
































Microsoft Word - RR_1_4669-Article Text-21197-1-18-20220510.docx


 Epidemiology and Society Health Review| ESHR 
Vol. 4, No. 2, 2022, pp. 40-47 ISSN 2656-6052 (online) | 2656-1107 (print) 
 
 

         10.26555/eshr.v4i2.4669   

  
 

 

40 

Research Article 
 
Biofilm Formation by Uropathogens and Its Impact on 
Antimicrobial Susceptibility Pattern 
 
Priyanka Sharma1, Sandeep Dogra1*, Bella Mahajan2, Shashi Sudhan Sharma3 

 
1 Department of Microbiology, Govt. Medical College, Jammu, India 
2 Department of Microbiology, ASCOMS & Hospital, Jammu, India  
3 Department of Microbiology, Govt. Medical College, Jammu, India 
 
* Correspondence: sandeepdogra@gmail.com. Phone: (+91) 9906091412 
 
Received 14 August 2021; Accepted 06 October 2021; Published 2 August 2022 
 

ABSTRACT 

Background: Out of all Hospital-Associated Infections (HAIs),Urinary Tract 
Infection (UTI) is the second most common infection that accounts for approximately 
34%, and 80% are associated with indwelling catheters and hence with biofilm 
formation, which invites multi-drug resistant microorganisms. The present study was 
designed to study in-vitro biofilm forming uropathogens and their antimicrobial 
susceptibility in a tertiary care hospital in north India. 
Method:The present cross-sectional study consisted of 200 urine specimens 
collected over one year from patients with symptoms of urinary tract infection. 
Following their isolation and identification, all the isolates were subjected to 
screening for biofilm formation by Congo Red Agar (CRA) and the Tube Adherence 
(TA) methods. Subsequently, the Kirby Bauer-disk diffusion method performed the 
antimicrobial susceptibility test. 
Results: Out of the total samples (n = 200), a total of 46 (23%) were positive by the 
CRA method, while 33 (16.5%) were positive by the TA method. Twenty-one (21%) 
isolates came positive by both methods. Biofilm formation was seen more commonly 
in females (82%). Biofilm-forming uropathogens develop a significantly higher 
resistance to antimicrobial drugs than non-producers. 
Conclusion: The correlation was significant between biofilm production and 
multidrug resistance. Also, it was concluded that the CRA method could be 
employed to detect biofilm formation in resource-limited countries. 

Keywords: Urinary tract infections; Tube adherence method; Congo red agar method; 
Antibacterial agents 

INTRODUCTION 

Talking of morbidity worldwide,Urinary Tract Infections (UTIs) is one of the leading causes of 
which is caused by different microorganisms. Worldwide, UTI has a prevalence of 11%(1), 



Sharma (Biofilm Formation by Uropathogens and Its Impact on Antimicrobial Susceptibility Pattern) 
 

 

Vol. 4, No. 2, 2022, pp. 40-47  10.26555/eshr.v4i2.4669 
 

41 

and according to an Indian study, it is 36.68% (2). These uropathogens tend to colonize the 
mucous membrane of the bladder and form micro bacterial communities called biofilms. The 
colonization by these microcolonies makes them impermeable to many antibiotics. It resultsin 
the evolution of multidrug-resistant strains, which is the leading causeof relapses in 
untreatable UTI. Biofilms consist of different layers of cells embedded in a matrix of 
extracellular exopolysaccharide – EEM (slime), which helps adhereto biomedical surfaces and 
protects them from the host immune system and antimicrobial therapy (3), and provides a 
survival strategy to the uropathogenic. The slimeconsists of extracellular DNA, proteins, 
polysaccharides, adhesin, and autolysin. It starts with the attachment of free-floating 
microorganisms to a surface. Initially,these areattached through weak van der Waal forces. 
Later, if left undisturbed, they anchor themselves more firmly via cell adhesion structures such 
as pili. Repulsion to water plays an essential role in determining the ability to form 
biofilms(4).Using his simple microscopes, Van Leeuwenhoekobserved microorganisms on 
tooth surfaces and can be regarded with the discovery of biofilms. Costertonet al., in 1978, 
explained the mechanisms ofmicroorganisms' adherence to living and nonliving materials and 
the help provided by ecologic niche (5). 

Biofilms aremainly formed in the prostate stones, urothelium, and implanted foreign bodies 
(6). Predisposing host factors are age, diabetes, long-term hospitalization, and catheterization 
(7). National Institute Health (NIH) says that among all the microbial infections, 80% are 
caused by biofilms (8). According to the Center for Disease Control and Prevention (CDC), 
USA, biofilms on indwelling medical devices consistof gram-positive or gram-negative bacteria 
or yeasts. The most common Gram-PositiveBacteria isolated are Enterococcus faecalis, 
Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus viridans, and Gram-
negative bacteriaEscherichia coli, Klebsiella pneumonia, Proteus mirabilis, and Pseudomonas 
aeruginosa. These bacteriacan originate from the skin of patients or healthcare workers or be 
some other source like the environment (9).Biofilms are composed of single or multiple 
species depending on the device and its duration of use in the patient.Biofilm on the urinary 
catheter is initially composed of single species, but with time, multispecies predominates(10). 
Biofilm-causing uropathogens have an inherent resistance to antibiotics, disinfectants, and 
antiseptics. Unlike planktonic populations, bacterial cells embedded in biofilmsshow intrinsic 
resistance to antibiotics which can be due to the inactivation of antimicrobial agents by 
exopolysaccharide (EPS), overexpression of stress-responsive genes, presence of oxygen 
gradients within the biofilm matrix, and differentiation of a subpopulation of biofilm cells into 
resistant dormant cells(11)(12).  

In this study, our goal was to detect the biofilm-forming uropathogens and study their 
antimicrobial susceptibility pattern among patients suffering from UTI in a tertiary care hospital 
in northern India. This study will help the clinicians to decide 

METHOD  

This cross-sectional study was performed for one year on 200 urine specimens from out-
patients (n=15) and in-patients (n=185) who were clinically diagnosed with UTI and fell under 
the inclusion criteria. Semi-quantitative urine culture was performed on UTI agar (HiMedia 
Labs) as per standardized SOPs of the department. 

As described by Freeman DJ et al., 1989 (13), CRA was performed. CRA medium was 
prepared by mixing brain heart infusion broth (Oxoid, UK) 37 g/L, sucrose 50 g/L, agar No. 1 
(Oxoid, UK) 10 g/L, and Congo red indicator (Oxoid, UK) 8 g/L. The Congo red stain (HiMedia 
Labs) was prepared separately as a concentrated aqueous solution and autoclaved (121°C 
for 15 min) from the rest of the other constituents. It was later added to the autoclaved brain 
heart infusion agar (HiMedia Labs) with sucrose at 55°C. CRA plates were then inoculated 
with test isolates and left for aerobic incubation at 37°C for 24 hours. For all positive isolates, 
CRA and TA methods detected biofilm formation. Black colonies with a dry crystalline 



Sharma (Biofilm Formation by Uropathogens and Its Impact on Antimicrobial Susceptibility Pattern) 
 

 

Vol. 4, No. 2, 2022, pp. 40-47  10.26555/eshr.v4i2.4669 
 

42 

consistency indicated biofilm formation, whereas no-biofilm formation was identified as red or 
pink crystalline colonies (Figure 1).  

 

Figure1. Congo Red Agar method shows biofilm producers (black crystalline colonies) & 
non-producers (pink colonies) 

Tube Adherencemethod, as described by Christensen GD et al. (14), 1982 is a quantitative 
method for biofilm detection. Test organisms were inoculated in 10 ml of trypticase soy broth 
with 1% glucose in test tubes and incubated at 37°C for 24 hours. After incubation, tubes were 
decanted, washed with phosphate-buffered saline (pH 7.3), and dried. Tubes were then 
stained with crystal violet (0.1%). The excess stain was washed with deionized water and 
dried. The scoring for the tube method was done according to the results of the control strains. 
Biofilm formation was considered positive when a visible film lined the wall and the bottom of 
the tube (Figure 2). 

 

Figure2. Tube Adherence method showing biofilm producer (tube A) & 
non-producer (tube B) 

 
Antimicrobial Susceptibility testing was performed using the Kirby Bauer Disk diffusion method 
on Mueller Hinton agar (HiMedia Labs) according to the Clinical Laboratory Standard Institute 



Sharma (Biofilm Formation by Uropathogens and Its Impact on Antimicrobial Susceptibility Pattern) 
 

 

Vol. 4, No. 2, 2022, pp. 40-47  10.26555/eshr.v4i2.4669 
 

43 

(CLSI 2019) guidelines (15). The antimicrobial discs used for Gram-positive isolates were 
Penicillin G 10 units, Gentamicin 10mcg, Ciprofloxacin 5mcg, Vancomycin 30mcg, Linezolid 
30mcg, Nitrofurantoin 300mcg, and Norfloxacin 10mcg. The antimicrobial discs used for 
Gram-negative isolates were Piperacillin-tazobactam 100/10mcg, Cefuroxime 30mcg, 
Cefepime 30mcg, Amikacin 30mcg, Imipenem 10mcg, Gentamicin 10mcg, Tobramycin 
10mcg, Ciprofloxacin 5mcg, Cotrimoxazole 1.25/23.5mcg, Chloramphenicol 30 mcg, 
Tetracycline 30 mcg, Nitrofurantoin 300mcg, Norfloxacin 10mcg, Amoxicillin/Clavulanic acid 
20/10mcg and Aztreonam 30mcg. 

 
RESULTS 

A total of 185 (92.5%) were indoor patients, while 15 (7.5%) were from the outdoor 
department. Out of 200 urine specimens, 64 (32%) were females, and 36 (18%) were males. 
Most patients belonged to 51 to 70 years (36%), followed by those above 70 years (29.5%). 
Of all, 102 (51%) patients were catheterized. Gram-negative dominated 70% of the positive 
isolates, whereas Gram-positive constituted 30%. Out of 200 uropathogens, 94 (47%) were 
Escherichia coli, followed by Enterococcus sp. 37(18.5%) (Table 1).  

Table 1. Bacterial uropathogens among UTI patients from Out Patient Department (OPD) 
and In-Patient Department 

 

S. No. Uropathogens OPD (n=15) IPD (n=185) Total (n=200) 

1 Escherichia coli 11 83 94 (47%) 

2 Enterococcus spp. 0 37 37 (18.5%) 

3 Klebsiella pneumoniae 0 28 28 (14%) 

4 Staphylococcus aureus 4 18 22 (11%) 

5 Acinetobacter spp. 0 7 7 (3.5%) 

6 Pseudomonas aeruginosa 0 6 6 (3%) 

7 Klebsiella oxytoca 0 4 4 (2%) 

8 Proteus mirabilis 0 1 1 (0.5%) 

9 Morganella morganii 0 1 1 (0.5%) 

 

Out of 102 catheterized patients, biofilm formation was observed in 72 (36%), which was way 
more than in community-acquired UTI cases 18 (9%). Table 2 shows biofilm production by 
different methods. CRA method detected 83 isolates(46%) as biofilm producers, whereas TA 
method detected only 59(33%) isolates as biofilm producers. 

Table 2. Biofilm production by different methods 
 

Methods Congo red agar 
method (%) 

Tube adherence method 
(%) 

Both methods (%) 

Total number of isolates 83 (46%) 59 (33%) 38 (21%) 
 



Sharma (Biofilm Formation by Uropathogens and Its Impact on Antimicrobial Susceptibility Pattern) 
 

 

Vol. 4, No. 2, 2022, pp. 40-47  10.26555/eshr.v4i2.4669 
 

44 

Amoxicillin (99%) and Amoxy-clavulanic acid (100%) were resistant in most biofilm-
positiveisolates. The highest degree of drug resistance was seen in biofilm-forming 
Acinetobacter spp. followed by Klebsiella sp. and Pseudomonas aeruginosa. Resistance to 
antibiotics like Cefuroxime, Aztreonam, Imipenem, Tobramycin, Norfloxacin, Cotrimoxazole, 
Chloramphenicol, Gentamicin and Tetracycline, Vancomycin was more in biofilm positive 
isolates (Table 3).  Biofilm-forming Gram-negative bacilli (GNB) uropathogenic developed 
significantly higher resistance towards antimicrobial drugs. 

DISCUSSION  

Urinary Tract Infectionspresenta severe health threat concerning antibiotic resistance, 
especially with biofilm production. During the period covered by our study, 200 samples were 
studied, 92% of which were received from different wards, Operation Theatre (OT), Cardiac 
Care Unit (CCU), Intensive Care Unit (ICU), while OPD samples were only 15% of the total 
and maximum samples fell into the age group of 51 – 70 years. This finding was also depicted 
in Madigan E & Neff D (16). Our study observed that the infected patients were primarily 
women (82%), which can be because of anal proximity and the shorter length of the urethra. 
A similar finding was also reported by Kashef N et al. (17).The age group of 61 – 75 years 
predominated in catheterized patients(n=102). The maximum number of patients were on 
catheterization for  >4 days, a similar finding by Niveditha S et al. (18). The detection of 
bacteriuria within one week of the catheterization in this study pertains to the inadequate 
precautions taken while handling catheters.  
 
Escherichia coli was isolated from 94 (47%) specimens, followed by Klebsiella pneumonia 
(16%) and Enterococcus spp. (18.5%), Pseudomonas aeruginosa (3%), Staphylococcus 
aureus (11%), Acinetobacter spp. (3.5%), Proteus mirabilis and Morganella morganii (0.5% 
each). These findings were similar to the studies conducted by Noor AF et al. (19). Escherichia 
coli was responsible for the maximum number of UTI casesbecause of the ability of 
UropathogenicEscherichia coli (UPEC) to express a variety of virulence factors like adhesins 
(e.g., type 1 and P fimbriae) and toxins like hemolysin.  Biofilm detection by CRA and TA 
methods was (46%) and (33%) respectively,and this correlates with the study of Hassan A et 
al.(20). CRA is a rapid, sensitive, and reproducible method and can be recommended in 
resource-limited countries. A similar finding was reported by Rewatkar AR and Wadher BJ et 
al. (21). Quantification of biofilms done by the TA method showed that only 9% were strong 
producers, whereas 20% were moderate producers. The rest of the isolates (71%) were weak 
producers. This was also observed by Panda PS et al. (22). Biofilm formation on CAUTI was 
observed more than Community-acquired UTI because bacteria survive on catheters easily 
as CAUTI creates an ideal environment for bacterial attachment and biofilm production. 

Antibiotic resistance was more among biofilm producers in comparison to non-producers. 
Similar results were obtained by Rewatkar AR and Wadher BJ et al. (21). A possible 
explanationis the persistence of the organism, decreased bacterial growth rate in a biofilm, 
and increased expression of resistance genes. Restricted penetration of antibiotics into the 
biofilm and the proximity of cells within a biofilm results in plasmid exchange and leads to the 
spread of antimicrobial resistance. 

 

 



Sharma (Biofilm Formation by Uropathogens and Its Impact on Antimicrobial Susceptibility Pattern) 
 

 

Vol. 4, No. 2, 2022, pp. 40-47  10.26555/eshr.v4i2.4669 
 

45 

Table 3.  Antibiogram of biofilm and non-biofilm producing isolates 
 

 
Antimicrobial agent 

Percentage resistance p-value 

Biofilm 
producers 

Non-biofilm 
producers 

Amoxicillin 72/73 (99%) 50/53 (94%) 0.019 

Amoxy Clavulanic acid 73/73 (100%) 51/53 (96%) 0.024 

Piperacillin-tazobactam 40/77 (52%) 27/64 (42%) 0 

Cefuroxime 71/73 (98%) 44/53 (83%) 0.004 

Cefepime 77/97 (80%) 56/64 (87%) 0.004 

Aztreonam 66/76 (87%) 39/56 (70%) 0 

Imipenem 67/73 (92%) 35/53 (66%) 0 

Amikacin 64/77 (84%) 54/64 (85%) 0.022 

Tobramycin 47/77 (61%) 17/64 (27%) 0 

Norfloxacin 94/103 (92%) 67/88 (77%) 0.002 

Cotrimoxazole 62/74 (84%) 28/61 (46%) 0 

Cefoxitin 58/100 (58%) 25/85 (30%) 0 

Chloramphenicol 46/73 (63%) 26/53 (50%) 0.022 

Gentamicin 98/104 (94%) 77/96 (80%) 0.022 

Tetracycline 64/74 (86%) 35/61 (57%) 0 

Ciprofloxacin 23/30 (77%) 31/35 (88%) 0.449 

Penicillin 24/27 (89%) 27/32 (84%) >0.5 

Vancomycin 10/27 (37%) 5/32 (17%) 0 

Nitrofurantoin 14/27 (52%) 6/32 (19%) 0 

Linezolid 0/27 (0%) 0/32 (0%) 0.001 

 
In the case of Escherichia coli, biofilm producers showed maximum resistance 
toamoxyclavulanic acid followed by cephalosporins, gentamicin, co-trimoxazole amikacin, and 
least resistance to piperacillin-tazobactam (37%). It was similar to the finding observed by 
Tiwari AA &Ghnawate N et al. (23). In the case of Klebsiella pneumonia, resistanceto multiple 
antibiotics was observed in biofilm producers, which also correlates with the study of Tiwari 
AA &Ghnawate N et al. (23).  Drug tobramycin was more effective in the case of non-biofilm 
producers with 83% sensitivity, while it was 25% sensitive for biofilm producers. Our study 
concluded that Klebsiella sp. was maximum resistant to antibiotics, maybe because of the 
high prevalence of resistant strain in our region or the exhaustive use of antibiotics. 
Pseudomonas aeruginosa was highly sensitive to tobramycin in the case of biofilm producers 
and non-producers. So it may be considered the antibiotic of choice for Pseudomonas 
aeruginosa. Our study had only one strain of Acinetobacter baumannii, a biofilm producer and 



Sharma (Biofilm Formation by Uropathogens and Its Impact on Antimicrobial Susceptibility Pattern) 
 

 

Vol. 4, No. 2, 2022, pp. 40-47  10.26555/eshr.v4i2.4669 
 

46 

resistant to all the antibiotics. On the other side, no biofilm-producing strain was isolated in the 
case of Morganella morganii and Proteus mirabilis. However, the insufficient sample size 
makes it impossible to draw practical conclusions from this data. In the case of Gram-positive 
cocci, linezolid was 100% sensitive in both biofilm producers and non-producers, which shows 
that it can be a good reservoir. This finding correlates well with the study of Panda PS et 
al.(22). 86% of Staphylococcus aureusisMRSA strains, a finding similar to a study by Yousefi 
M et al. (24). Our study highlights a broad range of uropathogens and Multi-Drug Resistant 
(MDR) isolates among biofilm-forminguropathogens.  

This study was concerned with a single tertiary setting. Thereforebroader surveillance is 
needed to determine the local resistance profiles of prevalent biofilm-forminguropathogens so 
that an optimal empirical therapy can be documented. 

CONCLUSION 

This study showed a considerable opportunity foruropathogens to form biofilms. We observed 
asignificant correlation between biofilm production and multi-drug resistance compared to 
non-biofilm-forming isolates. Finally, the CRA method can be employed as the routine 
laboratory test for in-vitro biofilm detection as it is cost-effective also.  

Authors' contribution 

BM and SSS contributed to the data collection and interpretation. PS, SD contributed to the 
data article writing and its publication. 

Funding Statement 

This research has not received external funding. 

Conflict of interest 

There is no conflict of interest in this research. 

REFERENCES 

1.  Chu CM, Lowder JL. Diagnosis and treatment of urinary tract infections across 
age groups. Am J Obstet Gynecol [Internet]. 2018;219(1):40–51. Available from: 
https://doi.org/10.1016/j.ajog.2017.12.231 

2.  M. Mehta, S. Bhardwaj  and JS. Screening of urinary isolates for the prevalence 
and antimicrobial susceptibility of Enterobacteria other than Escherichia coli. Int 
J Life Sci Pharma Res. 2013;3(1):100–4.  

3.  O′Gara JP and Humphreys H. Staphylococcus epidermidis biofilms: Importance 
and implications. J Med Microbiol. 2001;50(582):7.  

4.  V S, Ali F, Lokare N, Matew J. Diabetic foot ulcers and biofilm formation- The 
culprits. Int J Biomed Adv Res. 2016;7(9):428.  

5.  Heukelekian H HA. Relation between food concentration and surface for 
bacterial growth. J Bacteriol. 1940;40:547–58.  

6.  Costerton JW, Geesey GG CK-J. How bacteria stick. Sci Am. 1978;238:86–95.  
7.  Tenke P, Kovacs B JM and NE. The role of biofilm infection in urology. World J 

Urol. 2006;24(1):13–20.  
8.  Joo H.S. OM. Molecular basis of in vivo biofilm formation by bacterial pathogens. 



Sharma (Biofilm Formation by Uropathogens and Its Impact on Antimicrobial Susceptibility Pattern) 
 

 

Vol. 4, No. 2, 2022, pp. 40-47  10.26555/eshr.v4i2.4669 
 

47 

Chem Biol. 2012;19(12):1503–13.  
9.  L K. Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001;45:999–

1007.  
10.  Donlan RM. Biofilms and Device-Associated Infections. Emerg Infect Dis. 

2001;7(2):277–81.  
11.  Stickler DJ. Bacterial biofilms and the encrustation of urethral catheters. 

Biofouling. 1996;94:293–305.  
12.  Keren I, Kaldalu N, Spoering A, Wang Y LK. Persister cells and tolerance to 

antimicrobials. FEMS Microbiol Lett. 2004;230:13–8.  
13.  Freeman DJ, Falkiner FR KC. New method for detecting slime production by 

coagulase-negative staphylococci. J Clin Pathol. 1989;42(8):872–4.  
14.  Christensen GD, Simpson WA, Bisno AL BE. Adherence of slime-producing 

strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun. 
1982;37(1):318–26.  

15.  Institute CLS. Performance Standard for Antimicrobial Susceptibility Testing. 
24th Information Supplement. NCCLS Document M100-S24. Wayne, 
Pennsylvania. USA Clin Lab Stand Inst. 2014. 

16.  Madigan E and Neff DF. Care of the patients with long-term indwelling urinary 
catheters. Online J Issues Nurs. 2003;8(3):7.  

17.  Kashef N, Djavid GE SS. Antimicrobial susceptibility patterns of community-
acquired uropathogens in Tehran. Iran J Infect Dev Ctries. 2010;14(4):202–6.  

18.  Niveditha S, Pramodini S, Umadevi S, Kumar S SS. The Isolation and the Biofilm 
Formation of Uropathogens in the Patients with Catheter-Associated Urinary 
Tract Infections (UTIs). J Clin Boil Res. 2012;6(9):1478–82.  

19.  Noor AF, Shams F, Munshi SK, Hassan M NR. Prevalence and antibiogram 
profile of uropathogenic isolated from hospital and community patients with 
urinary tract infections in Dhaka City. J Bangladesh Acad Sci. 2013;37(1):52–
63.  

20.  Hassan A, Usman J, Kaleem F, Omair M, Khalid A IM. Evaluation of different 
detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis. 
2011;15(4):305–11.  

21.  Rewatkar AR and Wadher BJ. Staphylococcus aureus and Pseudomonas 
aeruginosa- Biofilm formation Methods. J Pharm Biol Sci. 2013;8(5):36–40.  

22.  Panda PS, Chaudhary U DS. Study of biofilm production and antimicrobial 
sensitivity pattern of uropathogens in a tertiary care hospital in North India. Int J 
Community Med Public Heal. 2016;3(9):2421–6.  

23.  Tiwari AA and Ghnawate N. Detection of Biofilm Forming Bacterial Communities 
from Urinary Catheter of Patients with Change in Its Antibiotic Susceptibility 
Pattern and Triclosan Effect from Different Hospitals of Amravati City 
Maharashtra, India. Open J Med Microbio. 2017;7(3):51–66.  

24.  Yousefi M, Pourmand MR, Fallah F, Hashemi A, Mashhadi R N-AA. 
Characterization of Staphylococcus aureus Biofilm Formation in Urinary Tract 
Infection. Iran J Public Heal. 2016;45(4):485–93.  

 


