ORIGINAL ARTICLE

Bali Medical Journal (Bali MedJ) 2023, Volume 12, Number 1: 1014-1020
P-ISSN.2089-1180, E-ISSN: 2302-2914

The association between biofilm formation ability

and antibiotic resistance phenotype in
clinical isolates of gram-negative bacteria:
a cross-sectional study

Irbasmantini Syaiful1, Agung Dwi Wahyu Widodo2*, Pepy Dwi Endraswari2,

Lindawati Alimsardjono2, Budi Utomo3, Muhammad Vitanata Arfijanto4

1
Study Program of Clinical Microbiology,
Faculty of Medicine, Universitas
Airlangga, Surabaya, Indonesia;
2
Department of Clinical Microbiology,
Faculty of Medicine, Universitas
Airlangga, Dr. Soetomo General
Academic Hospital, Surabaya, Indonesia;
3
Department of Epidemiology,
Biostatistics, Population Studies and
Health Promotion, Faculty of Public
Health, Universitas Airlangga, Surabaya,
Indonesia;
4
Department of Internal Medicine,
Faculty of Medicine, Universitas
Airlangga, Dr. Soetomo General
Academic Hospital, Surabaya, Indonesia;
*Corresponding author:
Agung Dwi Wahyu Widodo;
Department of Clinical Microbiology,
Faculty of Medicine, Universitas
Airlangga, Dr. Soetomo General
Academic Hospital, Surabaya, Indonesia;
agungimunologi@gmail.com
Received: 2023-01-09
Accepted: 2023-03-02
Published: 2023-03-24
ABSTRACT
Backgrounds: Antimicrobial-resistant pathogens that form biofilms cause treatment failure, increased mortality, and
morbidity. Gram-negative bacteria (GNB) infections have become an emerging issue in antimicrobial resistance. This study
analyzes the association between GNB biofilm formation ability and the antibiotic resistance phenotype in GNB clinical
isolates.
Methods: From June to August 2022, this cross-sectional study was conducted at Dr. Soetomo General Academic Hospital.
Isolates derived from clinical specimens, including blood, urine, and respiratory tract specimens, were identified for antibiotic
resistance phenotypes along with a quantitative biofilm formation assay. The antibiotic resistance phenotype was studied in
relation to the GNB isolates’ ability for biofilm formation.
Results: Of the 271 isolates studied, 95 (35.1%) were non-MDR, 143 (52.8%) were MDR, and 33 (12.2%) were XDR. On the
biofilm formation assay, 34 (12.5%) were non-biofilm producers, 237 (87.5%) were biofilm producers, consisting of 101
(37.3%) weak, 131 (48.3%) moderate, and 5 (1.8%) strong biofilm producers. Biofilm formation and antibiotic resistance
in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii were not significantly
associated (p > 0.05). However, Escherichia coli isolates resistant to the antibiotics amikacin, imipenem, meropenem, and
amoxicillin-clavulanate were found to have a higher predisposition to form biofilms. Klebsiella pneumoniae isolates resistant
to gentamycin were also positively associated with biofilm formation.
Conclusions: Gram-negative bacteria’s ability to form biofilms is not associated with their antibiotic resistance phenotype;
therefore, routine biofilm monitoring is critical for enhancing the quality of treatment strategy for biofilm-associated
infection.
Keywords: biofilm formation, antimicrobial resistance, MDR, XDR, Gram-negative bacteria.
Cite This Article: Syaiful, I., Widodo, A.D.W., Endraswari, P.D., Alimsardjono, L., Utomo, B., Arfijanto, M.V. 2023. The association
between biofilm formation ability and antibiotic resistance phenotype in clinical isolates of gram-negative bacteria: a cross-
sectional study. Bali Medical Journal 12(1): 1014-1020. DOI: 10.15562/bmj.v12i1.4101

INTRODUCTION
The worldwide spread of antimicrobial-
resistant bacteria has reached an
emergency level. Over 2.8 million illnesses
and over 35,000 fatalities were attributed to
antimicrobial-resistant bacteria annually
between 2012 and 2017 in the US. AMR
has killed a large number of people all over
the world (about 700,000 deaths per year).1
Antimicrobial resistance has become
an urgent issue for global health and
the economy in recent years. Infections
caused by antimicrobial-resistant
pathogens or multidrug-resistant (MDR)
microorganisms are on the rise, impacting
healthcare costs, patient outcomes, and
the ability to control the spread of disease.2
Bacterial resistance to antibiotics is
caused by various mechanisms, including
uptake limitation, target change, enzyme
inactivation, and the efflux pump.3 The
previously stated mechanisms of antibiotic
resistance are not the only reasons for the
failure of MDR infection treatment. The
ability to create biofilms, communities of
microorganisms connected between cells
and surrounded by an exopolysaccharide
(EPS) matrix on a surface, is one resistance
mechanism employed by bacteria to live
in an environment containing antibiotic
chemicals. Biofilm settings can foster a
thousand times more antibiotic resistance
in sessile bacteria than in planktonic
microorganisms.4,5 The pathogenesis
of infectious diseases that involve the
mechanism of biofilm formation is called
biofilm-related infection or biofilm-
associated infection (BAI).6,7
Biofilm involvement is responsible for
approximately 65% of infectious diseases
and approximately 80% of chronic
infections due to medical equipment and
biological surfaces.8 Major pathogens
with biofilm-forming ability include
Gram-positive Staphylococcus aureus,
Staphylococcus epidermidis, Enterococcus
faecalis, Streptococcus viridans, and
Gram-negative Escherichia coli, Klebsiella

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| doi:
www.balimedicaljournal.org
10.15562/bmj.v12i1.4101

pneumoniae, Acinetobacter baumannii,
Proteus mirabilis, and Pseudomonas
aeruginosa.9 The most significant Gram-
negative bacteria (GNB) with biofilm-
forming ability include Escherichia coli,
Klebsiella pneumoniae, Pseudomonas
aeruginosa, and Acinetobacter
baumannii.10 This is consistent with the
WHO’s classification of MDR pathogen
priority, where the four species occupy
critical priority.11 Due to their unique
features, GNBs have a distinct ability
to form biofilms compared to Gram-
positive bacteria. GNB is a didermic cell
with an envelope composed of the inner
and outer membranes (IM and OM)
and the periplasmic space between the
two. The peculiarity of GNB’s envelopes
and extracellular components biofilm
creation.9,10 Changes in gene expression of
microbes occur in the biofilm community,
with genes involved in glycolysis, carbon
metabolism, and ribosomal proteins
being down-regulated. Also, compared to
planktonic cells, sessile cells have more
active efflux pumps, produce more of
their cell wall, and have greater rates of
peptidoglycan gene transcription.12,13
The urine strain Klebsiella pneumoniae
correlated biofilm production and
resistance profiles, suggesting that isolates
showing broad Anti-microbial resistance
appeared to have superior biofilm
formation capacity compared to sensitive
strains.14 Biofilm-forming Staphylococcus
aureus isolated from inpatient wounds had
higher resistance levels than those from
non-biofilm-forming strains.15 However,
it is plausible that the same plasmids
that carry genes for antibiotic resistance
also contain genes involved in biofilm
formation, explaining why multi-resistant
bacteria are also biofilm producers.14
Additionally, quorum-sensing, a bacterial
cellular communication system unique
to biofilms, allows for the spreading
of antibiotic resistance genes among
community members. Low antimicrobial
penetration due to the presence of matrix,
changes in microbial multiplication rates,
and physiological changes in bacteria may
all play a role in microbial resistance in
biofilms.16 Treatment failure is possible
because of the increased bacterial survival
afforded by the biofilms formed by MDR
and XDR bacterial isolates. To better
Bali Medical Journal 2023; 12(1): 1014-1020 | doi: 10.15562/bmj.v12i1.4101
ORIGINAL ARTICLE
protect against infections caused by drug-resistant (XDR), and pan-drug
MDR or XDR bacteria, it is important resistance (PDR) are the categories that
to understand the precise link between define the resistance phenotype according
antibiotic resistance and biofilm-forming to the International Expert Proposal for
abilities in bacteria. Many studies in Interim Standards Guidelines (PDR).19,20
recent years have looked for a link Multidrug-resistant (MDR) bacteria refer
between biofilm production and antibiotic to those that show resistance to at least one
resistance patterns, but due to inconsistent agent spanning three or more classes of
findings, no definitive conclusions could antimicrobials. Extensively drug-resistant
be drawn until now. (XDR) bacteria show resistance to three
Based on those mentioned above, this or more antibiotics (i.e., bacterial isolates
study aims to determine if gram-negative remain susceptible to only one or two
bacteria antibiotic resistance phenotypes categories).21
in clinical isolates are associated with the
capacity to form biofilms. Biofilm formation
The tissue culture plate (TCP) method with
METHODS crystal violet staining, as described earlier
by O’Toole et al., was used to quantify
This cross-sectional study was conducted
the capability of biofilm formation in
at Dr. Soetomo General Academic Hospital
isolates with certain modifications.22
from June to August 2022. This study
In the beginning, these strains were
used a consecutive sampling technique;
inoculated onto Mueller-Hinton agar
isolates from urine, blood, and respiratory
for one night at 370 C. After that, the
tract specimens with identifiable Gram-
concentration of the isolates was adjusted
negative bacteria such as Escherichia coli,
to 0.5 McFarland ((~1.5×108 CFU/ mL)
Klebsiella pneumoniae, Pseudomonas
by adding 0.85% sodium chloride to
aeruginosa, and Acinetobacter baumannii
normal saline. A 10-liter aliquot of each
were employed as inclusion criteria
solution was diluted 1:200 in 190 liters
during the study period. Isolates from
of tryptic soy broth (TSB) containing 1%
specimens other than blood, urine, or
glucose. These dilutions were performed
respiratory tract specimens and isolates
on polystyrene microtiter plates with 96
with identifiable bacteria other than the
wells. After overnight incubation at 370 C,
Gram-negative bacteria were excluded.
the plates were washed three times with
Bacteria were identified utilizing the BD
phosphate-buffered saline (PBS), fixed by
PhoenixTM automated identification and
adding 200 mL of methanol to each well
susceptibility testing system (Becton-
and stained with 200 mL of crystal violet
Dickinson Diagnostic Systems, Sparks,
solution to each well for 20 minutes (CV).
MD, USA) at the Clinical Microbiology
After the plates had been washed three
Unit at Dr. Soetomo Hospital.
times to eliminate any excess stain, the
remaining CV was dissolved in 200 liters
Antibiotic resistance phenotypes
(L) of ethanol at a 95% concentration for
In this study, micro-dilution and disk
ten minutes. A sterility control (culture
diffusion methods were used to assess
medium without inoculum) and growth
the antibiotic resistance phenotype of
control (control media with inoculum)
bacterial isolates. The micro-dilution
are included on all plates. To assess the
method utilized by the BD PhoenixTM
ability for biofilm formation, an ELISA
automated identification and susceptibility
plate reader (iMark; BioRad Instruments)
testing system determined the minimal
was used to quantify the optical density at
inhibitory concentration (MIC). In
595 nm (OD595) in each well. The ability
contrast, disk diffusion methods measured
to form biofilm is classified according
the inhibition zone diameter of antibiotics
to the optical density cutoff (ODc),
against the isolates tested. The resulting
calculated using the following formula:
MIC and inhibition diameter zone were
ODc = ODavg negative control + (3 x the
then classified as sensitive, intermediate,
negative control’s standard deviation OD).
or resistant based on the breakpoint,
The following criteria were taken into
according to CLSI (2022) guidelines.17,18
consideration: No biofilm producers are
Multiple drug resistance (MDR), extremely
1015
 ORIGINAL ARTICLE

indicated by an OD595 value below ODc; Table 1. Characteristics and distribution of Gram-negative bacteria isolates.
weak biofilm producers are indicated by Gram-negative bacteria isolates (%)
an OD595 value greater than ODc but Characteristic p-value
EC n (%) KP n (%) PA n (%) AB n (%)
not twice as high as ODc; an OD595 value Gender
indicates moderate biofilm producers Male 56 (65.10) 35 (40.20) 26 (49.10) 21 (46.70) 0.029*
between 2 to 4 times than ODc, and an Female 30 (34.90) 52 (59.80) 27 (50.90) 24 (53.30)
OD595 value indicates strong biofilm Age
producers beyond 4 times than ODc.23 0-5 years 12 (14.00) 25 (28.70) 13 (24.50) 5 (11.10) 0.089
Each test was performed three times to 6-12 years 2 (2.30) 2 (2.30) 6 (11.30) 3 (6.70)
ensure accuracy. 13-25 years 9 (10.50) 2 (2.30) 5 (9.40) 2 (4.40)
26-45 years 17 (19.80) 17 (19.50) 9 (17.00) 9 (20.00)
46-65 years 28 (32.60) 26 (29.90) 14 (26.40) 21 (46.70)
Statistical analysis
>65 years 18 (20.80) 15 (17.30) 6 (11.40) 5 (11.10)
The obtained data were entered into a
Clinical specimen
spreadsheet created in Excel 2010. Version
Blood 24 (27.90) 19 (21.80) 2 (3.80) 16 (35.55) <0.001**
23 of the Statistical Package for the Social Urine 45 (52.30) 18 (20.70) 12 (22.60) 4 (8.90)
Sciences (SPSS) was used to export and Respiratory tract 17 (19.80) 50 (57.50) 39 (73.60) 25 (55.55)
analyze the data (IBM Corp., Armonk, Categories
NY, USA). After obtaining descriptive Non-MDR 16 (18.60) 37 (42.50) 21 (39.60) 21 (46.7) 0.015*
statistics, the data was presented in tables MDR 65 (75.60) 48 (55.20) 25 (47.20) 5 (11.1)
and figures. For intergroup comparisons, XDR 5 (5.80) 2 (2.30) 7 (13.20) 19 (42.2)
specific comparisons of biofilm formation Note: EC = Escherichia coli; KP = Klebsiella pneumoniae; AB = Acinetobacter baumannii; PA =
among GNB isolates, the Kruskall-Wallis Pseudomonas aeruginosa; MDR= multidrug-resistance organisms; XDR= extensively drug-
test was utilized. The correlation between resistant organisms; *significant <0.05; **significant <0.001.
characteristics and distribution of Gram-
negative bacteria isolates was analyzed
using Chi-square. The correlation
between biofilm formation and phenotype
resistance categories was analyzed
using Spearman’s rank correlation test.
A Pearson correlation test was used to
compare biofilm formation between
isolates susceptible and non-susceptible
to each antimicrobial category. Statistical
significance was assumed at p < 0.05 in all
analyses.

RESULTS
During the study period, 271 clinical
isolates originated from the inpatients.
Of the total 271 isolates, GNB isolates
identified as Escherichia coli were 86
(31.70%), Klebsiella pneumoniae were 87
(32.10%), Pseudomonas aeruginosa was
53 (19.6%), and Acinetobacter baumannii
were 45 (16.60%). The isolates from blood
specimens were 61 (22.50%), those from
urine specimens were 79 (29.20%) isolates,
and respiratory tract specimens were 131
(48.30%) isolates. 138 (50.90%) isolates
came from female patients, and 133
(49.10%) were from male patients. Isolate
distribution by age category, 55 (20.30%)
isolates came from patients aged 0–5 years;
13 (4.80%) isolates came from patients
aged 6–12 years; 18 (6.60%) isolates
came from patients aged 13–25 years;
Figure 1. Optical density (OD595) from the biofilm formation assay on each species of
GNB isolates (p<0.001).
52 (19.20%) isolates came from patients
aged 26–45 years; 89 (32.80%) isolates
came from patients aged 46–65 years and
44 (16.20%) isolates came from patients
aged >65 years. Of all the Gram-negative
rod isolates, 95 (35.10%) isolates were
non-MDR, 143 (52.80%) isolates were
MDR, and 33 (12.20%) isolates were XDR.
Acinetobacter baumannii has the highest
proportion of XDR compared to the other
GNB isolates studied in this study. The
distribution of isolates based on patient
characteristics, including gender, age
category, specimen type, room category,
and MDR categories, are summarized in
Table 1.
Resistance Phenotype and Sensitivity
Pattern of Gram-negative bacteria
isolates
Escherichia coli totaled 86 isolates, with
65 (75.60%) MDR isolates, 5 (5.80%)
XDR isolates, and 16 (18.60%) non-MDR
isolates. The resistance phenotype profile
of Escherichia coli isolates is as follows:
ampicillin (AMP) 95.30%, piperacillin

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 ORIGINAL ARTICLE

(PIP) 90.60%, tetracyclin (TE) 74.10%,
cephazolin (KZ) 72.10%, aztreonam
(ATM) 69.80%, cefotaxime (CTX) 69.80%,
ceftazidime (CAZ) 69.80%, trimethoprim-
sulfamethoxazole (SXT) 61.60%, cefepime
(FEP) 59.30%, moxifloxacin (MXF)
58.10%, ciprofloxacin (CIP) 56.90%,
levofloxacin (LEV) 56.90%, gentamicin
(CN) 43.00%, ampicillin-sulbactam
(SAM) 40.70%, chloramphenicol (C)
35.30%, amoxicillin-clavulanic acid
(AMC) 17.40%, meropenem (MEM)
14.10%, imipenem (IMP) 10.50%,
piperacillin-tazobactam (TZP) 9.30%, and
amikacin (AK) 1.20%.
Klebsiella pneumoniae totaled 87
isolates, with 48 (55.2%) MDR isolates, 2
(2.3%) XDR isolates, and 37 (42.5%) non-
MDR isolates. The resistance phenotype
profile of Klebsiella pneumoniae isolates
are as follows: ampicillin (AMP) 100%,
piperacillin (PIP) 62.80%, cephazolin
(KZ) 52.90%, cefepime (FEP) 50.60%,
aztreonam (ATM) 49.40%, cefotaxime
(CTX) 49.40%, ceftazidime (CAZ)
49.40%, trimethoprim-sulfamethoxazole
(SXT) 46.00%, ampicillin-sulbactam
(SAM) 40.20%, levofloxacin (LEV)
36.80%, ciprofloxacin (CIP) 32.20%,
chloramphenicol (C) 30.90%, tetracyclin
(TE) 30.20%, gentamicin (CN) 25.30%,
piperacillin-tazobactam (TZP) 23.30%,
meropenem (MEM) 20.70%, amoxicillin-
clavulanic acid (AMC) 16.10%, imipenem
(IMP) 16.10%, moxifloxacin (MXF)
15.30%, and amikacin (AK) 0.00%.
Pseudomonas aeruginosa totaled 53
isolates, with 25 (47.2%) MDR isolates, 7
(13.20%) XDR isolates, and 21 (39.60%)
non-MDR isolates. The resistance
phenotype profile of Pseudomonas
aeruginosa isolates is as follows:
aztreonam (ATM) 66.00%, cefepime
(FEP) 52.80%, piperacillin (PIP) 50.90%,
ciprofloxacin (CIP) 50.00%, levofloxacin
(LEV) 49.10%, gentamicin (CN) 42.30%,
ceftazidime (CAZ) 39.60%, piperacillin-
tazobactam (TZP) 20.80%, amikacin (AK)
17.00%, meropenem (MEM) 17.00% dan
imipenem (IMP) 13.20%.
Acinetobacter baumannii totaled 45
isolates, with 19 (42.20%) XDR isolates, 5
(11.10%) MDR isolates, and 21 (46.70%)
non-MDR isolates. The resistance
phenotype profile of Acinetobacter
baumannii isolate is as follows: piperacillin
(PIP) 65.10%, cefepime (FEP) 56.80%,
cefotaxime (CTX) 56.80%, ciprofloxacin
(CIP) 52.30%, levofloxacin (LEV) 51.10%,
tetracyclin (TE) 50.00%, gentamicin
(CN) 48.90%, ceftazidime (CAZ) 46.70%,
imipenem (IMP) 46.70%, meropenem
(MEM) 46.70%, piperacillin-tazobactam
(TZP) 46.70%, ampicillin-sulbactam
(SAM) 45.50%, amikacin (AK) 42.20%
dan trimethoprim-sulfamethoxazole
(SXT) 35.60%.

Biofilm Formation Ability in Gram-

negative bacteria isolates
The ability of biofilm formation in the
four species of GNB isolates in this study
is summarized in the graph in Figure
Figure 2. Distribution of resistance phenotype categories among different biofilm 1. Escherichia coli totaled 86 isolates,
formation abilities (p=0.101).
Table 2. Association between biofilm formation ability and resistance phenotype categories of GNB isolates.
Biofilm formation ability (%)
Bacteria Categories Non-producer Weak Strong p
Moderate n (%)
n (%) n (%) n (%)
EC Non-MDR 6 (37.50) 10 (62.50) 0 (0.00) 0 (0.00) 0.131
MDR 23 (35.40) 32 (49.20) 10 (15.40) 0 (0.00)
XDR 0 (0.00) 4 (80.00) 1 (20.00) 0 (0.00)
KP Non-MDR 0 (0.00) 13 (35.10) 24 (64.90) 0 (0.00) 0.177
MDR 2 (4.20) 8 (16.70) 35 (72.90) 3 (6.30)
XDR 0 (0.00) 1 (50.00) 1 (50.00) 0 (0.00)
PA Non-MDR 0 (0.00) 4 (19.00) 17 (81.00) 0 (0.00) 0.075
MDR 1 (4.00) 6 (24.00) 18 (72.00) 0 (0.00)
XDR 1 (14.30) 3 (42.90) 3 (42.90) 0 (0.00)
AB Non-MDR 0 (0.00) 8 (38.10) 13 (61.90) 0 (0.00) 0.352
MDR 1 (20.00) 1 (20.00) 2 (40.00) 1 (20.00)
XDR 0 (0.00) 11 (57.90) 7 (36.80) 1 (5.30)
Note: EC = Escherichia coli; KP = Klebsiella pneumoniae; AB = Acinetobacter baumannii; PA = Pseudomonas aeruginosa; MDR= multidrug-resistance
organisms; XDR= extensively drug-resistant organisms; *significant <0.05.

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 ORIGINAL ARTICLE

Table 3. Association between biofilm formation ability and specific bacteria species: Escherichia coli (p =
antimicrobial resistance. 0.131), Klebsiella pneumoniae (p = 0.177),
p-value Pseudomonas aeruginosa (p = 0.075), or
Antibiotics Acinetobacter baumannii (p = 0.352). Table
EC KP PA AB
Amikacin <0.001** ND 0.990 0.767 2 shows the association between biofilm-
Gentamycin 0.304 0.006* 0.972 0.585 forming ability and phenotypic resistance
Imipenem 0.046* 0.727 0.248 0.697 categories among GNB isolates.
Meropenem 0.030* 0.205 0.328 0.533 Meanwhile, we also investigated the
Amoxicilin-clavulanate 0.041* 0.278 ND ND association between biofilm formation and
Ampicilin-sulbactam 0.210 0.671 ND 0.521 specific antibiotic resistance in Escherichia
Piperacilin-tazobactam 0.174 0.180 0.762 0.671 coli and Klebsiella pneumoniae isolates. The
Cephazolin 0.542 0.304 ND ND results demonstrated that Escherichia coli
Cefotaxime 0.509 0.219 ND 0.703 isolates resistant to amikacin, imipenem,
Ceftazidime 0.877 0.219 0.728 0.637
meropenem and amoxicillin-clavulanate
Cefepime 0.491 0.209 0.928 0.961
tend to form stronger biofilms than
susceptible or intermediate resistance,
Trimethoprim-sulfamethoxazole 0.867 0.525 ND 0.230
being indicative of a positive correlation
Ciprofloxacin ND ND 0.870 0.612
between biofilm quantity and specific
Levofloxacin ND ND 0.519 0.584
resistance phenotype (p<0.05; Table 3).
Ampicillin 0.401 ND ND ND
Analysis of the association between the
Piperacillin 0.185 0.851 0.163 0.869
ability to form biofilms and resistance to a
Aztreonam 0.877 0.219 0.792 ND
specific antibiotic in Klebsiella pneumoniae
Chloramphenicol 0.752 0.248 ND ND
isolates showed a positive correlation
Tetracycline 0.290 0.604 ND 0.578
for the antibiotics gentamycin (p<0.05;
Note: EC = Escherichia coli; KP = Klebsiella pneumoniae; PA = Pseudomonas aeruginosa; AB =
Table 3), with resistant isolates showing
Acinetobacter baumannii; R = Resistant; I = Intermediate; S = Sensitive; ND = not determined.
*significant <0.05; **significant <0.001.
a higher ability to form biofilms. Analysis
of the association between the biofilm
with 29 (33.70%) categorized as non- Association between biofilm formation ability and resistance to each
producers, 46 (53.50%) weak, 11 (12.80%) formation ability and resistance antibiotic in Pseudomonas aeruginosa and
moderate, and no isolates classified as phenotype in Gram-negative bacteria Acinetobacter baumannii isolates showed
strong biofilm producers. There were 87 isolates no significant relationship (p>0.05).
Klebsiella pneumoniae isolates, with 2 The biofilm formation ability in each
(2.30%) categorized as non-producers, resistance phenotype category is DISCUSSION
22 (25.30%) weak, 60 (69.00%) moderate, summarized in Figure 2. Of the 95 non- Antibiotic resistance among Gram-
and 3 (3.40%) strong biofilm producers. MDR isolates, 6 (6.30%) were non- negative bacteria (GNB) is a major
Pseudomonas aeruginosa totaled 53 producers, 35 (36.60%) were weak, 54 global health concern. The current study
isolates, with 2 (3.80%) categorized as non- (56.80%) were moderate, and no isolates looked at four of the most common and
producers, 13 (24.50%) weak, 38 (71.70%) were categorized as strong biofilm significant GNB strains found in hospitals,
moderate, and no isolates classified as producers. Of the 143 MDR isolates, including Escherichia coli, Klebsiella
strong biofilm producers. Acinetobacter 27 (18.90%) were non-producers, 47 pneumoniae, Pseudomonas aeruginosa,
baumannii totaled 45 isolates, with 1 (32.90%) were weak, 65 (45.50%) were and Acinetobacter baumannii. In this
(2.20%) categorized as non-producers, moderate, and 4 (2.80%) were strong study, 87.5% of isolates were detected as
20 (44.40%) weak, 22 (48.90%) moderate, biofilm producers. Of the 33 XDR isolates, biofilm-forming. This study had a higher
and 2 (4.40%) strong biofilm producers. 1 (3.00%) isolate was a non-producer, 19 proportion of biofilm-forming isolates
Statistical analysis using the Kruskal- (57.60%) were weak, 12 (36.40%) were than earlier studies by Di Domenico EG et
Wallis test gave a p < 0.001. Thus the ability moderate, and 1 (3.00%) was a strong al. (64.28%), Dumaru R et al. (62.73%), and
to form biofilms in the four species of biofilm producer. Statistical analysis using Qian W et al. (71.8%).24-26 The percentages
GNB isolates studied in this study differed the Spearman correlation test gave p = of biofilm-forming GNB isolates studied
significantly in each group. Escherichia 0.101, indicating no significant association were Escherichia coli (66.3%), Klebsiella
coli had the weakest biofilm-forming between biofilm formation ability and the pneumoniae (97.7%), Pseudomonas
ability, while Pseudomonas aeruginosa resistance phenotype of GNB isolates. aeruginosa (96.2%), and Acinetobacter
had the most vital biofilm-forming ability Resistance phenotypic categories were baumannii (97.8%). This study
compared to the other GNB isolates not significantly associated with biofilm- demonstrated that Escherichia coli has
studied in this study. forming ability in any of the four tested the weakest ability to form biofilms, and

1018 Bali Medical Journal 2023; 12(1): 1014-1020 | doi: 10.15562/bmj.v12i1.4101


Pseudomonas aeruginosa has the strongest
ability to form biofilms compared to the
other GNB isolates studied.
We analyze the obtained isolates to see
if there is an association between biofilm
formation and the antibiotic resistance
phenotype. Although no association was
discovered, we identified a comparable
level of biofilm-forming ability in non-
MDR, MDR, and XDR isolates, with no
significant differences between these
groups, similar to prior studies.24 This
confirms the findings of an earlier study
that found no correlation between
antibiotic resistance and biofilm formation
in Escherichia coli, Klebsiella pneumoniae,
and Pseudomonas aeruginosa isolates; the
latter found that MDR isolates had no
greater ability to become stronger biofilm
producers than non-MDR isolates.25
Meanwhile, various studies have presented
contrasting conclusions based on this
research, in which Qian W et al. discover
an association between biofilm formation
and antibiotic resistance, with more robust
biofilm-formation strain populations likely
harboring greater proportions of XDR
for Escherichia coli isolates.26 Previous
research indicated that most biofilm-
forming Pseudomonas aeruginosa strains
that also possessed biofilm-associated
genes were not multidrug-resistant.27
Dumaru R et al. also concluded that most
of the biofilm-producing GNB were multi-
drug resistant.28
Even though this study did not uncover
a connection between biofilm-formation
ability and categories of antibiotic
resistance phenotypes, an association
between biofilm-forming ability and
resistance to specific antibiotics was
discovered. Escherichia coli isolates with
resistance phenotype to the antibiotics
amikacin, imipenem, meropenem, and
amoxicillin-clavulanate were found
to have a greater potential to produce
biofilms. In Klebsiella pneumoniae isolates,
gentamycin resistance was positively
correlated with biofilm formation. Several
other studies have reached the same
conclusion as this: that the ability to create
biofilms is linked to resistance to certain
antibiotics. Isolates of Escherichia coli with
amoxicillin and amoxicillin-clavulanate
resistance phenotype are also stronger
biofilm formers.29 A study concluded
Bali Medical Journal 2023; 12(1): 1014-1020 | doi: 10.15562/bmj.v12i1.4101
an association between resistance to
meropenem and the ability to form
biofilms in Acinetobacter baumannii.30
Also, gentamicin resistance is linked
to biofilm formation in Escherichia coli
isolates, according to research by Cepas V
et al.25
It is worth to note that all of the
isolates in our investigation were tested for
antimicrobial susceptibility as planktonic
cells, as opposed to biofilms, where the
cells adhere to one another. Thus, the MDR
phenotype was not caused by multiple
biofilm mechanisms and architectural
features, like the glycocalyx matrix, outer
membrane structure, metabolic and
growth rate heterogeneity, factors such
as stress reactions, quorum sensing, and
the development of persister cells from
existing cells.16,31 Biofilm-based antibiotic
resistance mechanisms in microorganisms
are distinct from conventional antibiotic
resistance. It is hypothesized that biofilms’
protective layer of biomaterials causes
poor antibiotic penetration, adaptive
responses to stress, and the formation
of persistent cells, which together form
a multi-layered defense system, making
eradication particularly challenging when
combined with the bacteria’s resistance.32,33
In this study, the absence of an association
between the ability to form biofilms and
the phenotype of antibiotic resistance
phenotype categories reflects the two
distinct mechanisms of biofilm formation
and antibiotic resistance phenotype, which
can be investigated further by examining
the characteristics of the genes involved in
the two mechanisms.
The limitation of this study is that
no genotypic examination was carried
out to detect genes related to antibiotic
resistance and biofilm formation in each
isolate. Detection of genes implicated
in biofilm-forming ability can be an
alternative diagnostic modality in treating
biofilm-associated infection. Further
analysis through gene mapping related
to antibiotic resistance and biofilm
formation is needed to determine the exact
association between these two variables.
The high proportion of GNB isolates with
MDR and XDR strains and their ability to
form biofilms highlight the importance of
biofilm involvement in the pathogenesis
of infectious diseases. Both qualitatively
and quantitatively, routine monitoring of
ORIGINAL ARTICLE
biofilms can be recommended clinically
in conjunction with other strategies to
improve the quality of treatment for
biofilm-associated infections, including
stringent implementation of infection
control and prevention activities.
CONCLUSION
The GNB isolates tested in the current
study included Escherichia coli, Klebsiella
pneumoniae, Pseudomonas aeruginosa,
and Acinetobacter baumannii. Biofilm
formation was found to be unrelated to the
resistance phenotype. However, a positive
correlation between biofilm formation
and resistance to specific antibiotics was
observed in isolates of Escherichia coli
and Klebsiella pneumoniae. To establish
the precise association between these
two factors, further genomic research
is required, in addition to routine
surveillance of biofilm formation ability
and antimicrobial resistance profiles of
GNB isolates.
CONFLICTS OF INTEREST
All authors – none to declare.
ETHICAL CLEARANCE
The Clinical Research Ethics Committee
has given their clearance to this study at
Dr. Soetomo General Hospital No. 0959/
LOE/301.4.2/VII/2022 prior to the study
being conducted.
FUNDING
None to declare.
AUTHORS’ CONTRIBUTIONS
All authors contributed equally to this
manuscript. The final manuscript was read
and approved by all authors.
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