|Year : 2016 | Volume
| Issue : 4 | Page : 204-209
Escherichia coli biofilms: Accepting the therapeutic challenges
Trupti Bajpai1, M Varma2, GS Bhatambare3, M Pandey4
1 Department of Microbiology, Sri Aurobindo Institute of Medical Sciences Medical College and PG Institute, Indore, MP; Department of Biochemistry, SOS, IGNOU, New Delhi, India
2 Department of Biochemistry, Sri Aurobindo Institute of Medical Sciences Medical College and PG Institute, Indore, MP, India
3 Department of Microbiology, Sri Aurobindo Institute of Medical Sciences Medical College and PG Institute, Indore, MP, India
4 Department of Biochemistry, SOS, IGNOU, New Delhi, India
|Date of Web Publication||15-Nov-2016|
Assistant Professor, Department of Microbiology, Sri Aurobindo Institute of Medical Sciences Medical College and PG Institute, MR-10 Crossing, Indore-Ujjain Highway, Indore, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Background: Urinary tract infections (UTI's) are a major public health concern globally. Recurrent UTI's that are predominantly caused by uropathogenic Escherichia coli's forms biofilm that is an intracellular, structured bacterial community, enclosed in a self-produced matrix, adherent to an inert, or living surface. Biofilm physiology is characterized by increased tolerance to stress, antibiotics, and immunological defenses, which is at the origin of their resilience in most medical and industrial settings. Materials and Methods: The present prospective study was carried out from December 2013 to May 2014 in the Department of Microbiology of a Teaching Tertiary Care hospital located in central India. A total of 100 consecutive, nonrepetitive E. coli isolates were subjected to biofilm formation study by Christensen's tube adherence method. All the isolates were also subjected to antimicrobial susceptibility testing by Kirby-Bauer disc diffusion method in accordance with the Clinical Laboratory Standard Institute 2013) guidelines. Results and Discussion: Out of the 100 E. coli isolates studied, 62 (62%) were positive for biofilm formation. High percentage of resistance was detected in isolates among the male inpatient group. Overall drug resistance was found to be very high among both biofilm as well as nonbiofilm forming isolates indicating excessive drug resistance among both community and hospital organisms. Conclusion: A greater understanding of the nature of biofilm organisms in chronic UTI's would help in the development of novel and more effective treatments for these problematic diseases.
Keywords: Biofilms, Escherichia coli, urinary tract infection
|How to cite this article:|
Bajpai T, Varma M, Bhatambare G S, Pandey M. Escherichia coli biofilms: Accepting the therapeutic challenges. Int J Health Allied Sci 2016;5:204-9
|How to cite this URL:|
Bajpai T, Varma M, Bhatambare G S, Pandey M. Escherichia coli biofilms: Accepting the therapeutic challenges. Int J Health Allied Sci [serial online] 2016 [cited 2017 Nov 17];5:204-9. Available from: http://www.ijhas.in/text.asp?2016/5/4/204/194081
| Introduction|| |
Recurrent urinary tract infections (UTI's) are frequently associated with significant morbidity and mortality. ,, Community-acquired UTI (CAUTI) and hospital-acquired UTI (HAUTI) are among the most encountered infections predominantly caused by uropathogenic Escherichia More Details coli (UPEC). Uropathogenic strains of E. coli act as an opportunistic pathogen leading to conditions such as cystitis and pyelonephritis. These extraintestinal organisms form intracellular bacterial communities, thereby posing biofilm-like properties within the bladder epithelium ,, or on the indwelling devices such as catheters. Persisting infections due to biofilm formation are certainly a novel but an additional burden to the treating clinicians. 
Biofilm notion is based on the unicellular planktonic cells, which gather irreversibly on biotic and abiotic surfaces to form a sedentary but dynamic community with a complex structure. Bacterial cells in the biofilm that is embedded in the self-produced extracellular polysaccharide matrix display spatial and functional heterogeneity. , It allows them to outlast a strong host immune response and antibiotic therapy thereby surviving hostile conditions. ,,, The biofilm makes it possible for the concerned bacteria to undergo dormancy and hibernation, enabling them to survive the environmental stresses of urinary tracts, and disseminate their genomes. , During the last decade, the negative impact of biofilm development in human activities has stimulated research, aimed at providing multiple clues for combating detrimental biofilm. 
The in vitro detection of biofilm of UPEC's and antimicrobial susceptibility pattern among UTI patients have been documented across the globe. Due to the paucity of data on the prevalence of biofilm-producing E. coli among UTI patients in our region, we have made an effort to guide health-care providers to treat biofilm-associated infections among UTI patients. In this context, the study was aimed to perform in vitro detection of biofilm formation by E. coli strains isolated from urine cultures and to determine their susceptibility patterns to 19 commonly used antibiotics.
| Materials and Methods|| |
The present prospective and analytic study was carried out in the Department of Microbiology of a Teaching Tertiary Care Hospital located in central India. A total of 100 consecutive, nonrepetitive E. coli isolates obtained from the urine specimen of patients suspected of UTI attending our hospital from December 2013 to May 2014 were subjected to biofilm formation study. Urine sample from outpatients department (OPD) and inpatients department (IPD) of both the genders was included in the study. Ethical clearance was obtained by the Institutional Ethical and Research Committee. Identification of the strain was based on colony characteristics on blood agar, MacConkey agar, and UTI chromogenic agar media (nonmucoid and mucoid colonies) [Figure 1]a and b as well as by the reactions in standard biochemical tests and simultaneously by automated vitek-2 compact system (Biomerieux, France). All E. coli isolates included in the study were simultaneously analyzed for antimicrobial resistance pattern.
|Figure 1: (a) Nonmucoid colonies of Escherichia coli on chromogenic media. (b) Mucoid colonies of Escherichia coli on chromogenic media|
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A qualitative assessment of biofilm formation was determined by adherence of bacterial biofilms on borosilicate tubes as described by Christensen's tube method.  Brain-heart infusion broth was inoculated with a loop full of E. coli isolates from overnight culture plates and incubated for 48 h at 37°C aerobically. Then, the supernatants were discarded, and the glass tubes were stained with 0.1% safranin solution. They were washed with distilled water thrice and were dried finally in an inverted position. Each isolate was tested in triplicate. Biofilm formation was considered as positive when a visible film lined the wall and bottom of the test tube. The exclusive observation of a stained ring at the air-liquid interface was considered as negative [Figure 2]. Biofilm producer Staphylococcus epidermidis American Type Culture Collection (ATCC)-35984 and nonbiofilm producer S. epidermidis ATCC 12228 were used as positive and negative controls. Antimicrobial resistance profile of all the isolates was determined by the Kirby-Bauer disk diffusion method on Muller-Hinton agar.  Selection of the drugs and interpretation of results were done in accordance with the Clinical Laboratory Standards Institute (CLSI)-2013 and CLSI-2014 guidelines. , Extended spectrum beta-lactamase (ESBL) production among the various E. coli isolates was done through phenotypic confirmatory disk diffusion method, and carbapenemase production among the various isolates was determined by the modified Hodge test. E. coli ATCC 25922, Klebsiella pneumoniae ATCC BAA-1705, and K. pneumoniae ATCC BAA-1706 were used as control strains for antibiotic sensitivity testing.
|Figure 2: Borosilicate test tubes showing positive and negative biofilm production|
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| Results|| |
A total of 100 consecutive and nonrepetitive E. coli isolated from cases of UTI were studied for biofilm formation. Out of these, 90 (90%) isolates produced nonmucoid, and 10 (10%) isolates produced mucoid colonies when cultured on different media. Among them, 57 out of 90 (63.3%) nonmucoid and 5 (50%) out of 10 mucoid isolates were found to be positive for biofilm formation. A total of 41 and 59 E. coli isolates from OPD and IPD while 43 and 57 isolates from male and female patients, respectively were considered for biofilm study. During the study, 37 out of 62 (59.6%) biofilm forming isolates and 26 out of 38 (68.4%) nonbiofilm forming isolates showed at least one type of drug resistance mechanism (ESBL or carbapenemase production or both). The results of the biofilm production based on the differences in gender and hospitalization status and the various drug resistance mechanisms exhibited by the isolates have been mentioned in [Table 1]. Antibiotic resistance profile of all the isolates has been mentioned in [Table 2].
|Table 1: Biofilm formation and drug resistance results of various Escherichia coli isolates |
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|ntblTable 2: Antibiotic resistance profile (%) of the various biofilm and nonbiofilm forming Escherichia coli |
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| Discussion|| |
In the present prospective study, E. coli have been chosen as a model organism for biofilm study. E. coli is known to be a highly versatile bacterium ranging from harmless gut commensals to extra- or intra-intestinal pathogens, including common colonizers of medical devices and the primary causes of recurrent urogenital infections. Literature reveals that the most predominant uropathogen that is capable of causing complicated and uncomplicated UTI is Gram-negative Bacilli, with E. coli accounting for the highest prevalence in most instances. This trend is in agreement with the previous studies ,, where UPEC's are the primary cause of CAUTI (70%-90%) and HAUTI (50%). , Its frequent community lifestyle and the availability of wide array of genetic tools contribute to establish E. coli as a relevant model organism for biofilm study. 
The pathogenic potential of E. coli is thought to be dependent on the presence of virulence factors such as autotransporter proteins, curli fimbriae, and F-conjugative pilus responsible for initial adhesion and capsule exopolysaccharide for the formation of microcolonies in which bacteria multiply. ,,
Christensen's tube method was chosen for biofilm detection as it has been recommended by several authors in their studies as a reliable and more accurate qualitative tool for determining biofilm formation by clinical isolates. It is based on the enhancement of exopolysaccharide production using enriched medium. ,, The method used for the detection of biofilm production produced consistent results when the isolate was tested in triplicate during the biofilm study.
Bacteria that invade bladder cells and grow into biofilm may be responsible for many recurrent UTI's.  In vitro susceptibility tests performed by several authors have shown considerable increase in resistance of biofilm to killing. ,, Bacteria in the biofilm display dramatically increased resistance to antibiotics. First, the presence of bacterial exopolysaccharides prevents the access of antibacterial agents, antibodies, and white blood cells into the biofilm. Usually, the planktonic cells can be easily eradicated by antibiotics; then, the sessile bacterial cells in biofilm can even withstand host immune responses. , Second, it may be because the drug concentration obtained may be insufficient in certain areas of the film. The bacteria located at the base of biofilm are metabolically inactive and are thus resistant to several antibiotics that contribute to avoid the accumulation of effective drug concentration. Thus, biofilm can be regarded as universal strategy for bacterial survival. , Furthermore, the proximity of cells within the biofilm can facilitate plasmid exchange and hence the spread of antimicrobial resistance. ,
As opposed to the study made by other authors, ,, our study comprised the antimicrobial resistance data with respect to the differences in gender and hospital status of the patients. As a result, we were able to reach an interesting finding in which all IPD and OPD isolates of both the gender groups were resistant to beta-lactams as compared to nonbiofilm producers. It is because the concentration of antibiotics needed to kill bacteria in sessile phase is much often than those required for bacteria in planktonic phase. Resistance noticed may be due to the production of antibiotic hydrolyzing enzymes such as beta-lactamases that accumulate within glycocalyx and produce concentration gradients thereby protecting the underlying cells.  This can also be confirmed from the studies made by several authors. 
Another important finding of our study was that biofilm producer isolates were less susceptible to antimicrobial agents than the nonbiofilm producers, especially with reference to aminoglycosides in both IPD gender groups and OPD male group. Resistance to aminoglycosides which have trouble penetrating the biofilm is a shared feature of the biofilm bacteria. The penetration of aminoglycosides is strongly restricted by exopolymer matrix.  The biofilm isolates from OPD female groups might be more susceptible to antibiotics, being community-acquired.
Our biofilm organisms were more susceptible to fluoroquinolones and carbapenems, especially in both OPD gender groups and IPD female groups. Fluoroquinolones and carbapenems are indeed very effective in stopping the growth of biofilm. However, at the same time, retarded diffusion at the certain stage may again protect the biofilm from its antimicrobial activity. Increasing concentration of both these drugs can cause an initial decrease in the number of viable biofilm cells, but the remaining small population becomes insensitive to further increase in drug concentration. Furthermore, the low levels of resistance may become high due to selective pressure of exposures with constant use as a result of drug abuse and arbitrary presentation of this agents.  Biofilm isolates from IPD male groups might be less susceptible to these drugs, being hospital acquired.
Microbial biofilms are known to be associated with persistent infections, which respond poorly to conventional antibiotic therapy. This helps in spread of antibiotic resistance traits among nosocomial pathogens by increasing mutation rates and genome exchange which are responsible for antibiotic resistance. Antibiotic therapy against device associated biofilm organisms often fails without the removal of infected implant. An elevated pressure of efflux pumps and physiological heterogeneity is further responsible for resistance among biofilm bacteria.  This can be attributed to high rates of resistance among male IPD biofilm forming isolates in our study. Biofilm bacteria from IPD male patient groups revealed higher resistance as compared to nonbiofilm bacteria with respect to all antibiotic groups (including 18 antibiotics), except nitrofurantoin. Such resistance was not observed in female IPD groups. This trend may be because the source of E. coli responsible for UTI in both the patient groups might be different. In case of female IPD groups, they might be less resistant fecal while in case of male IPD groups; they might be more resistant hospital organisms originating from hospital environment including indwelling catheters. In addition, it is well established that catheters provide attractive sites for bacterial colonization and biofilm bacteria thrive in their matrix gel and gently flowing warm, nutritious urine. 
Nonbiofilm forming E. coli isolates were found to be more resistant as compared to biofilm forming isolates in all of our study groups except IPD male groups. Not only biofilm forming isolates (59.6%) but also large number of nonbiofilm isolates (68.4%) showed one of the mechanisms of resistance. These results were indeed alarming indicating that heavy resistance has already been developed among the E. coli isolates of our hospital and community. If not controlled, within no time, they would disseminate their drug resistance genomes among the hospital and community non-E. coli organisms, through horizontal transfer mechanisms.
To conclude, a significant correlation between biofilm formation and multidrug resistance was observed among the male IPD group in our study. Higher rates of resistance were noticed not only among the biofilm forming isolates but also among the nonbiofilm forming isolates. In addition, a greater understanding of the nature of biofilm organisms in chronic UTI's would help in the development of novel and more effective treatments for these problematic diseases.
The authors wish to thank the Chairperson and Dean of the institute for providing laboratory facilities and healthy working atmosphere during the study period. The authors are also thankful to the technical staff of the institute for providing necessary helping hand during the endeavour.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]