**3. Observation and results**

Hence, the present study was undertaken to detect the biofilm producing organisms, isolated

The present study was conducted from 2009 to 2012. A total number of 350 bacterial and 50 Candida strains were studied. The microbial strains were isolated from different clinical specimens like urine, blood, pus and wound swab, endotracheal aspirate, urinary catheter tip, central venous catheter tip etc. All the microbial strains were identified by conventional methods [55]. We used microtitre plate biofilm assay to detect microbial attachment to an

**1.** The microbial cells were grown in Brain heart infusion broth overnight.

incubated at optimal growth temperature [56] for 48 hours

**4.** Then the wells were washed twice to remove planktonic cells.

**2.** On next day, the cultures were diluted 1:100 using the brain heart infusion broth.

**3.** 100μl of each diluted culture was inoculated into each of three wells in a microtiter plate which has not been tissue culture treated. The plates were covered by the lid and was

**5.** Microbial cells which were adhered to the wells were subsequently stained with crystal violet solution that allowed visulisation of the attachment pattern. 125 μl of 0.1% crystal violet solution was added to each well and stained for 10 minutes at room temperature.

**7.** The plates were washed successively twice with distilled water. Any crystal violet that is not specifically staining the adherent microbial cells were removed by this washing step.

**8.** The microtiter plates were then inverted and tapped vigorously on tissue paper to remove

**9.** The microtiter plates were then air dried. The dried microtiter plates may be stored at

**10.** This surface associated dye was solubilized by adding ethanol or any other solvent for semiquantitative assessment of biofim formed. 200μl of 95% ethanol or other appropriate solvent [56] was then added to each stained well and was kept for 10 to 15 minutes

**11.** The contents of each well were mixed by pipetting and then 125μl of the crystal violet / ethanol solution from each well was transferred to a separate well of another 96 well

**6.** The microtiter plates were shaken and the crystal violet solution was removed.

from different clinical specimens in our laboratory.

**2. Material and methods**

abiotic surface [56].

any excess liquid.

room temperature for several weeks.

microtiter plate maintaining the same sequence.

**Steps:**

64 Infection Control

Out of 350 bacterial strains studied, 90 were *Pseudomonas aeuginosa*, 80 were *E. coli*, 35 were *Klebsiella pneumoniae*, 80 were *Coagulase positive Staphylococci*, 30 were Coagulase negative 35 included Proteus sp(5), *Vibrio cholerae*(3), *Acinetobacter baumanii*(4), Enterococcus sp.(23). Out of 50 Candida strains 23 were *Candida albicans*, 16 were *Candida tropicalis*, 2 were *Candida dubliensis*, 6 were *Candida krusei* and 3 were *Candida glabrata*. Amongst 350 bacterial strains, 153(43.7%) and out of 50 *Candida* species 28(56%) were biofilm producers respectively. Amongst 50 Candida species, 11 (22%) were strong biofilm producers, and 6/11 (54.5%) were *Candida albicans*.

**Figure 1.** Microtitre plate biofilm assay for detection of microbial attachment

Maximum 65( 72.2%) of *Pseudomonas aeruginosa* strains produced biofilms. 51(33.3% ) biofilm producing bacterial strains were isolated from catheterized urine samples or patients having other medical devices. 108(70.6% ) bacterial strains producing biofilms were isolated from patients having chronic infections eg persistent or recurrent UTI, Chronic obstructive airway disease, cystic fibrosis etc.

producing Pseudomonas aeruginosa 28 (49.1%) were strongly adherent biofilm producers, compared to only 5/33 (15.1%) non β – lactamase producers. Amongst the 120 Enterobacter‐ iaceae strains studied. 82 (68.3%) were newer β – lactamases producers, whereas 48/82 (58.5%) were strong biofilm producers and only 3/38 (7.9%) non β – lactamase producing strains were

**Organisms Methicillin resistant Methicillin sensitive**

Table 2 shows amongst the Methicillin Resistant Staphylococcus aureus (MRSA) strains, 14/34 (41.2%) and Methicillin Resistant Coagulase negative Staphylococci (MR – CONS) 4/11 (36.4%) were strong biofilm producers compared to 2/46 (4.3%) *Methicillin sensitive Staphylococcus*

Out of 23 Enterococcus species 13/23 (56.5%)were High level Aminoglycoside Resistant (HLAR) strains and it was also found that 8/13 (61.5%) HLAR strains were strong biofilm

Our Hospital is a tertiary care centre in a rural setup. Though CLSM is the best phenotypic method, it could not be used as it is very costly. We did a pilot study with Staphylococci in 2008 and found 33% of Staphylococcus aureus and 44.7% of Coagulase Negative Staphylococci (CONS) were biofilm producers and amongst the 3 phenotypic methods tissue culture plate method gave the best results [58]. The present study correlated well with reports of other authors that Extended Spectrum β-Lactamase (ESBL) producing strains, Methicillin Resistant Staphylococcs aureus(MRSA) were more adherent to microtitre plate than Non ESBL and Non

Lee et al in 2008 have also reported a positive correlation between biofilm formation and ESBL producing Acinetobacter baumanii [59]. Norouzi et al in 2010 have reported that in their study 14% ESBL producing Pseudomonas aeruginosa has formed strongly adherent biofilm com‐ pared to only 4% of non-ESBL producing Pseudomonas aeruginosa [60]. It has also been

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Biofilms: A Challenge to Medical Fraternity in Infection Control

http://dx.doi.org/10.5772/55649

67

11 19

4 2

Coagulase positive Staphylococcus [80] 34 46

**Table 2.** Incidence of strong biofilm producing Methicillin resistant Staphylococcus strains.

*aureus* (MSSA) and 2/19 (10.5%) Methicillin sensitive CONS.

producers compared to only 2/10 (20%) of non HLAR strains.

strong biofilm producers.

Strongly adherent biofilm producing Coagulase positive Staphylococcus

Coagulase negative Staphylococci

Strongly adherent biofilm producing Coagulase negative Staphylococci

(CONS) [30]

(CONS)

**4. Discussion**

MRSA strains (Figure 2).

In our study the cut off OD(ODc) was 0.003. The biofilm forming organisms are grouped into weak group (OD ≥ 0.003 to 0.006), moderate group (OD ≥ 0.006 to 0.012) and strong group (OD > 0.012).


**Table 1.** Incidence of strong biofilm producers amongst newer β – lactamases producing strains

It was observed that out of 57 newer β – lactamases (Extended spectrum β – lactamases i.e. ESBL, Amp C β – lactamases and Metallobetalactamases i.e. MBL only and in combination) producing Pseudomonas aeruginosa 28 (49.1%) were strongly adherent biofilm producers, compared to only 5/33 (15.1%) non β – lactamase producers. Amongst the 120 Enterobacter‐ iaceae strains studied. 82 (68.3%) were newer β – lactamases producers, whereas 48/82 (58.5%) were strong biofilm producers and only 3/38 (7.9%) non β – lactamase producing strains were strong biofilm producers.


**Table 2.** Incidence of strong biofilm producing Methicillin resistant Staphylococcus strains.

Table 2 shows amongst the Methicillin Resistant Staphylococcus aureus (MRSA) strains, 14/34 (41.2%) and Methicillin Resistant Coagulase negative Staphylococci (MR – CONS) 4/11 (36.4%) were strong biofilm producers compared to 2/46 (4.3%) *Methicillin sensitive Staphylococcus aureus* (MSSA) and 2/19 (10.5%) Methicillin sensitive CONS.

Out of 23 Enterococcus species 13/23 (56.5%)were High level Aminoglycoside Resistant (HLAR) strains and it was also found that 8/13 (61.5%) HLAR strains were strong biofilm producers compared to only 2/10 (20%) of non HLAR strains.
