|Year : 2003 | Volume
| Issue : 3 | Page : 161-165
Characterization of biofilm formed on intrauterine devices
V Pruthi , A Al-Janabi , BM J Pereira
Reproductive Biology Laboratory, Department of Biotechnology, IIT Roorkee, Roorkee-247 667, UP, India
Reproductive Biology Laboratory, Department of Biotechnology, IIT Roorkee, Roorkee-247 667, UP, India
PURPOSE: Intrauterine device (IUD) is one of the most convenient contraceptive procedures used by women of Asian and African countries. Previous surveys have revealed that 75% of the IUDs recovered from patients suffering from reproductive tract infections (RTIs) were covered with a consortium of microbes. This study was designed to characterize these microbes and recommend remedial measures. METHODS: Quantitative measurement of biofilm formation was assessed by a microtitre plate assay on 86 samples of microorganisms dislodged from IUDs of patients with RTIs. Susceptibility of biofilm to various antimicrobial agents was also quantified. Scanning electron microscopy (SEM) was used to scrutinize the microorganisms adherent to IUDs. RESULTS: The organisms associated with IUDs were predominantly composed of Staphylococcus aureus (16%), Staphylococcus epidermidis (18%), Pseudomonas aeruginosa (5%), Escherichia coli (27%), Neisseria gonorrhoeae (2%), Candida albicans (20%) and Candida dubliniesis (12%). SEM studies indicated that these organisms were organized into biofilms. Studies on the in vitro adherence pattern by crystal violet staining on 96 well microtitre plates revealed that the biofilms were stably established after 60 hours. These biofilms are resistant to an array of antibiotics tested. CONCLUSION: Biofilm formation may be one of the major causes for persistent infection and antibiotic resistance in IUD users.
|How to cite this article:|
Pruthi V, Al-Janabi A, Pereira BJ. Characterization of biofilm formed on intrauterine devices. Indian J Med Microbiol 2003;21:161-5
|How to cite this URL:|
Pruthi V, Al-Janabi A, Pereira BJ. Characterization of biofilm formed on intrauterine devices. Indian J Med Microbiol [serial online] 2003 [cited 2019 Sep 20];21:161-5. Available from: http://www.ijmm.org/text.asp?2003/21/3/161/8008
In developing countries IUDs are the most preferred means of contraception in women. The tiny IUDs that are fitted inside the uterus are now available in several shapes and sizes. The present generation of IUDs is made up of a variety of materials ranging from copper to all plastic. More innovation in design (Novo T) and choice of materials (T-Cu 380 series, T-Cu-220 series and multiload-375) has since been done. IUDs that steadily release hormones (Progestasert, LNG-20) are also available. It is known that the insertion of IUDs stimulate inflammatory or foreign body responses, which in turn cause cellular/biochemical changes in the endometrium and uterine fluid. These changes are believed to be responsible for the contraceptive effects. Although studies have shown that IUDs effectively prevent fertilization, provide high degree of sexual satisfaction and are cost effective, they are known to be associated with a risk of pelvic infection, heavier periods, menstrual cramps and above all complication associated with colonization of microbes on these implanted devices. Most often the medical practitioner is left with little option but to remove the IUD when antibiotics cannot deal with the insult of pathogens adequately. In this study, we have screened the microbes attached to IUDs recovered from patients with RTIs and report here some of their characteristics.
| ~ Materials and methods|| |
Infected IUDs removed from the patients with RTIs were collected from local nursing homes and the hospital at IIT Roorkee under aseptic conditions and brought to the laboratory for investigation.
Quantitative measurement of adherent microorganisms
The thread that is attached to keep the device in place was found to harbor microbes, which were screened and characterized as described below. Briefly, infected thread pieces (0.5cm) from the devices were placed in a 10 mL of 0.15M PBS that contained 0.1% Tween-80 and sonicated in an ultrasonic cleaner waterbath (Branson 2200, USA) for 30 minutes at room temperature to detach adherent microorganisms. The microbial suspension was vortexed vigorously for 15 seconds to break up clumps. Ten fold serial dilutions of each suspension were plated on 5% blood agar base (Himedia, India) using spread plate technique, incubated at 30ºC for 18 hours and the mean number of colony forming units (CFU) was determined. Identification of isolates was done according to standard procedures., All microbial strains so obtained were maintained on slants of Luria-Bertani (LB) agar medium and sub-cultured monthly. After every two months, cultures were replaced from freeze-dried stocks.
Determination of biofilm dry weight
The microbes that detached from a unit size of infected IUD after sonication were centrifuged at 10,000 rpm for 5 minutes at room temperature. The pellet so obtained was transferred to preweighed cellulose nitrate paper (0.45 µm pore size; 25mm diameter), dried at 80ºC overnight and weighed. This weight was taken as a measure of the dry biomass.
Monitoring biofilm formation
Biofilm formation was monitored by the ability of cells to adhere to the wells of microtitre plate (MTP) made of PVC (Beckon Deckson labware). The biofilm forming microorganisms recovered from IUDs were grown in LB medium and samples were drawn at 12 hour intervals. The quantification measurements were carried out in the mixed population of organisms listed in [Table - 1].
One hundred microlitre (1:100 diluted LB broth) of these samples were inoculated in MTP and incubated at 30ºC for 10 hours. MTP wells were then rinsed thoroughly thrice with 0.15M PBS to remove free-floating organisms. Crystal violet (100µL of 1% solution) was then added to each well (this dye stains the cells but not the PVC) and the plates were incubated at room temperature for 15 minutes. Excess of stain was removed by rinsing with distilled water. The stain that was taken up by biofilm forming organisms was extracted twice in 200µL aliquots of 95% ethanol, 100µL of which was transferred to a new MTP (Costar) and the absorbance was determined in a plate reader at 600nm (Metertech Microplate Reader Model 960).
Susceptibility of biofilm to antimicrobial agents
The biofilm identified on IUDs comprised of a consortium of microorganisms that included fungi, gram positive and gram negative bacteria [Table - 1]. Therefore, the susceptibility testing was carried out with representative antimicrobial agents that were active against each of these classes of microorganisms. The antimicrobial agents used were penicillin, tetracycline, amphotericin-B, fluconazole and nystatin. The susceptibility was tested using Baillie and Douglas protocol. Briefly, MTP wells were inoculated with 100µL/well of 107 cells obtained after 60 hours as detailed earlier and allowed to form biofilm. Antimicrobial agents at varying concentration (2µg/mL-128µg/mL depending upon the MIC) were added to these wells, incubated for 5 hours and then stained with crystal violet. Cells detached from MTP wells were rinsed and the residual biofilm was quantified as percentage reduction in absorbance at 600nm.
Scanning Electron Microscopy (SEM)
Microbial biofilms formed on IUDs were fixed with 2.5% (v/v) glutaraldehyde in 0.15 M PBS for 1 hour at room temperature. They were then treated with 1% (w/v) osmium tetra oxide for 1 hour, washed thrice with distilled water, treated with 1% (w/v) uranyl acetate for 1 hour and washed again with distilled water. The samples were then dehydrated in ethanol. All samples were dried to critical point, gold coated and viewed under SEM (LEO-435, England).
| ~ Results and Discussion|| |
The vagina and surrounding regions of the reproductive tract are known to support a large number of bacteria and fungi. The migration of these to the upper part of the female urinogenital tract often leads to discomfort and infection. [Table - 1] shows the identity and percentage distribution of organisms in 86 samples recovered from patients with IUD infection. The microbial flora obtained from the vaginal swabs and IUDs matched to a large extent. IUDs removed from women were shown to harbour S. epidermidis, Streptococci spp., Corynebacterium spp., Micrococcus spp., Enterococcus spp., and anaerobic lactobacilli., Majority of the infected IUDs have been reported to be due to gram positive bacteria notably Staphylococci, but infections due to gram negative bacteria and fungi tend to be of more serious concern.,, The cord/ thread attached to the tail of the IUDs is perhaps one of the routes of microbial migration from the vagina to the uterus. A previous study has indicated that there was less incidence of biofilm formation on IUDs that did not have a tail protruding into the cervical region. Besides, microbial load was heaviest on the IUD when the distal portion of the tail was directly exposed to the vaginal flora. Although the uterine secretions under normal conditions actively deal with such migrations, the presence of the IUD gives a solid surface for attachment and an ideal niche for the biofilm to form and flourish.
SEM analyses of the biofilm topography formed on these devices revealed a dense network of mono or multi layer of cells from same or different species embedded within a matrix of extra cellular polymer material [Figure - 1]. Such organization is typical of biofilms found on medical devices and implants. This provides an opportunity for the microorganisms to accommodate each other and also to thrive under hostile conditions of pH, oxygen availability and redox potential. Our results are in agreement with the suggestion that the heterogeneous mosaic architecture of biofilms could be in response to environmental stress. Besides, the complex structure could provide protection against host defense mechanisms.,
[Figure:2a], [Figure:2b] show that maximum microbial adherence to the 96 wells MTP was obtained after 60 hours at 30°C. Dry biomass (2.3g/L) was also reported to be highest at this stage [Figure:2c]. It may be clarified that this dry biomass represents a mixture of organisms listed in [Table - 1] in various combinations. Similar results were obtained during studies on biofilms and infections associated with other implanted devices by other research groups. It would be relevant to note that the temperature of the uterine environment is also conducive for the formation of biofilms.
Data obtained on the influence of antimicrobial agents (penicillin, tetracycline, amphotericin-B, fluconazole and nystatin) indicates that treatment with antibiotics far in excess of recommended minimum inhibitiory concentration (MIC) level reduced biofilm cell counts by approximately 30-40% compared with the control sample [Figure:3]. Other research groups also obtained similar results., Our investigations strongly support the contention that infections due to biofilm formation on IUDs are difficult to resolve, as they can counter both host defense mechanism and antibiotic therapy. We believe that release of these microbes from the IUDs would initiate a sequence of events leading to chronic infection on account of antibiotic resistance. The situation is more complicated due to the mixed nature of the flora found in biofilms. Several possibilities including phenotypic changes resulting from nutrient limitation, possible protective effect of the matrix, differential expression of drug resistance genes can be considered as the mechanisms contributing to increased resistance of antimicrobial agents to biofilm formed on infected IUDs. The strategies involving antiseptic bonded biomaterials are now in common use and future work on such biomaterials is likely to yield improved devices. Therapeutic approaches which interfere with the expression of or activity of gene and gene products involved in microbial biofilm formation are likely to provide novel and potential beneficial alternatives to current therapies. The clinical usefulness of these strategies remains to be determined.
| ~ Acknowledgement|| |
Research fellowship from Indian Cultural Council for Relations (ICCR), Government of India, to Abbas Al-Janabi is thankfully acknowledged.
| ~ References|| |
|1.||World Health Organization (WHO) Geneva. Mechanism of action, safety and efficacy of intrauterine devices. Technical Report Series 1987;753:91. |
|2.||Gupta PK, Malkani PK, Bhasin K. Cellular response in the uterine cavity after IUD insertion and structural changes of the IUD. Contraception 1971;4:375-384. |
|3.||Newton JR. Copper intrauterine devices: evaluation for long term use-a review. Br J Obset Gynaecol 1982;4:20-31. |
|4.||Lee NC, Rubin GL, Ory HW, Burkman RT. Type of intrauterine device and the risk of pelvic inflammatory diseases. Obstet Gynecol 1983;62:1-6. [PUBMED] |
|5.||Krieg NR, Holt JG. Bergey's manual of systematic bacteriology, 2nd ed. (William and Wilkins, Baltimore) 1984. |
|6.||Cowan ST, Steel KJ. Manual for the identification of medical bacteria. 2nd ed. (Cambridge University Press, Cambridge) 1974. |
|7.||Baillie GS, Douglas LJ. Candida biofilms and their susceptibility to antifungal agents. Methods Enzymol 1999;310:644-656. |
|8.||Lewis R. A review of bacteriological culture of removed intrauterine contraceptive devices. Br J Fam Plan 1988;24:95-97. |
|9.||Marrie TJ, Costerton JW. A scanning and transmission electron microscopic study of the surfaces of intrauterine contraceptive devices. Am J Obstet Gynecol 1983;146:384-394. |
|10.||Wolf AS, Kreiger D. Bacterial colonization of intrauterine devices (IUDs). Arch Gynecol 1986;239:31-37. |
|11.||Costerton JW, Stewart PS, Greenberg EP. Bacterial Biofilms: a common cause of persistence infection. Science 1999;284:1318-1322. |
|12.||O'Toole GA, Kaplan HB, Kloter R. Biofilm formation as microbial development. Ann Rev Microbiol 2000;54:49-79. |
|13.||Reynold TB, Fink GR. Baker's yeast, a model for fungal biofilm formation. Science 2001;291:878-881. |
|14.||Donlan RM, Costerton JW. Biofilm survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002;15:167-193. |
|15.||Bank HL, Williamson, HO. Scanning electron microscopy of Dalkon Shield tails. Fertil Steril 1983;40:334-339. |
|16.||Lewandowski Z. Structure and function of biofilms. In: Biofilms: recent advances in the study and control. Evans, LV Ed (Harwood Academics Publishers, Amsterdam, The Netherlands) 2000:1-17. |
|17.||Baillie GS, Douglas LJ. Matrix polymer of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J Antimicrob Chemother 2000;46:397-408. |
|18.||Donlan RM. Biofilm and device associated infections. Emerging Infectious diseases 2001;7:277-281. |
|19.||Brown MRW, Collier PJ, Gilbert P. Influence of growth rate on susceptibility to antimicrobial agents: Modification of the cell envelope and batch & continuous culture studies. J Antimicrob Chemother 1990;34:1623-1628. |
|20.||Duguid IG, Evans E, Brown MRW, Gilbert P. Susceptibility of S. epidermidis biofilms towards ciprofloxacin: Influence of specific growth rate and cellular division cycle. J Antimicrob Chemother 1992;30:791-802. |