European Journal of Cardiovascular Medicine

Acinetobacter seems to be one of the most resilient pathogens due to its increasing prevalence in hospitals, its resistance to treatment and its association with elevated mortality rates. Being a potent nosocomial pathogen, Acinetobacter tends to persist for months on clothing, bedclothes, bed rails, ventilators, and other environmental surface such as sinks and doorknobs [1]. The factors which contribute to its virulence include outer membrane proteins (OMPs)[2] and biofilm formation that allows the bacteria to grow in unfavourable conditions with the help of the protein called BAP (biofilm associated protein) [3]. Biofilm formation is a major factor in pathogenesis of Acinetobacter as it protects the organism from the action of hosts immune system as well as from antimicrobial agents [4]. In view of increasing prevalence and evolution of multi drug resistant Acinetobacter isolates as potential nosocomial bug with limited treatment options, further research in this field is call of the hour.

A cross-sectional study was conducted in the Department of Microbiology at GMC, Kota from January 2023 to December 2023 which included 100 samples received from adult ICU patients.

Ethical clearance:

This study protocol was proceeded after the approval from Institutional Ethical Clearance Committee and Research Review Board [F.3() NO.62/dated 15/12/22].

Inclusion criteria:

Patients above 18 yrs of age, both male & female which were admitted in various ICUs, with or without prolonged catheterization, prosthesis, shunts etc. were included.

Exclusion criteria:

Patients below 18yrs of age and not admitted in ICU were excluded.

Sample processing:

The samples collected were processed according to the standard procedures which included direct microscopic examination of Gram-stained smear and inoculation of samples on MacConkey agar and blood agar which were then incubated aerobically at 37℃for 18-24hrs.After preliminary identification of growth, isolates were subjected to Gram staining and conventional biochemical tests for further identification. Antimicrobial susceptibility testing was done by Kirby-Bauer Disc diffusion method as per CLSI(M100ED32) guidelines. Isolates resistant to Cefotaxime (CTX) were further tested for confirmation of ESBL production by Combined disc diffusion test. Biofilm formation was detected by Microtiter plate method.

Combined disc diffusion test [5]: Principle:

The activity of extended spectrum Cephalosporins against ESBL producing organisms is enhanced by the presence of Clavulanic acid. Procedure: Prepared a standard suspension (0.5 McFarland standards) of Cefotaxime resistant Acinetobacter isolates and made a lawn culture on MHA (Muller- Hinton agar) plate. Then paired discs of Ceftazidime(30µg) and Cefotaxime were each placed at distance of 20mm (center to center) from Combined discs of Ceftazidime-clavulanate (30/10 µg) & Cefotaxime-clavulanate (30/10 µg) respectively and incubated aerobically at 37℃ for 18-24hrs. Interpretation: ≥ 5mm increase in the zone of inhibition of the Ceftazidime or Cefotaxime used in combination with Clavulanate than the antibiotic used alone is considered positive.

Microtiter plate method [6]:

Each isolate was grown overnight in trypticase soy broth (TSB) with 0.25% glucose at 37^oC (total 31 isolates). The overnight growth was diluted in a ratio of 1:40 in TSB with 0.25 % glucose. 200 µl of diluted suspension of each isolate (total 31) was then poured in a sterile ,96 welled, flat-bottomed polystyrene microtiter plate along with a negative control and incubated for 24 hrs. After 24 hrs of incubation, the wells were decanted & gently washed three times with 200 µl of phosphate buffered saline (PBS), then dried in an inverted position and stained with 1% crystal violet for 15 min. The wells were rinsed again with 200 µl of ethanol-acetone (80:20 v/v) to solubilise crystal violet. The optical density of stained adherent biofilm was determined by using ELISA reader at 490nm wavelength. The interpretation of biofilm formation was done according to the criteria of Stepanovic et al [7] as follows-

1. Mean OD ≤0.120 (None/ weak biofilm production).
2. Mean OD 0.120 – 0.240 (Moderate biofilm production).
3. Mean OD ≥ 0.240 (Strong biofilm production).

Statistical analysis:

Descriptive statistical methods like percentage distribution and graphical presentation were used for the analysis of categorical variables in the study. The Chi-square test was used to test the association between ESBL production and biofilm formation.

Among 100 samples, 31(36.04%) isolates of Acinetobacter were identified out of which 68% were A. baumannii,29% were A. lowffii & 3.22% were A.haemolyticus(Table 1). Antimicrobial susceptibility test revealed that 26 (83.8%) isolates were Cefotaxime resistant, among which 19 (61.3%) were confirmed as ESBL producers by combined disc diffusion test (Table 2). Among 21 Acinetobacter baumannii isolates, 8(38%) were biofilm producers and among 9 Acinetobacter lwoffii isolates, 1(11%) was biofilm producer (Table 3). Among total 31 Acinetobacter isolates, 9(29%) were biofilm producers and 22(71%) were non-producers. None of the Acinetobacter haemolyticus showed Biofilm formation. Among 13 ESBL producing Acinetobacter baumannii isolates ,8 (61.5%) was showing biofilm formation and among 5 ESBL producing Acinetobacter lwoffii isolates, 1(20%) was biofilm producer (Table 4). p-value was found statistically significant i.e. less than 0.05 in ESBL (p-value 0.016) producing isolates of Acinetobacter baumanii. However, p-value for the Acinetobacter lwofii isolates which were ESBL producers was not significant i.e. more than 0.05.

Table 1: Species distribution of Acinetobacter isolates (n=31)

Table 2: Percentage of ESBL producers among Acinetobacter isolates

Table 3: Biofilm production in Acinetobacter isolates by microtiter plate assay

Table 4: Correlation between biofilm formation & ESBL production

Acinetobacter is a significant problem in Indian healthcare settings, particularly in intensive care units (ICUs) due to its ability to form biofilm on various surfaces leading to emergence of multi drug resistance. The prevalence of Acinetobacter in the present study in various ICUs was 36.04% (31 Acinetobacter out of 100 samples). Studies done by Garima Gautam et al [8] and Aarti Sangale et al[9] had almost similar prevalence of 33.02% and 38.7% respectively. In two other studies conducted by Tuhina Banerjee et al[10] and Tanvir Kaur et al [11], prevalence of Acinetobacter was little higher (42.9% & 42% respectively) than the present study which could be due to geographical & climatic differences favouring longer survival of organism, inadvertent use of higher antibiotics and larger sample size in their studies. Overall, it was observed that Acinetobacter had high prevalence among ICU patients owing to the fact that most of these patients were on mechanical ventilators, had endotracheal tube in situ, longer ICU stay and presence of underlying comorbid illness which made them vulnerable target for this notorious nosocomial pathogen.

Acinetobacter baumannii (68%) was the most common species isolated in the present study followed by Acinetobacter lwoffii(29%) and Acinetobacter haemolyticus(3.22%) which was similar to the study done by S R Sharma et al [12] [Acinetobacter baumannii (71%), Acinetobacter lwoffii(17.5%), Acinetobacter haemolyticus(7.2%)]. Similarly, in other studies, conducted by MV Yadav et al [13], M Soni et al [14], and A K Sharma et al [15], Acinetobacter baumannii (92%,60%, 84.5% respectively) was the most prevalent species followed by Acinetobacter lwoffii (7%, 29%, & 7.3% respectively) and Acinetobacter haemolyticus (1%, 11%,4.5% respectively). The reason behind this could be due to its higher virulence (attributed to high antibiotic resistance) and its ability of prolonged survival in hospital environment.

In the present study, percentage of ESBL producing Acinetobacter isolates was high (61.3%) which was similar to the studies done by PM Ambili et al [16] and N Chaudhary et al [17] which showed 51% & 31% ESBL producers. However, it was in contrast with the study done by MV Yadav et al [13] in which ESBL producers were only 14%. Increased prevalence of ESBL producers could be due to inadvertent use of higher antibiotics and failure to follow local antibiogram in the respective hospital. In the present study, out of 19 ESBL producers, biofilm formation was seen in 9(47.4%) isolates, which was similar to the studies done by DM Khan et al (61.5%) [18] and Punia et al (76%) [19]. In their studies, it was seen that ESBL production was corelated with biofilm formation. Biofilm formation interferes with the penetration of the antibiotics and hence is associated with higher antimicrobial resistance.

In the present study, it is evident that biofilm formation is significantly associated with ESBL production. Biofilm enhances the bacterial ability to establish and maintain the infection for longer period of time by acting as a shield against host’s immune system and antimicrobial therapy. It might be the reason of chronic and persistent infections, treatment failure and increased mortality in hospitalized patients. Early detection of biofilm formation and its correlation with ESBL production will be very helpful in early and appropriate treatment of patients as well in forming antibiogram for the hospital, thus aid in preventing further development of drug resistance and chronicity of infections.

Acknowledgement:

We acknowledge the efforts of Ms Rifa Parveen, PhD scholar, Department of microbiology, GMC, Kota for her valuable support during the research work.

Financial support and sponsorship: Nil

Conflicts of interest: There are no conflicts of interest.

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