Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium capable of causing a wide array of infections, particularly in individuals with compromised immune systems. ^[1] These infections range from superficial skin and soft tissue infections to severe conditions such as pneumonia, urinary tract infections, and bacteremia. ^[2] The organism’s ability to adapt to diverse environments and its intrinsic resistance mechanisms make it a formidable pathogen in both community and healthcare settings. ^[3] 

The problem of multidrug resistance (MDR) has escalated significantly in recent years, with P. aeruginosa at the forefront. MDR strains exhibit resistance to three or more classes of antibiotics, severely limiting therapeutic options. ^[4] This resistance is mediated through various mechanisms, including efflux pumps, enzymatic degradation of antibiotics, and alterations in target sites. ^[5] Moreover, P. aeruginosa is known for its capacity to form biofilms—structured communities of bacteria encased in a self-produced extracellular polymeric matrix. ^[6] Biofilms enhance bacterial survival by providing a protective barrier against antibiotics and the host immune response, further complicating treatment outcomes. ^[7]

The clinical implications of MDR P. aeruginosa infections are profound. Patients with these infections often experience prolonged hospital stays, increased healthcare costs, and higher mortality rates. ^[8] Effective management of these infections requires a comprehensive understanding of the interplay between antibiotic resistance and biofilm formation. ^[9] This study aims to explore these aspects by analyzing the antibiotic susceptibility patterns and biofilm-forming abilities of MDR P. aeruginosa isolates obtained from clinical samples.

This is a prospective and observational study was conducted in the Department of Microbiology, Tertiary Care Teaching Center over a period of 1 year.

Bacterial Isolates

Clinical isolates of P. aeruginosa were collected from various sources, including blood, urine, and respiratory samples, from a tertiary care hospital. Sample collection spanned six months, ensuring a diverse and representative set of isolates. All isolates were identified based on colony morphology, Gram staining, and biochemical tests, including oxidase and citrate utilization tests.

Antibiotic Susceptibility Testing

The disk diffusion method was employed to determine susceptibility to antibiotics, including ceftazidime, ciprofloxacin, imipenem, and piperacillin-tazobactam, following Clinical and Laboratory Standards Institute (CLSI) guidelines. Zones of inhibition were measured and interpreted. In addition, the minimum inhibitory concentration (MIC) was determined for select isolates using the broth microdilution method to confirm resistance patterns.

Biofilm Formation Assay

Biofilm formation was quantified using a microtiter plate assay. Briefly: Overnight cultures of isolates were diluted 1:100 in tryptic soy broth (TSB) supplemented with 1% glucose. Aliquots (200 µL) were transferred into 96-well flat-bottom plates and incubated at 37°C for 24 hours. Wells were washed three times with phosphate-buffered saline (PBS) to remove planktonic cells. The adherent biofilm was stained with 0.1% crystal violet for 15 minutes. Excess stain was removed, and wells were washed with distilled water. The dye bound to the biofilm was solubilized using 33% acetic acid, and optical density (OD) was measured at 570 nm.

Statistical Analysis

All data were entered into SPSS software version 29 for analysis. Descriptive statistics, including means and standard deviations, were calculated. Inferential statistics, such as Pearson’s correlation coefficient and Chi-square tests, were used to evaluate the relationship between biofilm formation and antibiotic resistance. A p-value < 0.05 was considered statistically significant.

Table 1: Patient Age Distribution

Table 2: Gender Distribution

Table 3: Sources of Isolates

Table 4: Hospital Departments

Table 5: Associated Infections

Table 6: Risk Factors

Table 7: Summarizes the resistance rates observed among the 100 P. aeruginosa isolates

This table highlights the resistance rates of Pseudomonas aeruginosa isolates to key antibiotics. Ciprofloxacin exhibits the highest resistance (80%), followed by ceftazidime (70%), imipenem (60%), and piperacillin-tazobactam (50%). These results underline the significant challenge posed by MDR strains in clinical treatment, emphasizing the need for alternative therapeutic strategies.

Table 8: Isolates were categorized based on biofilm biomass

The isolates are categorized based on their biofilm-forming abilities. Strong biofilm-formers represent 40% of the isolates, followed by moderate formers at 35%, and weak/non-formers at 25%. This distribution demonstrates that a significant proportion of isolates have robust biofilm-forming capabilities, which likely contribute to their antibiotic resistance.

Table 9: Comparison of Biofilm Formation and Antibiotic Resistance

This table correlates biofilm-forming ability with resistance to ciprofloxacin, ceftazidime, and imipenem. Strong biofilm-formers have the highest resistance rates (e.g., 90% resistance to ciprofloxacin), while weak/non-formers exhibit the lowest resistance. This positive correlation indicates that biofilm formation significantly contributes to the resistance of P. aeruginosa isolates.

Table 10: Minimum Inhibitory Concentration (MIC) Distribution

The MIC data shows the range of concentrations required to inhibit the growth of resistant isolates. Ciprofloxacin exhibits the widest MIC range (4–64 µg/mL), reflecting its diminished efficacy. Ceftazidime and imipenem also show high resistance rates within their respective MIC ranges. These findings further reinforce the complexity of treating MDR infections.

Table 11: Biofilm Inhibition by Anti-Biofilm Agents

This table presents the effectiveness of agents like DNase, Dispersin B, and EDTA in reducing biofilm biomass. Dispersin B is the most effective, reducing biofilm OD by 70%, followed by EDTA (60%) and DNase (50%). These results suggest that biofilm-disrupting agents can enhance treatment strategies when combined with antibiotics.

Table 12: Statistical Analysis of Correlations

The statistical analysis demonstrates significant correlations between biofilm OD and resistance to ciprofloxacin (r = 0.68), ceftazidime (r = 0.72), and imipenem (r = 0.65), all with p-values < 0.05. These results confirm that higher biofilm-forming ability is associated with greater antibiotic resistance, providing a strong basis for targeting biofilm mechanisms in therapeutic interventions.

The findings reveal the formidable adaptability of MDR P. aeruginosa, combining high resistance to antibiotics with robust biofilm-forming capabilities. The observed positive correlation between biofilm formation and resistance highlights a critical challenge in clinical management. Strong biofilm formers exhibited resistance rates exceeding 90% for ciprofloxacin and ceftazidime, underscoring the protective role of biofilms in limiting antibiotic penetration and facilitating bacterial survival. ^[10]

In this study isolates are categorized based on their biofilm-forming abilities. Strong biofilm-formers represent 40% of the isolates, followed by moderate formers at 35%, and weak/non-formers at 25%. This distribution demonstrates that a significant proportion of isolates have robust biofilm-forming capabilities, which likely contribute to their antibiotic resistance.

Biofilm-associated resistance is a multifaceted phenomenon influenced by several factors, including the presence of an extracellular polymeric substance (EPS) matrix, altered metabolic states of cells within the biofilm, and the upregulation of efflux pumps. ^[11] The EPS matrix acts as a physical barrier, impeding the diffusion of antibiotics. Furthermore, cells embedded within biofilms often exhibit a dormant state, rendering them less susceptible to antibiotics that target active cellular processes. ^[12-15]

This study presents the effectiveness of agents like DNase, Dispersin B, and EDTA in reducing biofilm biomass. Dispersin B is the most effective, reducing biofilm OD by 70%, followed by EDTA (60%) and DNase (50%). These results suggest that biofilm-disrupting agents can enhance treatment strategies when combined with antibiotics.

The efficacy of anti-biofilm agents, such as Dispersin B and EDTA, demonstrated significant biofilm disruption, with reductions in biofilm biomass by up to 70%. These findings suggest that incorporating biofilm-disrupting strategies into treatment regimens could enhance the efficacy of conventional antibiotics. DNase, another agent tested, showed moderate effectiveness, indicating its potential as an adjunct therapy. ^[16]

In current study the MIC data shows the range of concentrations required to inhibit the growth of resistant isolates. Ciprofloxacin exhibits the widest MIC range (4–64 µg/mL), reflecting its diminished efficacy. Ceftazidime and imipenem also show high resistance rates within their respective MIC ranges. These findings further reinforce the complexity of treating MDR infections.

In this study effectiveness of agents like DNase, Dispersin B, and EDTA in reducing biofilm biomass. Dispersin B is the most effective, reducing biofilm OD by 70%, followed by EDTA (60%) and DNase (50%). These results suggest that biofilm-disrupting agents can enhance treatment strategies when combined with antibiotics.

Clinical implications of these findings are significant. The high prevalence of MDR strains coupled with robust biofilm formation necessitates a paradigm shift in treatment approaches. Strategies targeting both planktonic and biofilm-associated bacteria are imperative. ^[17] Combination therapies involving antibiotics and biofilm disruptors, as well as the development of novel agents targeting biofilm-specific pathways, could pave the way for more effective management of MDR P. aeruginosa infections. ^[18,19]

Further research should focus on unraveling the molecular mechanisms underlying biofilm formation and resistance in MDR strains. Investigating quorum sensing inhibitors, which disrupt bacterial communication and biofilm formation, represents a promising avenue for future therapeutic development. Additionally, exploring host immune modulators that enhance the clearance of biofilm-associated infections could provide synergistic benefits

MDR P. aeruginosa isolates demonstrate extensive antibiotic resistance and significant biofilm-forming potential. Addressing these challenges necessitates innovative therapeutic approaches and a deeper understanding of resistance mechanisms. Results indicated a high resistance to commonly used antibiotics, with a significant proportion of isolates demonstrating strong biofilm-forming ability. The findings underscore the critical need for novel therapeutic strategies to combat MDR P. aeruginosa infections.

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