Materials and Methods
Study design and subjects
This cross-sectional study was conducted in the Department of Pulmonary Medicine and Microbiology Department of a tertiary care hospital, Lucknow, between January 2011 and May 2012. Two hundred and fifty patients from indoor and outdoor patient departments of pulmonary medicine, with confirmed diagnoses of lower respiratory tract infections and who did not receive either of the antibiotics in previous 72 hrs, patients of ages ≥ 18 years with symptoms which were suggestive of LRTI, following fever (>100F), cough, production of sputum, leukocytosis (>12000 wbc/mm3 or >15% bands) who gave written informed consents for their participation and their sputum samples for examination, were enrolled for the study. The study was approved by the institutional ethics committee.
Sample collection and processing
Sputum samples were collected in sterile, wide mouthed containers and then transferred to Microbiology Laboratory for further processing. Samples were cultured onto Pseudomonas isolation agar plates (Hi-media). Colonies with an appropriate colonial morphologies were classified presumptively as P. aeruginosa and they were further identified by conventional biochemical tests [6]. Antimicrobial susceptibility was done by Kirby Bauer disk diffusion method as per Clinical Laboratory Standard Institute (CLSI) 2010 guidelines [7]. P. aeruginosa isolates were stored in 1% nutrient agar stabs at 4°C for doing further analyses.
Qualitative and Quantitative biofilm formation assays
1. Tube method (TM)
The qualitative assay for biofilm formation was noted according to the method which was described by Christensen et al., [5].
2. (a) Tissue culture plate (TCP) method
(b) Modified Tissue culture plate (MTCP) method
Both these tests were performed as has been described by Dheepa et al., [8]. The reading was performed two times:
Before addition of glacial acetic acid, as in standard microtitre-plate test and
After glacial acetic acid was added, as in modified microtitre-plate test.
The wells were then treated with 160 μL of 33% glacial acetic acid for 15 min at room temperature, to solubilize the dried crystal violet which was adherent to any biofilms.
P. aeruginosa PAO1 (Biofilm producing) and P. aeruginosa ATCC 27853 (non-biofilm-producing) were used as controls.
For the purpose of doing a comparative analysis of test results, the adherence capabilities of the test strains were classified into four categories: non-adherent (0), weakly adherant (+), moderately adherant (++), or strongly (+++) adherent, based upon the ODs of bacterial films. The cut-off optical density (OD) for a tissue culture-plate is defined as three standard deviations above the mean OD of the negative control. Strains were classified [Table/Fig-1].
Classification of bacterial adherence by microtitre plate method
| OD values of Tissue Culture Plate method | OD values of modified Tissue Culture Plate method |
|---|
| Mean OD value | Adherence | Biofilm formation | Mean OD value | Adherence | Biofilm formation |
| <0.062 | Non | Non | <1.33 | Non | Non |
| 0.062-0.124 | Weak | Weak | 1.33-2.66 | Weak | Weak |
| 0.124-0.248 | Moderate | Moderate | 2.66-5.32 | Moderate | Moderate |
| >0.0248 | Strong | Strong | >5.32 | Strong | Strong |
For all P. aeruginosa isolates, the strength of biofilm formation was calculated as per the given formula.
Average OD value Biofilm production
OD ≤ ODc Non-adherent
ODc<OD ≤ 2 × ODc Weakly adherent
2×ODc <OD ≤ 4 × ODc Moderately adherent
4 × ODc < OD Strongly adherent
Optical density cut-off value (ODc) = average OD of negative control + 3x standard deviation (SD) of negative control.
Statistical Analysis
The results have been presented in means (±sd) and percentages. The Mann-Whitney U-test was used to compare the continuous variables. The sensitivity, specificity, positive and negative predictive and accuracy values of the tests were calculated. A p-value of <0.05 was considered as significant. STATA, version 8.0 was used for all the analysis [Table/Fig-2].
Spectrophometric assay of bioflim formation among isolates of P. aeruginosa under two experimental conditions
| Experimental condition | OD570 Mean+ SD | No.(%) biofilm producers | No. (%) Strong biofilm producer isolates | No. (%) Weak/non biofilm Producer isolates | p-value |
|---|
| With GAA | 1.06±0.71 | 80 | 19 (23.6) | 61 (76.4) | p=0.0001* |
| Without GAA | 0.18±0.17 | 80 | 16 (20) | 64 (80) |
| p-value | 0.001* | | 0.0001** | | Moderate |
For the data calculation, we did classification of bacterial adherence, based on three standard deviations above the mean OD of the negative control [8].
Results
Out of 250 sputum samples, 80 were confirmed as P. aeruginosa isolates. Biofilm productions assessed by qualitative tube method revealed 16 (20%) strong biofilm producers, 17 (21.25%) moderate producers, and 47 (58.75%) weak or non-biofilm producers [Table/Fig-3].
Overall results of the isolates for biofilm in TM and MTCP test
| Biofim foramtion | TM (%) | TCP(%) | MTCP(%) |
|---|
| No of isolates (80) | High | 16(20%) | 5(6.25%) | 7 (8.75 %) |
| Moderate | 17(21.25%) | 11(13.75%) | 12(15%) |
| Weak | 29(36.25%) | 48(60%) | 30(37.5%) |
| None | 18(22.5%) | 16(20%) | 31(38.75%) |
Quantitative biofilm formation assay done on 80 isolates of P. aeruginosa showed that all isolates were better biofilm producers (OD was more than 0.0248) in presence of glacial acetic acid than in absence of glacial acetic acid.
The overall percentage of resistance which was observed among all the P. aeruginosa isolates, including those for biofilm positive and biofilm negative isolates and that for resistance to 14 antibiotics which were tested, has been given in [Table/Fig-4]. This table shows that the resistance to antibiotics was more for most of the biofilm positive isolates as compared to that for the biofilm negative isolates. Isolates showed maximum resistance to amoxyclav (97%), followed by ampicillin (95%) and levofloxacin (74%).
Antibiotic susceptibility results (percentage) of P. aeruginosa isolates
| S. No. | Resistance |
|---|
| Antibiotics | Biofilm positive Isolate (n=44) | Biofilm negative Isolates (n=36) | Resistance of all isolates (n=80) |
|---|
| 1 | Ampicillin | 90.9 | 100 | 95 |
| 2 | Amikacin | 31.8 | 33.3 | 33 |
| 3 | Amoxiclav | 93.1 | 97.2 | 97 |
| 4 | Aztreonam | 43.1 | 41.6 | 43 |
| 5 | Ciprofloxacin | 25 | 25 | 25 |
| 6 | Ceftazidime | 70.4 | 55.6 | 64 |
| 7 | Cefepime | 36.3 | 36.1 | 37 |
| 8 | Ceftriaxone | 63.6 | 69.4 | 67 |
| 9 | Gentamicin | 47.7 | 44.4 | 47 |
| 10 | Imipenem | 6.81 | 0 | 4 |
| 11 | Meropenem | 47.7 | 38.8 | 44 |
| 12 | Pipercillin/ Tazobactum | 15.9 | 13.8 | 15 |
| 13 | Tobramycin | 29.5 | 33.3 | 32 |
| 14 | Levofloxacin | 79.5 | 66.6 | 74 |
Mean level of OD570 was significantly (p=0.001) higher among isolates with GAA (1.06±0.71) than in those without GAA (0.18±0.17). The percentage of strong biofilm producer isolates was 23.6% with GAA and it was 20% without GAA. However, the percentage of weak/non biofilm producer isolates was 76.4% with GAA and it was 80% without GAA. The differences were statistically significant (p=0.0001). Sensitivity and specificity of TM were 84.3% and 58.6% respectively, whereas for MTCP, both these parameters had higher values: 88.6% and 63.9% respectively [Table/Fig-5].
Diagnostic parameters of TM and MTCP method for biofilm detection
| Screening method | Sensitivity | Specificity | Positive predictive value (%) | Negative predictive value (%) | Accuracy (%) |
|---|
| TM | 84.3 | 58.6 | 78.2 | 68.0 | 75 |
| MTCP | 88.6 | 63.9 | 75.0 | 82.1 | 77.5 |
Discussion
Multidrug-resistant P. aeruginosa has been reported worldwide and it has now been recognized to cause one of the healthcare-associated infections which are most difficult to control and treat. We evaluated biofilm formation capability of originate from the sputum of the patients of LRTI.
In the TCP method, the number of isolates which showed strong biofilm formations was 5 (6.25%) and weak biofilm producers were 48(60%).
Our findings differed from those of Hassan et al., who reported that the TCP method had shown the biofilm formation to be 70 (64.7%), and non or weak biofilm producers to be 40 (36.3%)[9].In our study, biofilm producers showed increased resistance to levofloxacin (79.5%), followed by ceftazidime (70.4%) and meropenem (47.7%) and non-biofilm producers showed increased resistance only for ampicillin and ceftriaxone. A recent study showed significantly higher resistance to ceftazidime, cefepime and pipercillin [8].
By modified TCP method, strong biofilm procedures were found to be 7(8.75%), 12(15%) were moderate producers and 30 (37.5%) isolates were weak biofilm producers. Biofilm production, studied particularly by doing a qualitative biofilm assay by tube method, showed only 20% isolates to be strong biofilm producers. Thus, TM cannot be recommended as a general screening test to identify biofilm-producing isolates [5,10]. Looking simply at percentages, we could arrive at conclusion that TM was the most effective method, but after doing extensive statistical analysis, it was found that modified tissue culture plate method with glacial acetic acid had better sensitivity and specificity as compared to TM.
Our data indicated that MTCP method was an accurate and a reproducible method which could be used for biofilm detection and that this technique could serve as a reliable quantitative tool for determining biofilm formation.
Conclusion
We can conclude from given study, that most of the P. aeruginosa isolates obtained from sputum samples of LRTI patients in vitro, had capacities to produce biofilms. MTCP method was found to be the most quantitative, reliable and accurate screening method as compared to TM, for studying biofilm formations.