 20.00
2. 21–40 02 06.66
3. 41–60 14 46.66
4. 61–80 08 26.66
5. >80 00 00.00
6. Total 30 99.99
Table 3 Distribution of specimens of P. aeruginosa
clinical isolates.
Sr. no. Name of specimen Number %
1. Pus/wound swab 19 63.33
2. Urine 06 20.00
3. Sputum 04 13.33
4. Body fluid 01 03.33
5. Total 30 99.99
Table 4 Number of P. aeruginosa isolates forming biofilm.
Biofilm formation No. of isolates %
Strong 10 33.33
Weak 03 10.00
Negative 17 56.66
Total isolates 30 100.00
Table 5 Multiple drug resistant P. aeruginosa isolates
showing biofilm formation.
Biofilm formation No. of MDRPA isolates %
Strong 9 60.00
Weak 1 06.66
Negative 5 33.33
Total MDRPA isolates 15 100.00
Fig. 1 Strong biofilm producer.
Fig. 2 Week biofilm producer.
Fig. 3 Non-biofilm producer.

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J. D. Andhale
piperacillin 36.66%, amikacin 33.33%, ciprofloxacin
33.33%.
P. aeruginosa
was least resistant to imipenem 10%
followed by gentamicin 30% which can be considered
as effective drugs in this present review.
DISCUSSION
P. aeruginosa
is is a major and well-recognised nosocomial
pathogen. It can survive and multiply even with mini-
mal nutrients. It can cause severe infection in hospital-
ised patients. Despite advances in sanitation facilities and
the introduction of a wide variety of antibiotic agents
with antipseudomonal activities, life-threatening infections
caused by
P. aeruginosa
continue to be hospital infections.
In this study, we aimed to determine prevalence, bio-
film formation and antimicrobial susceptibility pattern
of
P. aeruginosa
isolated from various clinical samples.
In this study, the isolation rate of
P. aeruginosa
was 10%.
Similar prevalence rate of 9.7 and 10.17 was reported
by Dash et al. and Siham et al., respectively17,18. In com-
parison, higher prevalence rate of 32.1 and 20.3% was
reported by Rajat et al. and Javiya et al. in Gujarat, India,
respectively19,20. In similar studies, lower rates of isola-
tion is reported by Srinivas et al. (9.3%) and Pathi et al.
(8.43%)21,22. This varied prevalence of
P. aeruginosa
in dif-
ferent places may attribute to the type of clinical speci-
mens received for examination, population studied, type
of hospitals and geographic allocations. In this study, the
rate of isolation in male was 23/30 (76.66%) higher
than females 7/30 (23.33). Ahmed et al
.
(77.7%) and
Raakhee et al. (66.78%) reported higher incidence in
males than females respectively23,24. In our study, most
of the patients belonged age group of 41–60 (46.66%).
Ahmed et al. reported a higher prevalence rate among
61–80 years (43.92%)23. This could be explained due
to decreased immunity and prolonged hospital stay and
other associated co-morbidities in these age groups.
In our study, the commonest sample was Pus/wound
swab 19/30 (63.33%), urine 6/30 (20%) and sputum
4/30 (13.33%). Similar findings are also reported by
Pathi et al.22. In our study, biofilm production was found
in 13/30 (43.33%). Strong biofilm producer was shown
by 10/30 (33.33%) and weak biofilm producer in
3/30 (10%). Gurung et al. have reported 33% (16/49)
of biofilm production by
P. aeruginosa
25. In our study, we
found higher antibiotic resistance in strong biofilm pro-
ducers as compared to the negative biofilm producers.
66.66% (10/15) of
P. aeruginosa
producing biofilm for-
mations showed MDR pattern. In a similar study, Dardi
et al. reported 65% (39/60)26. The resistance profiles of
P. aeruginosa
to the nine anti-microbial agents tested varied
among the isolates investigated. The most important fea-
ture was that 90% of
P. aeruginosa
isolates were found to
be sensitive to imipenem followed by 70% gentamicin,
66.66% amikacin, 66.66% ciprofloxacin and 63.33%
piperacillin. Imipenem proved to be the most effective
antibacterial agent for routine use among the
P. aeruginosa
strains investigated in this study. Susceptibility pattern of
other antibiotics tested was as piperacillin 63.33%, cef-
tazidime + tazobactum 60%, carbenicillin 53.33%, cef-
tazidime + clavulinic acid 53.33% and ceftazidime 50%.
Ceftazidime was found to be the least effective drug when
tested. Raakhee et al. have reported 79.19 susceptibility
to imipenem, 74.45% amikacin and 62.77% ciproflox-
acin, 71.53% piperacillin and ceftazidime 41.97%24.
Arora et al. reported 96.3% sensitivity to imipenem,
27% gentamicin, 58.5% amikacin, 26.8% ciprofloxacin,
piperacillin 56% and ceftazidime 37%27.
CONCLUSION
P. aeruginosa
is one of the most frequently isolated patho-
gen from the clinical specimens. Imipenem proved to
be the most effective drug against
P. aeruginosa
. Use of
ceftazidime should be restricted as it found to be least
effective against
P. aeruginosa
. In this study, antibiotic resis-
tance was significantly higher among biofilm producing
P. aeruginosa
than non-producer. The percentage of bio-
film production seen in individual hospital along with
their antibiotic sensitivity pattern could inform choice
of antibiotic. To avoid rapid emergence of drug-resistant
strains, periodic testing of biofilm formation and antibi-
otic sensitivity should be carried out to detect the resis-
tance trends. As this is a hospital-based epidemiological
Table 6 Antimicrobial susceptibility patterns of P. aeruginosa clinical isolates.
Sr. no. Antibiotic Sensitive no. (%) Resistant no. (%)
1. Amikacin (30 mcg) 20 (66.66) 10 (33.33)
2. Ceftzazidime + clavulinic acid (30 + 10 mcg) 16 (53.33) 14 (46.66)
3. Ceftazidime + tazobactum (30 + 10 mcg) 18 (60.00) 12 (40.00)
4. Ceftazidime (30 mcg) 15 (50.00) 15 (50.00)
5. Carbenicillin (100 mcg) 16 (53.33) 14 (46.66)
6. Ciprofloxacin (5 mcg) 20 (66.66) 10 (33.33)
7. Gentamicin (10 mcg) 21 (70.00) 09 (30.00)
8. Imipenem (10 mcg) 27 (90.00) 03 (10.00)
9. Piperacillin (100 mcg) 19 (63.33) 11 (36.66)

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Isolation of
P. aeruginosa
from clinical samples
data, this will help for implementation of better infec-
tion control strategies and for better patient manage-
ment. This study will help to improve the knowledge
of antibiotic resistance patterns among physicians. Every
effort should be made to prevent selection and spread of
resistant organism. Frequent hand washing to prevent
spread of organism should be encouraged. Better sur-
gical and medical care should be provided to patients
during hospital stay.
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