Role of Bacterial Biofilm in Resistant Cases of Chronic Rhinosinusitis and Otitis externa View PDF

Biofilm is a 3D specialized population of adherent microorganisms, surrounded by an extracellular polymeric substance (EPS). In most environments, including human illnesses biofilm communities tend to be polymicrobial. Biofilms have numerous advantages, such as passive resistance, metabolic cooperation, by-product influence, quorum sensing systems, an increased gene pool with more efficient DNA sharing, and many other synergies that provide them with a competitive advantage, by providing multiple bacterial and/or fungal species in a single community. The greater the variety, the better the biofilm is in terms of its longevity [1].

The high antibiotic resistance and to host immune mechanisms, such as less vulnerability to phagocytosis and opsonization , is one of the most important features of biofilms [2,3].

In vitro studies have shown that, owing to the physical barrier imposed by the polysaccharide matrix that prevents the spread of compounds or inactivates the biocide action of certain agents, certain strains of bacteria in the biofilm state may be more than 1000 times more resistant to antibiotics in relation to the minimum resistance mechanisms [4,5].

There have been several different approaches to examining and appreciation the complex existence of biofilms.

The conventional techniques involved both scanning electron microscope and transmission electron microscopy (SEM and TEM, respectively).

These approaches provide comprehensive imaging of biofilms’ complex architecture, growth phases, and polymicrobial nature.

Nevertheless, owing to difficulties in fixing, the presence of artifacts during fixation processes and the difficulties in recognizing individual bacterial organisms, both methods may be limited to clinical usefulness. The distinction among mucus, clot, and biofilm is also hard for SEM, though TEM only makes a two-dimensional portion of the biofilm [6].

Fluorescent in situ hybridization (FISH) with confocal laser scan microscopy (CLSM) is used to surmount these problems and to simplify the method in biofilm recognition. In addition to being able to make three-dimensional biofilm structures, CLSM with FISH also has the benefit of speciating the visualized bacteria. Crystal violet stain is a low-cost assay to evaluate the growth of in-vitro biofilm but does not represent in-vivo biofilm growth.

One of the most popular chronic disorders is chronic rhinosinusitis. This disease greatly affects the life quality of its patients and is a social and economic burden on the society. In contrast to the general public, sufferers with persistent or chronic rhinosinusitis have a worsening sense of overall health and vitality. CRS reflects a variety of inflammatory and infectious processes that affect the nose and paranasal sinuses at the same time and is distinguished by at least two symptoms.

Nasal congestion or nasal discharge, face pain and reduced sense of smell (anterior/posterior nasal drip) are all involved. Furthermore, one of the major presentations being examined is the existence of polyps and mucosal edema. It seems that the period of the illness is more than 12 weeks [7].

Otitis externa, also known the ear of the swimmer, includes diffuse inflammation of the external ear canal which could extend to the pinna distally and to the tympanic membrane proximally [8]. Otitis externa, referred to as chronic otitis externa, persists for 3 months or longer and is mostly a product of allergies, chronic dermatological disorders, or acute otitis externa that is inadequately treated.

40 patients were included in this study. Culture of forty cases, twenty cases of otitis externa and twenty cases of chronic rhinosinusitis. All the blood samples, MacConkey Agar, are inoculated. Plates incubated overnight at 37?. After bacterial identification and storage. Quantitative determination of biofilm formation [9].

Exclusion criteria

• Patients that receive antibiotics 48 hrs. before the collection of samples.
• Patients that have clinical proof of fungal infection.
• Patients with mixed infection i.e., otitis media and otitis externa.

This technique has been used for all isolated bacterial pathogens. Overnight culture with fresh TSB was diluted at 1:100 in the tryptic Soya Broth (TSB). Three wells with a lid filled with 0.2 ml per bacterial suspension of a sterile 96-well flat-bottomed plastic tissue culture plate (Cellstar, greiner bio-one). Sterile broth with no bacteria was used for a negative control [10].

At 37°C, for 24 hours the plates have been covered and incubated aerobically. Following incubation, the contents of the wells have been aspirated and each well with 25 ml of sterile physiological saline was washed three times. For the removal of all non-adherent bacteria, the plate was vigorously shaken. The residual bacterial attached were fixed for fifteen min utilizing 0.2 ml of absolute methanol each well followed by elimination of methanol and air drying. With 0.2 ml of 0.2% Hucker crystal violet solution, the plates were stained for 5 min. With flowing tap water, the residual stain was rinsed.

Table 1: Distribution of the studied cases according to diagnosis (n=40).

Table 2: Distribution of the studied organisms for CRS cases (n=20).

The plates have been permitted to dry in the air. With 160 pl of 30% (v/v) glacial acetic acid, the dye attached to the adherent cells was resolubilized. Every well’s optical density (OD) was calculated at 590 nm that used an automated plate reader.

The cut-off OD (ODc-) was measured as 3 standard deviations above the negative control wells’ average OD.

Results were interpreted as follow [11]:

Table 3: Distribution of the studied organisms according to level of biofilm for CRS cases (n=20).

Table 4: Distribution of the studied organisms for OE ^ cases (n=22).

Table 5: Distribution of the studied organisms according to level of biofilm for OE cases (n=22).

Most common isolates from CRS patients were MRSA. Forty samples have been collected in Karmouz health and staphylococcus aureus representing 65% of isolates. insurance hospital collected between 11/2016 to 4/2017. Twenty cases of CRS and twenty cases of OE.In our study biofilm forming bacteria in CRS were foundin (11 of 20) about 55% using microtiter plate assay. Results shows different organisms in CRS and different organisms in OE with two cases with mixed infection. Another study, in the largest series to date [12], did not directly observe the presence of biofilms in patient samples but instead assessed the ability of bacteria recovered from CRS patients to grow biofilms in vitro using the Calgary biofilm assay. Of 157 samples obtained in a tertiary rhinology clinic, they noted a biofilm formation rate of 28.6%.

Speciation of the cultures showed that S. aureus was the most isolated organism (33%), but that 20% of patients had either purely pseudomonal infections or poly-microbial infections containing P. aeruginosa.

In a prospective study by Psaltis AJ, et al. (2007) 40 patients undergoing FESS for CRS had mucosal samples taken intraoperatively and were analyzed for mucosal biofilms using CLSM showing 50% of their study population with evidence of biofilms (20/40) [13].

In another study by used CLSM and FISH analysis to examine intraoperative samples taken from 18 patients with CRS and five controls undergoing septoplasty [14]. The analysis found 78% (14/18) of patients with detectable bacteria in a biofilm matrix.

Ferguson BJ, et al. (2005) used TEM in conjunction with bacterial cultures to show biofilms on 50% (2/4) of patient samples taken intraoperatively in presumed CRS, both of which grew out P. aeruginosa [6].

The other 2 patients were discovered to have a nonbacterial etiology to the CRS. Ramadan HH, et al. (2005) obtained intraoperative samples from the ethmoid bullae from five patients undergoing functional endoscopic sinus surgery (FESS), all of which showed morphological criteria of biofilms on SEM [15]. The mucosal surface of all specimens also showed various degrees of abnormality that ranged from disarrayed cilia to a complete absence of cilia and goblet cells.

A follow-up study by Sanclement JA, et al. (2005) observed biofilms in 80% (24/30) of patients [16].

The discrepancy in the above-mentioned results might exist or could be a result of the different detection methods used and/or differences in the patient populations studied. Furthermore, this inconsistency of data could be due to the fact that the collection of small samples was not representative of the entire sino-nasal cavity, Regardless of these discrepancies, the consistent demonstration of biofilms on the sino-mucosal samples of patients with CRS suggests that these complex structures might play a role in either the pathogenesis or persistence of chronic rhinosinusitis.

On the other hand, we will discuss biofilm in Otitis Externa. Using microtiter plate assay, most common isolates from OE patients were Pseudomonas 45% and Proteus 27% and staphylococcus Aureus 22.7%.

In another study, microbiological results for OE swabs shows that the incidences of Pseudomonas have varied from a low 12% to a high of 80% and the incidence of staphylococcus has been reported as low as 8.5% and as high as 29% [17,18].

In our study biofilm were identified in about 19 of 22 isolates representing 86.3% of chronic otitis externa patients. Biofilms were identified in 23 of the 25 patients (92%) with chronic otitis externa. On the contrary, in the acute otitis externa group, biofilms were isolated only in three cases (20%). The difference in frequency was significant from a statistical point of view. This evidence suggests that biofilm is a determining factor in the development and maintenance of chronic external otitis [19,20].

Table 6: Relation between level of biofilm and organism for CRS cases (n=20).

Table 7: Relation between level of biofilm and organism for OE cases (n=22).

Bacterial biofilms are highly ordered structures inside a protective extracellular matrix consisting of bacterial communities. One of the potential etiologies for the incidence and persistence of inflammation in CRS and OE is bacterial biofilms.

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