Comparision of biofilm production and multiple drug resistance in ...

Research article
Int J Pharm Biomed Sci 2011, 2(4), 103-107
ISSN No: 0976-5263
Research Drops
PharmaInterScience Publishers
Comparision of biofilm production and multiple drug resistance in
clinical isolates of Acinetobacter baumanii from a tertiary care
hospital in South India
M. Dheepa*,
Vinitha L Rashme,
B. Appalaraju
Department of Microbiology, PSG Institute
of Medical Sciences & Research Centre,
Peelamedu, Coimbatore- 641004, Tamil
Nadu, India
*Correspondence:
Dr. M. Dheepa
Tel: +91 9843009211
E-mail: dheepamicro@gmail.com
The present work is aimed at to determine biofilm formation in clinical isolates
of Acinetobacter baumannii and to determine the antibiotic susceptibility pattern of
seventeen different antibiotics and to correlate between biofilm production and
multidrug resistance. A total of 50 isolates were screened for biofilm production by
both qualitative and quantitative method in Acinetobacter baumannii isolates.
Biofilm production is demonstrated with standard tube test method, in which
bacterial film lining a culture tube is stained with a cationic dye and visually scaled.
In the second microtitre plate method, the optical density of the stained bacterial
film is determined spectrophotometrically. In biofilm production, both qualitative
(tube method) and quantitative (microtitre plate) method showed 30 isolates (60%)
as biofilm producers. Resistance to antibiotics such as ceftazidime, cefepime and
pipercillin was comparatively higher among biofilm producers than non-biofilm
producers .Our investigations showed a simultaneous emergence of resistance to
many antimicrobial agents available and represent a severe threat in the treatment
of hospitalized patients. This study demonstrates a high propensity among the
clinical isolates of A. baumannii to form biofilm and a significant association of
biofilm with multiple drug resistance.
Key words: Acinetobacter baumanii, biofilm detection, multidrug resistance
Received: 05 Dec 2011 / Revised: 08 Dec 2011 / Accepted: 11 Dec 2011 / Online publication: 28 Dec 2011
1. INTRODUCTION
Acinetobacter baumannii has emerged as an important
and problematic human pathogen as it is the causative agent
of several types of infections including pneumonia,
meningitis, septicemia, and urinary tract infections. It is
ranked second after Pseudomonas aeruginosa among the
nosocomial, aerobic, non-fermentative, gram negative bacilli
pathogens. Furthermore, this organism frequently causes
infections associated with medical devices, e.g., vascular
catheters, cerebrospinal fluid shunts, foley catheters etc.
Multidrug-resistant Acinetobacter baumannii has been
reported worldwide and is now recognized as one of the most
difficult healthcare-associated infections to control and to
treat [1].
Multi drug resistant is witnessed only in Acinetobacter
baumannii (A. baumannii) and not in any other species of
Acinetobacter. Multidrug-resistant (MDR) Acinetobacter
baumannii is not a new or emerging phenomenon, but
Acinetobacter baumannii has always been an organism
inherently resistant to multiple antibiotics [2]. The potential
ability of Acinetobacter baumannii to form biofilms might
also explain its outstanding antibiotic resistance, survival
properties and increased virulence.
Study of microbial bioflms has received significance
attention over the past decades. Therefore, studies and
diagnostic methods identifying virulent bacterial strains, i.e.,
strains with a capacity for slime production and consequent
biofilm formation are necessary to develop effective
strategies for biofilm control and improvement of patient care
[3]. We chose to investigate biofilm formation by clinical
isolates of A. baumannii and multiple drug resistance among
them and tried to correlate them in order to understand how
this pathogen persists in the hospital environment and causes
outbreaks.
2. MATERIALS AND METHODS
A total of 50 Acinetobacter baumannii isolates from
samples of sputum, urine, wound swab, tracheal tip, tracheal
aspirate, central line tip, ear swabs, blood, pus, bronchial
lavage and endotracheal tube which were received for a
period of six months in the microbiology laboratory of PSG
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M. Dheepa et al., Int J Pharm Biomed Sci 2011, 2(4), 103-107
104
hospitals, Coimbatore, South India, were included in the
study.
2.1 Isolation and identification
Blood agar and MacConkey agar were used for isolation
of Acinetobacter baumannii from the samples. For
identification, a battery of biochemical tests were done which
included catalase test, oxidase test, carbohydrate fermentation
tests (glucose, lactose, sucrose, and mannitol), citrate &
urease tests, OFdextrose test and growth at 44°C.
2.2 Antimicrobial suceptibility testing
1
2 3
Antimicrobial susceptibility testing was performed using
14 different therapeutically relevant antibiotics by Kirby
Bauer disk diffusion method according to norms of Clinical
Laboratory Standards institute (CLSI) and the disks were
supplied by Bio-Rad Laboratories, Mumbai.
Fig.1. Biofilm formation on glass surfaces under static growth conditions.
Tube 1- non adherent, tube 2- weakly adherent, tube 3- strongly adherent.
2.3 Detection of biofilm
2.3.1 Tube method
The quantitative assay for biofilm formation was
performed according to the method described by Christensen
et al 4. Glass tubes filled with 3 mL of trypticase soy broth (Hi
media, Mumbai) were inoculated with a loopful of a pure
culture of a strain of Acinetobacter baumannii grown
overnight from blood agar plate. After 48 h incubation at
37°C, the content of each tube was decanted. The tubes were
then stained with 1% safranin for 7 min. Then the tubes are
washed with distilled water for 5 min. A positive result was
indicated by the presence of an adherent film of stained
material on the inner surface of the tube. Presence of stained
material at the liquid-air interface alone was not regarded as
indicative of slime production. Tubes containing trypticase
soy broth only were included in the test as negative controls.
2.3.2 Microtitre-plate method
Three wells of a sterile 96-well flat bottomed plastic
tissue culture plate (Tarson, Kolkata) were filled with 200 µL
of bacterial suspension in trypticase soy broth inoculated at
37°C for 24 h. Negative control wells contained broth only.
The plates were covered and incubated aerobically for 24
hours at 37°C.Then, the content of each well was washed
three times with 250 µl of sterile physiological saline. The
plates were vigorously shaken in order to remove all nonadherent
bacteria. The remaining attached bacteria were fixed
with 200 µL of 99% methanol per well, and after 15 min
plates were emptied and left to dry. Then, plates were stained
for 5 min with 0.2 mL of 2% crystal violet per well. Excess
stain was rinsed off by placing the plate under running tap
water. After the plates were air dried, the dye bound to the
adherent cells was resolublized with 160 µL of 33% (v/v)
glacial acetic acid per well. The OD of each well was
measured at 578 nm using ELISA reader (Tulip diagnostics,
India).The reading was performed two times: (i) before
addition of glacial acetic acid, as in standard microtitre-plate
test and (ii) after glacial acetic acid was added as in modified
microtitre-plate test.
For the purpose of comparative analysis of test results, the
adherence capabilities of the test strains were classified into
the following under four categories: non-adherent (0), weakly
(+), moderately (++), or strongly (+++) adherent, based upon
the ODs of bacterial films. The cut-off optical density (ODc)
for the microtitre-plate is defined as three standard deviations
above the mean OD of the negative control.
Strains were classified as follows:
OD ≤ ODc
non-adherent
ODc < OD ≤ 2 x ODc weakly adherent
2 x ODc < OD ≤ 4 x ODc moderately adherent
4 x ODc < OD strongly adherent
All the tests were carried three times and the results were
averaged [5].
3. RESULTS
Twelve Acinetobacter baumannii strains (24 %) were
isolated from tracheal aspirates. The source of each isolate is
shown in Table 1. Qualitative tube method of biofilm
screening showed 30 isolates positive for biofilm production.
In the quantitative assay for the biofilm production, they were
classified as strongly adherent, moderately adherent, weakly
adherent and non adherent. Some of the isolates showing
biofilm formation are shown in the Fig.1.
For the purpose of data calculation, we used classification
of bacterial adherence (Table 2 and Table 3) based on three
standard deviations above the mean OD of the negative
control [5].
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M. Dheepa et al., Int J Pharm Biomed Sci 2011, 2(4), 103-107
Table 1
Source of acinetobacter baumannii isolates
Culture No (%)
Tracheal aspirate 12 (24)
Sputum 8 (16)
Endotracheal tube 6 (12)
Wound swab 6 (12)
Tracheal tip 5 (10)
Ear swab 3 (6)
Blood 3 (6)
Pus 3 (6)
Urine 1 (2)
Central line tip 1 (2)
Foley’ s catheter tip 1 (2)
Bronchial lavage 1 (2)
Total 50 (100)
105
The overall percentage of resistance observed among
biofilm positive and biofilm negative isolates and resistance
of all the isolates for 17 antibiotics tested, is given in Table 6.
This table shows that the resistance of antibiotics is more for
most of the biofilm positive isolates compared to the biofilm
negative isolates. Isolates showed maximum resistance to
aztreonam 82%, amikacin 78% and ofloxocin 76%.
The multi-drug resistance patterns of the biofilm
producing Acinetobacter baumannii isolates is shown in
Table 7. The maximum multiple resistances among the
isolates were noticed for anti microbial agents such as
aztreonam, ciprofloxacin and imipenam.
Table 2
Classification of bacterial adherence by microtitre plate method
Mean OD value Adherence Biofilm formation
0.36 Strong High
Table 3
OD values of modified microtitre-plate method
Mean OD value Adherence Biofilm formation
2.28 Strong High
Table 4
Screening of 50 Acinetobacter baumannii isolates for detection of biofilm
and the overall results of the tube and microtitre-plate test
Test
Number of strains showing
No Weak Moderate Strong
adherence adherence adherence adherence
(0) (+) (++) (+++)
Tube method 3 17 17 13
Standard microtitre-plate 1 19 28 2
method
Modified microtitre-plate
method
0 2 29 19
Quantitative microtitre assay for biofilm formation was
strongly positive in 2 isolates and moderately positive in 28
isolates while the remaining nineteen isolates were weakly
adherent and one non adherent. Nineteen weakly adherent
isolatesalong with onenon adherent strain were considered as
negative or non-biofilm producers. Both the methods for
biofilm detection thus showed similar results.
In modified microtitre plate method 19 isolates showed
strong adherence while 29 isolates showed moderate
adherence while others were weakly or non adherent. The
overall results for bofilm detection are given in the Table 4.
4. DISCUSSION
Acinetobacter baumannii infections present a global
medical challenge. They are opportunistic pathogens and are
particularly successful at colonizing and persisting in the
hospital environment. Multidrug-resistant Acinetobacter
baumannii has been reported worldwide and is now
recognized as one of the most difficult healthcare-associated
infections to control and treat.
It is also among the most common causes of devicerelated
nosocomial infection that results when the organism is
able to resist physical and chemical disinfection, often by
forming a biofilm. Thus infections due to bacteria that form
biofilms are a tenacious clinical problem. Only, few reports
have described the ability of clinical isolates of A. baumannii
to attach and form biofilms on glass surfaces. In our study,
the high isolation rates of Acinetobacter baumanii of about
24 % were from tracheal isolates and 16 % from sputum
samples. Another study done in Taiwan showed a high
isolation rate of Acinetobacter baumanii recovered from
sputum 71% and 10% from wound pus [6]. In the present
study done for biofilm production, both qualitative (tube
method) and quantitative (microtitre plate) method showed
30 isolates (60%) as biofilm producers. One of the recent
studies had 62% biofilm positive isolates among the 51
isolates studied, which was comparable with our results [7].
Imipenem resistance was 54 % in our study. Another
study done in Pondicherry India showed 100 % resistance to
imipenem [7]. A similar study done in Korea revealed 17%
of imipenem-resistant isolates [8]. Another study showed
53% of A. baumannii which were resistant to carbapenems
from a report of a citywide clonal outbreak in New York City
[9].
In the 50 isolates used in our study, highest sensitivity
was seen in netilmycin 72%, then in levoflox 62%,
doxycyline 52%, tobramycin 50%, piperacillin + tazobactum
50%, imipenam 46%, ampicillin+sulbactum 44%,
ciprofloxacin 38%, co-trimoxazole 32%, meropenem 32%,
ceftazidime 28%, pipercillin 28%, gentamicin 28%,cefepime
28%, ofloxocin 24%, amikacin 22% and aztreonam 18%
respectively.A study done in Bulgaria in 2001 showed
highest resistance to ciprofloxacin 97.1%, aztreonam 97%,
amikacin 97%, cefepime 96.7%, tetracycline 95%,
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M. Dheepa et al., Int J Pharm Biomed Sci 2011, 2(4), 103-107
Table 5
Antibiotic susceptibility pattern of the 50 A.baumannii isolates tested are
given in this table
Antibiotic
Number (% sensitive)
n=50
Amikacin 30 µg 22
Ceftazidime 30 µg 28
Ciprofloxacin 5 µg 38
Cefepime 30 µg 28
Imipenem 10 µg 46
Pipercillin 100 µg 28
Ampicilllin + sulbactum 10 µg +10µg 44
Gentamicin 10 µg 28
Co-trimoxazole 1.25µg +23.75 µg 32
Doxycyline 30 µg 52
Piperacillin + tazobactum 100 µg + 10 µg 50
Meropenem 10 µg 32
Tobramycin 10 µg 50
Levofloxacin 10 µg 62
Aztreonam 30 µg 18
Netilmycin 30 µg 72
Ofloxacin 5 µg 24
Table 6
Antibiotic susceptibility results (percentage) of A. baumannii isolates
Antibiotics
Resistance of
Biofilm positive
isolates N=20
Biofilm negative
isolates N=30
Amikacin 80 73.3 78
Ceftazidime 95 86.6 72
Ciproflox 85 80 62
Cefepime 95 86.6 72
Imipenem 65 70 54
Pipercillin 95 93.3 72
Ampicilllin + sulbactum 55 66 56
Gentamicin 70 70 72
Co-trimoxazole 75 76 68
Doxycyline 35 56 48
Piperacillin + tazobactum 40 50 50
Meropenem 70 66 68
Tobramycin 75 33 50
Levoflox 50 30 38
Aztreonam 85 76.6 82
Netilmycin 20 20 28
Ofloxocin 60 76 76
Table 7
Multiple drug resistant patterns of A. baumannii
Multiple drug combinations
Number of isolates
showing resistance
Resistance
of all
isolates
N =50
Percent
Ak, Ao, Cf, I, Of 31 62
Ak, Ao, I 31 62
Cf, I 33 66
Ak, I, Of 30 60
Ao, Cf, I 33 66
Ak- amikacin, Ao- aztreonam, Cf- ciprofloxacin, I- imepenem, Of –
ofloxacin
chloramphenicol 94.7%, amoxicillin/clavulinic acid 94.7%,
netilmycin 93.9%, ceftazidime 90.9%, gentamicin 87.9%,
pipercillin/tazobactam 84.8%, nalidixic acid 84.2%,
tobramycin 75%, imipenem 7.2%, colistin 7.1% [10].
106
Another study done in Estonia showed 60 % of A.
baumanii isolates sensitive to ampicillin + sulbactum, 95%
to imepenem and meropenum and 70 % to amikacin [11]. In a
study conducted in UK showed resistance to piperacillin +
tazobactum 39%, imipenam 7%, ciprofloxacin 46%,
meropenem 0.5%, ceftazidime 46%, pipercillin 72%,
gentamicin 43%, amikacin 21% respectively [12]. Similar
study conducted in Ankara in 2002 shows the resistance
rates were 31.2% for netilmicin, 44.6% for sulbactamcefoperazone,
53.6% for imipenem, 59.8% for amikacin,
59.9% for tobramycin, 74% for ciprofloxacin, 78% for
gentamicin, 79.5% for sulbactam-ampicillin, 82.3% for
cotrimoxazole, 84.8% for ticarcillin, 87.3% for piperacillintazobactam,
88.1% for ceftazidime and 92.1% for piperacillin
[13].
Another study was conducted in USA regarding the multi
drug resistance. The results showed, about 79.5% (66/83)
were multi-drug resistant (MDR) A. baumanii. Among these,
62 were resistant to ceftazidime and 66 were resistant to
imipenem. The imipenem resistant isolates (66/83) were also
resistant to kanamycin, amikacin, gentamicin, streptomycin,
tetracycline, ciprofloxacin and nalidixic acid [14].
In a study conducted in India, Hyderabad Acinetobacter
baumannii showed 77% resistance to piperacillin-tazobactam
[15]. In another study conducted in India, Pune 75% of the
isolates were multidrug resistant (MDR) and resistance to
aztreonam 60% and for imipenem 29% was noted [16].
In our study biofilm producers showed increased
resistance to Ceftazidime 95%, Cefepime 95% and Pipercillin
95%. A recent study showed significantly higher resistance to
cefotaxime, amikacin, ciprofloxacin and aztreonam among
biofilm producers. Non-biofilm producers showed increased
resistance only for netillin and ofloxacin [7].
5. CONCLUSIONS
In conclusion, continuous surveillance of the in-vitro
antimicrobial susceptibility of Acinetobacter baumannii
isolates in various geographical areas are necessary in order
to generate data useful for optimising empirical antimicrobial
treatment of patients with infections that are likely to be
caused by this pathogen. Our investigation reveals a
simultaneous emergence of resistance to many antimicrobial
agents when the organisms produce biofilm and represents a
severe threat in the treatment of hospitalized patients. It is a
challenge to treat and also to eradicate this multidrug
resistant organism because the overall healthcare costs which
are attributed to the treatment of biofilm associated infections
will be much higher. The high rate of invitro antibiotic
resistance of the Acinetobacter baumanii strains indicate the
importance of controlled antibiotic usage and appliance of
hospital infection control measures.
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M. Dheepa et al., Int J Pharm Biomed Sci 2011, 2(4), 103-107
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