Acinetobacter species are significant gram-negative opportunistic bacteria that have played a major role in hospital acquired illnesses, particularly in ICUs. There is a significant risk to safety of the patient and therapeutic outcomes due to the rising incidence of Acinetobacter baumannii infections in ICUs.[1]

Acinetobacter baumannii is notorious and have the ability to form biofilm, which not only raises the microbe’s survival in hospital environments but also contributes to its resistance to different antibiotics, making treatment more challenging and raising the rates of morbidity and death in patients who are seriously ill. By enhancing the organism’s resistance to antimicrobial therapies and enabling its persistence in hospital environments, this biofilm formation complicates therapy alternatives and increases illness and fatality rates in affected patients.[2,3]

Acinetobacter baumannii infections are particularly concerning due to their association with antibiotic resistance. Acinetobacter baumannii that is resistant to carbapenem has been identified by the WHO as a significant microbes that poses an urgent worldwide danger.[4]

Given the crucial role that Acinetobacter biofilms play in the development of infections and their link to multidrug resistance, it is important to investigate the generation of biofilms among Acinetobacter species, particularly those isolated from ICUs.[5]

According to research, a significant percentage of Acinetobacter baumannii strains isolated from the patients of intensive care unit which showing the strong biofilm production capacity, in fact some studies have reported that up to 98% of strains are capable of forming biofilms in clinical settings.[6]

Biofilms can develop on a variety of hospital surfaces, including sewage systems, medical equipment, and areas that are regularly touched. Biofilms are considered to be responsible for around 80% of chronic infections and over 65% of nosocomial illnesses.[7]

Treatment failures and chronic infections may arise from biofilms protecting bacterial cells from the effects of antibiotics. Research indicates that Acinetobacter baumannii in biofilm can exhibit resistance levels up to 1000 times higher than those of their planktonic counterparts.[8]

The investigation was carried out in the bacteriology section of the Department of Microbiology at TMMC and RC Moradabad, Uttar Pradesh, India, with clearance from the college’s CRC and IEC. The research was conducted on 97 clinical isolates of Acinetobacter spp. that were collected from different clinical sources.

Clinical samples from all patients older than eighteen who were admitted to ICU’s fulfilled the inclusion criteria were included in this study and exclusion criteria is patients denying consent, and non-ICU patients were not included.

The samples were grown in MacConkey and CLED agar according to the inclusion requirements. After 24 to 48 hours, growth was determined, gram staining was performed, and biochemical characteristics for gram-negative bacteria were determined in order to identify the species. biochemical test such as Oxidase, Catalase, Indole, Methyl Red, Triple Sugar Iron test, Citrate, Urease, and Oxidative Fermentative test were used to further identify the samples.

In accordance with CLSI guidelines 2023, antibiotic sensitivity to twelve therapeutically important antibiotics were determined using the Kirby-Bauer disc diffusion method and an automated system (VITEK-2 COMPACT).

The antibiotic discs were as follows: Ampicillin/Sulbactum(A/S), Cefotaxime (CTX), Ceftazidime (CAZ), Co-trimoxazole (COT), Gentamicin (GEN), Levofloxacin (LE), Amikacin (AK), Cefepime (CPM), Imipenem (IPM), Meropenem (MRP), Colistin (CL) and Polymyxin-B(PB). To produce biofilms, the Congo Red Agar process was used.[9-13]

Statistical analysis

When appropriate the data were submitted to Chi-square tests. Data interpretation was done through the use of SPSS software while through Excel 7.0 software, graph production was made easy.

The Institutional Ethical Committee at TMU  Moradabad  provided the ethical clearance Ref. No. TMU/IEC/2024-25/PG/130.

After the patient agrees then we fill out the informed consent form.

A total number of 97 isolates were incorporated in the present investigation, all were accounted for positive Acinetobacter spp. Isolation. There were 97 distinct clinical samples in all, with 48 (49.48%) coming from male and 49 (50.51%) from female With 20 cases, the age range highly impacted was 51-60 years old (20.61).

Table 1: Gender distribution by age group within the study population

Table 2: Sensitivity Pattern of Acinetobacter baumannii complex and Acinetobacter lwoffii

We found that the Acinetobacter baumannii Complex showed an antibiotic resistance pattern to CTX (84.52%), COT (83.33%), GEN (77.38%), and IPM (75%). This study found that one Acinetobacter baumannii was significantly susceptible to AMP (57.14%).

Table 3: Biofilm production distribution in Acinetobacter species

Biofilm production was positive in 58 (59.79%) of the 97 isolates, and high in 27 (27.83%) of them. When tested with the Congo red test, 39 (40.20%) isolates produced no biofilm

Table 4: Distribution of isolates resistance to Acinetobacter baumannii

Biofilm producers showed the greatest resistance to Ampicillin/Sulbactam (80%) and Amikacin (79.16%), followed by Cefepime (65.62%) and Meropenem (69.84%).

Studying biofilm production among Acinetobacter species isolated from ICUs and its correlation with MDR at TMU Hospital in Moradabad, Uttar Pradesh, was the goal of the current study.

Out of the 97 samples, the study group found that males (50.51%) were more likely to be impacted than females (49.48%). In their 2019 survey, Murugesh K et al. discovered that 54.45% of the population was male.[14] Females had a greater prevalence (54.20%), according to Rebic V et al. (2018).[15]

The maximum number of strains (20.61%) isolated from the age group of 51-60 years. This finding was similar to other researcher finding like as Gupta et al. (2015), reported the infections were most prevalent in people over 50 years, then in people ages 0-10.[16] In their study, Rani et al. (2022) discovered that the majority of  isolates came from the elderly population, with 22.58% of them being in the 61-70 age range.[17]

We found that the Acinetobacter baumannii Complex exhibited an antibiotic resistance pattern to CTX (84.52%), COT (83.33%), GEN (77.38%), and IPM (75%).Taneja N et al. (2011) found that Acinetobacter was resistant to GEN (79.5%). [18]

Similarly, Mostofi et al. (2011) demonstrated that strains of Acinetobacter baumannii were resistance to IPM (76%) and GEN (61%) in their study.[19] Rajkumari S. et al. (2020) did, however, report a substantial amount of resistance to AMP 100%.[20] In this investigation, Acinetobacter baumannii exhibited a moderate level of AMP sensitivity (57.14%).

A significant percentage of Acinetobacter baumannii isolates have been shown to be capable of producing biofilms. Out of the 97 isolates that were used to test for biofilm production, 58 (59.79%) had showing biofilm production, and 27 (27.83%) had high biofilm production. 39 strains (40.20%) tested negative for biofilm production using the Congo red test. In contrast to Acinetobacter Lwoffii 4 (4.12%), Acinetobacter baumannii Complex 23 (23.71%) had the greatest number of robust biofilm forms based on isolates. According to Dheepa et al. (2011), biofilm was generated by 60% of their 50 isolates of  Acinetobacter baumannii.[21] But according to Jeetendra Gurung et al. research, 50% of Acinetobacter baumannii can produce biofilm.[22]

Acinetobacter baumannii biofilm generation has been linked to multidrug resistance in various investigations. Ampicillin/Sulbactam (80%) and Amikacin  (79.16%) were the most  resistant  drugs that biofilm producers showed, followed by Co-Meropenem (69.84%) and Cefepime  (65.62%). Even 100% resistance to several antibiotics drugs has been documented in biofilm- forming  Acinetobacter species by Nahar et al. (2012).[23]

Our study highlights the pivotal biofilm formation’s function in the persistence of MDR of Acinetobacter species within ICU settings. MDR strains demonstrated significantly elevated antibiotic tolerance within biofilms. This dichotomy suggests that biofilms serve as a transient survival strategy for less-resistant isolates, whereas MDR strains exploit biofilms to amplify existing resistance traits.

LIMITATIONS

• Due to the lack of resources and time biofilm production cannot be identified by molecular methods in this study.
• The best technique for figuring out the minimum inhibitory concentration of colistin, which was not employed in this investigation, is broth microdilution (BMD) in conjunction with colistin antibiotics sensitivity testing (AST).

According to studies on Acinetobacter species isolated from intensive care units, biofilm development and multidrug resistance are significantly correlated. Multidrug resistance is significantly linked to studies on Acinetobacter species biofilm formation in intensive care units. These bacteria’s capacity to create protective biofilms makes them highly harmful in clinical settings.

This study establishes a strong relationship between biofilm production and antibiotic resistant in ICU’s  Acinetobacter isolates. While non-MDR strains depend on biofilms for transient tolerance, MDR strains leverage biofilms to amplify resistance. Generally speaking, understanding the connection between antibiotic resistance and biofilm development is crucial to developing efficient treatment plans and managing infections brought on by these opportunistic microorganisms. Future work should explore transcriptional regulation of biofilm genes and combinatorial therapies targeting biofilm disruption.

1. Howard A, O’Donoghue M, Feeney A, Sleator RD. Acinetobacter baumannii. Virulence. 2012 May;3(3):243–50.
2. Sharma M, Sapkota J, Jha B, Mishra B, Bhatt CP. Biofilm Formation and Extended-Spectrum Beta-Lactamase Producer among Acinetobacter Species Isolated in a Tertiary Care Hospital: A Descriptive Cross-sectional Study. J Nepal Med Assoc. 2019 ;57(220):424-428. (PMID: 32335654)
3. Pattanaik A, Banashankari GS. Characterisation of Acinetobacter with special reference to carbapenem resistance and biofilm formation. Trop J Path Micro. 2019;5(6):386-95.
4. Huang L, Tang J, Tian G, Tao H, Li Z. Risk Factors, Outcomes, and Predictions of Extensively Drug-Resistant Acinetobacter baumannii Nosocomial Infections in Patients with Nervous System Diseases. Infection and Drug Resistance. 2023 Nov 22;16:7327–37.
5. Authman SH, Ali FS, Al-Marjani MF. Biofilm formation in imipenem-resistant Acinetobacter baumannii from the intensive care unit. J Glob Pharma Technol. 2017; 10:404-11
6. Amin M, Pai V, Qayoom S, Arshi S, Khurshid S. Biofilm formation and multidrug resistance in nosocomial isolates of Acinetobacter.Indian J. Microbial. Res. 2018;5(3):425-29.
7. Assefa M, Amare A. Biofilm-Associated Multi-Drug Resistance in Hospital-Acquired Infections: A Review. Infection and Drug Resistance. 2022 Aug;Volume 15:5061–8.
8. Mendes S, Combo SI, Allain T, Domingues S, Buret AG, Silva G. Co-regulation of biofilm formation and antimicrobial resistance in Acinetobacter baumannii: from mechanisms to therapeutic strategies. European Journal of Clinical Microbiology & Infectious Diseases. 2023 Oct 28;
9. Kulkarni SS, Madalgi R, Ajantha GS, Kulkarni RD. Identification of genus Acinetobacter: Standardization of in-house PCR and its comparison with conventional phenotypic methods. Journal of Laboratory Physicians. 2017 Oct;9(04):279–82.
10. Hudzicki J. Kirby-Bauer Disk Diffusion Susceptibility Test Protocol. American Society for Microbiology. American Society for Microbiology; 2009 Dec.
11. M100 Performance Standards for Antimicrobial Susceptibility Testing A CLSI supplement for global application. Available from: https://clsi.org/media/tc4b1paf/m10033_samplepages-1.pdf
12. VITEK® 2 COMPACT. bioMérieux Website. 2021 Available from: https://www.biomerieux.com/tr/en/our-offer/clinical-products/vitek-2-compact.html
13. JCDR - Antibiotic resistance, Congo red agar method, Tissue culture plate method, Tube method. Jcdr.net. 2018
14. Murugesh K, Naik TB, Ravindranath C. Antibiotic susceptibility profile of Acinetobacter isolates from various clinical specimens at a tertiary care hospital in South Karnataka. Indian J Microbiol Res. 2019; 6(4): 280-283.
15. Rebic V, Masic N, Teskeredzic S, Aljicevic M, Abduzaimovic A, Rebic D. The Importance of Acinetobacter Species in the Hospital Environment. Medical Archives. 2018;72(3):330.
16. Gupta N, Gandham N, Jadhav S, Mishra RN. Isolation and identification of Acinetobacter species with reference to antibiotic resistance. J Nat Sc Biol Med. 2015 January; 6(1): 159-162.
17. Rani C, Sharma SR, Farooq U, Singh S, Sharma V, Ahamad I. Isolation of Acinetobacter and its antimicrobial resistance pattern in all lower respiratory samples from ICU. IP Int J Med Microbiol Trop Dis. 2022; 8(2): 118-122
18. Taneja N, Singh G, Singh M, Sharma M. Emergence of tigecycline & colistin resistant Acinetobacter baumannii in patients with complicated urinary tract infections in north India. The Indian Journal of Medical Research. 2011;133(6):681.
19. Sepideh Mostofi. Multi-drug resistance in Acinetobacter baumannii strains isolated from the clinical specimens of three hospitals in Tehran-Iran. African Journal of Microbiology Research. 2011 Nov 16;5(26).
20. Rajkumari S, Pradhan S, Sharma D, Jha B. Prevalence and antibiogram of Acinetobacter species isolated from various clinical samples in a tertiary care hospital. JCMS Nepal. 2020; 16(1): 26-32.
21. Dheepa M. Comparision of biofilm production and multiple drug resistance in clinical isolates of Acinetobacter baumannii from a tertiary care hospital in South India. Int J Pharm Biomed Sci, 2 (4) (2011), pp. 103-107
22. Bhattacharyya P, Gurung J, Khyriem AB, Banik A, Lyngdoh WV, Choudhury B. Association of biofilm production with multidrug resistance among clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa from intensive care unit. Indian Journal of Critical Care Medicine. 2013;17(4):214–8.
23. Nahar A, Anwar S, Saleh AA, et al. Virulence Factors and Antibiotic Susceptibility Pattern of Acinetobacter Species in a Tertiary Care Hospital in Bangladesh. Ibrahim Medical College Journal 2012; 6(1): 27-30.