European Journal of Cardiovascular Medicine

Pseudomonas aeruginosa is a ubiquitous, Gram-negative bacterium associated with nosocomial infections such as ventilator-associated pneumonia, urinary tract infections, and bloodstream infections.1 Its ability to form biofilms on both biotic and abiotic surfaces contributes to its persistence and resistance to antibiotics.2 Biofilms are structured microbial communities encased in a self-produced extracellular polymeric substance, which impedes antibiotic penetration and facilitates resistance mechanisms.3

Understanding the relationship between biofilm formation and antimicrobial susceptibility in P. aeruginosa is critical for effective infection control and management. This study investigates the biofilm-forming capacity of P. aeruginosa isolated from clinical specimens and its impact on antimicrobial resistance profiles in a tertiary care hospital setting.

A cross-sectional study, conducted at Microbiology laboratory in the Department of Microbiology, GMC, Kota from January 2023 to December 2023, included 225 samples of isolated P.aeruginosa invarious clinical samples.

INCLUSION CRITERIA- only isolates of P. aeruginosa from various clinical sample, patients above 18 years of age and male, female and transgender were included.

METHODS

Bacterial identification

All isolated samples were sub-cultured onnutrientagar for pure isolation which were processed for appropriate phenotypic characterization based onmorphology, culture and further tested by conventionalbiochemical tests including catalase test, oxidase test, OF test,lactose fermentation testandpigment production test and growth at 42°C leading to identification as P. aeruginosa.

Antibiotic susceptibility test-

A routine antibiotic susceptibility test was performed   for P.aeruginosa by Kirby-Bauer disk diffusion method.0.5 McFarland standard was used to compare the inoculum turbidity. The inoculated plates were incubated overnight at 37◦c.Various antibiotics such as Amikacin-AK 30mcg, Gentamicin-GEN 10mcg, Piperacillin75mcg /tazobactam 10mcg -PIT, Ciprofloxacin-CIP 5mcg, Imipenem-IPM10 mcg, Cefepime-CPM 30mcg, Aztreonam-AT 30mcg, Ceftazidime-CAZ 30mcg, Meropenem-MRP 10mcg and Amoxicillin clavulanic acid-AMC 30mcg were used. The results were recorded by measuring the inhibition zone as sensitive(S), intermediate(I) and resistant(R), as per CLSI 2022 guidelines.4 As a quality control, we were used P.aeruginosaATCC 27853.

Detection of Biofilm Productionby tissue culture plate method5-Organism isolated from fresh agar plates was inoculated in 10 mLTSB (Trypticase soya broth) with 1% glucose and incubated at 370C for 24 h. The culture was diluted 1:100 with fresh medium. 96 well flat bottom tissue culture plates were filled with 0.2 mL of diluted cultures individually. Uninoculated TSB was used as a control for sterility and non-specific binding of media. The plates were then incubated at 370C for 18-28h.After incubation, the plates were gently tapped for the removal of unbound content. The wells were then washed with 0.2mL of phosphate buffer saline (Ph7.4) four times. Biofilm formed by bacteria adherent to the wells was fixed by 2% sodium acetate and stained by crystal violet (0.1%). Excess stain was removed by using deionized water and plates were kept for drying. Optical density (OD) of stained adherent biofilm was obtained by using microELISA auto reader at wavelength 490 nm. These OD values was considered as an index of bacteria adhering to surface and forming biofilm.

1. Mean OD =< 0.120 (Non\Weak Biofilm production)
2. Mean OD = 0.120-0.240 (Moderate biofilm production)
3. Mean OD=>0.240 (Strong Biofilm production)

Image 1-Biofilm formation by Microtiter plate method

During the 1year period of study from January 2023 to December 2023, 225 isolates of P. aeruginosa were collected from various clinical samples (Table2). The phenotypic identification of the P. aeruginosa isolates was performed by bacteriological methods (Gram’s staining, colony morphology, and biochemical tests) using standard methodology. Out of 225 sample, 155 (68.88%) were from males and 70 (31.11%) were from females (Table1).Ps.aeruginosamost commonly isolated from  59-69 age group.

The maximum number of isolates were obtained from Sputum68 (30%), followed by Urine 59 (26%), pus57 (25%), Body fluids 26 (12%), and Endotracheal tube15 (7%). In the present study, P. aeruginosa showed resistance against most of thecommonly used antibiotics (Table 2). Out of 225 isolates, 110 (48.88%) of P. aeruginosa isolates were identified as MDR and 115 (51.11%) of isolates were susceptible to most commonly used antibiotics (Table 3). All 225 isolates were tested for biofilm production by MTPM. Among the MDR isolates, 53 (48.1%) were biofilm producers and 57 (51.8%) were biofilm non-producers.MDRisolates showed maximum positivity for biofilm formation in MTPM. There was a significant relationship between biofilm production and MDR.  Among 115 antibiotic susceptible isolates, 9 (7.82%) were biofilm producers and 106 (92.2%) were biofilm non-producers. There was a significant relation between biofilm production and resistance pattern of the isolates. Biofilm producing isolateswere more resistance as compared to non-biofilm producers (Tables 3 and, Figure 3).

This study shows that the antimicrobial susceptibility testing should be done for all the samples to the appropriate antibiotics to treat patients with P. aeruginosa infections.

Table 1: Age and sex wise biofilm production in isolated Pseudomonas aeruginosa (n=225)

Figure-1 Age and sex wise biofilm production in isolated Pseudomonas aeruginosa (n=225)

Table 2: Clinical samples showed biofilm formation in isolated Pseudomonas aeruginosa (n=225)

Table3: Antimicrobial resistance pattern of P. aeruginosa among biofilm producers and non-producers

Table 4: Association between multi drug resistant isolates and biofilm producers

MDR: Multidrug‑resistant

Pseudomonas aeruginosa is a significant opportunistic pathogen responsible for a variety of infections, particularly in immunocompromised individuals. Its ability to form biofilms and exhibit antimicrobial resistance makes it a critical focus of clinical and microbiological research.6

In the present study, 225 P. aeruginosa were isolated from various clinical samples. The maximum number of isolates were obtained from Sputum 68 (30%), followed by Urine 59 (26%), pus 57 (25%), Body fluids 26 (12%), and Endotracheal tube 15 (7%). Similar result was found by Karthic A et al. and Rodrigues et al.7,8\

Gender distribution shows male (66.2%) predominance over female (33.8%) in this study. Similar observation was made in other studies that reported a slight male preponderance, Golia S, Kamali E, Swapna M9,10,11 whereas Anil and Shahid have reported slight predominance of female patients 80 (55.17%) over males 65 (44.83%).12

Present study found that Pseudomonas aeruginosa infection mainly occurred in population aging between 59-68 years (27%). Similar results were observed by Shrestha R et al and Chandel et al in which maximum incidence (31%) was among patients >60 yrs of age.13,14 This can be explained by the fact that the patients in these age groups have decreased immunity, presence of other co-morbid conditions, prolonged hospitalization with indwelling medical devices, making them vulnerable to develop biofilm related infections.

In this study, out of a total of 225 isolates, the antibiotic susceptibility pattern of P. aeruginosa revealed that 110 (48.9%) isolates were MDR while 115 (51.1%) were sensitive to commonly used antibiotics. These results are similar with the study conducted by Ijaz et al.,15 which showed resistance pattern in 119 (58.6%) isolates. Contrarily, Obritsh et al,16was of the view that rates of isolation of MDRPA varied between 0.6- 32 and this variation was dependent upon the geographical location and the type of study. The higher resistance rates noted in our study may be because the organism in our study was isolated not only from one specimen but from others specimens like sputum, urine,pus, body fluid and ET.

In the present study, among a total of 225 isolates of P. aeruginosa, 62 (27.6%) were biofilm-producers and this finding is comparable with a study done by Shrestha et al 13and Kulkarni DM et al17 who showed (33%), (26.6%) but in contrast with the study done by Neopane et al which showed higher rate of biofilm production (83.33%).

In the present study, among 110MDR isolates, 53(48.2%) were biofilm producers and among 115 sensitive isolates, only 9(7.82%) were biofilm producers while majority of them are non-biofilm producers 106(92.12%). Biofilms create a microenvironment that enhances survival under antimicrobial pressure, contributing to the persistence of infections.18

In the present study, it is evident that Biofilm production significantly impacts expression of MDR in P. aeruginosa. Biofilm enhances the bacterial ability to establish as well as maintain the infection by protecting the microorganisms from host immune system and acting as a shield against antimicrobial agent. Early detection of biofilm production and its correlation with MDR will help the physician for proper treatment thus will prevent the further development of resistance and chronicity of infection.

Financial support and sponsorship: Nil.

Conflicts of interest: There are no conflicts of interest.

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