Association of Biofilm Formation with Antimicrobial Resistance Among the Acinetobacter Species in A Tertiary Care Hospital in Bangladesh

Purpose: The purpose of this study was to detect biofilm formation in clinical isolates of Acinetobacter species and to observe correlation between biofilm formation and antimicrobial resistance among Acinetobacter isolates. Methods: Two hundred fifty six clinical samples collected from patients who were admitted in Intensive Care Unit (ICU) and on device, patients from Surgery, Medicine, Gynae & Obs and Urology department of Bangabandhu Sheikh Mujib Medical University (BSMMU) and from Burn unit of Dhaka Medical College Hospital were included in this study. Biofilm formation and antibiotyping were performed for the isolates of Acinetobacter species recovered from clinical samples including tracheal aspirates, blood, urine, wound swab, pus, throat swab, endotracheal tubes, burn samples, ascitic fluid, sputum, aural swab, oral swab, cerebrospinal fluid, and catheter tip. Correlation of biofilm formation with antimicrobial resistance pattern among Acinetobacter isolates were also observed in this study. Result: A total of 256 various specimens were studied of which 95 Intensive Care Unit (ICU) and 161 Non ICU samples. Out of 95 ICU and 161 Non ICU samples, Acinetobacter species were isolated from 32 (33.7%) and 20(12.4%) respectively. From 32 ICU and 20 Non ICU Acinetobacter isolates, 28 (87.5%) and 11 (55%) were biofilm producers. Biofilm forming capacity of Acinetobacter species was significantly (p<0.008) greater in ICU than in Non ICU isolates. In both ICU and Non ICU isolates, biofilm forming Acinetobacter species were 100% resistant to amoxicillin, ceftriaxone, ceftazidime, cefotaxime, cefuroxime, and aztreonam. Resistance to antibiotics such as gentamicin, amikacin, netilmicin, ciprofloxacin and imipenem was higher among biofilm forming Acinetobacter isolates in ICU than Non ICU isolates. Susceptibility to colistin was 100% in Non ICU isolates but in ICU it showed 7.1% resistance. Conclusions: This investigation showed that most of the clinical isolates of Acinetobacter species were biofilm producers especially from ICU samples and they were multidrug resistant. Even polymixin resistant Acinetobacter isolates are slowly emerging. This is very alerming for us that biofilm forming multidrug resistant Acinetobacter species represents a severe threat in the treatment of hospitalized patients. So, antibiotic policy and guidelines are essential to eliminate major outbreak in future.


Introduction
Acinetobacter species has emerged as an important nosocomial pathogen as it is the causative agent of several types of infections including pneumonia, meningitis, septicaemia, and urinary tract infections and also responsible for causing intermittent outbreaks especially in ICU.They ranked second after Pseudomonas aeruginosa among the nosocomial, aerobic, non-fermentative, gram negative bacilli pathogens. 1,2Infections caused by Acinetobacter are associated with medical devices, e.g.vascular catheter, CSF shunt, Foley catheter, surgical interventions etc. [3][4][5] The presence and duration of invasive procedures, as well as exposure to broad spectrum antibiotics, have been identified as risk factors for acquisition of Acinetobacter in numerous studies. 6Infections caused by them are difficult to control due to multidrug resistance, which limit therapeutic options in critically ill and debilitated patients especially from the intensive care unit, where its prevalence is most noted. 2inetobacter infections are associated closely with surgery or the use of artificial devices.Patients become infected following initial colonization.This process is influenced by various risk factors, particularly in ICUs, where multiple manipulations following surgery, as well as the use of endotracheal tubes and intravascular, ventricular or urinary catheters, can result in colonization by opportunistic bacteria such as Acinetobacter. 6quisition of the ability to form biofilm could be a good strategy to enhance a microorganism's survival under J MEDICINE 2013; 14 : 28-32 .stressed conditions, e.g., during host invasion or following antibiotic treatment.This is because cells growing in biofilms are highly resistant to numerous types of antimicrobial agent.In addition, the ability of horizontal gene transfer of bacterial cells is enhanced within biofilm communities, thereby facilitating the spread of antibiotic resistance.The high colonizing capacity of A. baumannii, combined with its resistance to multiple drugs, contribute to the organism's survival and further dissemination in the hospital setting. 7he present study was undertaken on clinical isolates of Acinetobacter species to determine biofilm formation and to observe correlation between biofilm formation and antimicrobial resistance among Acinetobacter species.

Detection of biofilm
The ability of Acinetobacter isolates to form biofilm was performed as described by Toledo et al., 2001. 12Isolates were grown over night at 37 0 C in Brain Heart Infusion Broth (BHIB) with 0.25 % glucose.The culture was diluted at a ratio of 1:20 in fresh Brain Heart Infusion broth (BHIB) with 0.25% glucose.200 µl of this suspension was used to inoculate in sterile 96 well flat bottomed polystyrene microtiter plate.Then the plate was incubated at 37 0 C for 24 hours.Wells were washed with Phosphate buffer solution (PBS) three times.Non-adherent cells were removed by washing with phosphate buffer.Then the microtiter plate was dried in an inverted position.After that plate was stained with 0.5% Crystal violet (CV) for 15 minutes.Wells were rinsed once more.Then 200 µl ethanol/ acetone (80: 20, v/v) were added in each well to solubilize CV.The optical density (OD) was determined using a microtiter reader.Each assay was performed in triplicate & repeated twice.The average OD values were calculated for all tested strains and negative controls, since all tests were performed in triplicate and repeated three times.Second, the cut off value (ODc) was established.It was defined as three standard deviations (SD) above the mean OD of the negative control: ODc=average OD of negative controls + (3XSD of negative control).Final OD value of a tested strain was expressed as average OD value of the strain reduced by ODc value (OD= average OD of a strain -ODc).ODc value was calculated for each microtiter plate separately.Any negative value was presented as zero, while any positive value was indicated biofilm production. 13

Discussion
Acinetobacter infections present a global medical challenge.4][5] Biofilm formation is thought to be a key pathogenic feature, especially in relation to intravascular line infections and ventilated associated pneumonia.Generally, two properties are often associated with biofilm producing bacteria, namely, the increased synthesis of exopolysaccharide (EPS) and the development of antibiotic resistance. 14One can assume that increased production of EPS in Acinetobacter is likely to create a protective environment leading to difficulty in antibiotic penetration leading to development of resistance.In addition, there appears to be some differences in the cellular physiology of cells within the biofilm that also results in increased drug resistance. 15Thus infections due to bacteria that form biofilm is a tenacious clinical problem.In this work, biofilm formation by Acinetobacter isolates were tested and tried to correlate them with antimicrobial resistance.
In this current study, the high isolation rates of Acinetobacter species of about 100% from endotracheal tube, followed by 54.3% from tracheal isolates, 36.4% from central venous catheter blood in ICU and 50% from peripheral blood, 44.4% from urine and 25% from wound swab and 13.9% from pus in non ICU samples.In India, a study reported that, the high isolation rate of Acinetobacter species of about 24% were from tracheal aspirates, 16% from sputum, 12% from endotracheal tube, 12% from wound swab and 6% from blood. 16r study showed 28 (87.5%) and 11 (55.0%)isolates were biofilm producers from 32 ICU and 20 Non ICU Acinetobacter species.Higher rate of biofilm production was found in patients on device in ICU.Present results showed that biofilm plays a role in the pathogenesis of some device-associated Acinetobacter infections.Other study showed that, more than 60% of A. baumannii isolates from clinical samples formed biofilm, and these isolates were associated mainly with device-associated infections. 17 this study, Acinetobacter species showed higher biofilm production in central venous catheter blood 100%, peripheral blood 100%, urine 100% and tracheal aspirates 84.2% but endotracheal tube showed 66.7% biofilm production in ICU.

Conclusion
In conclusion, the data obtained in the present work showed that most of the clinical isolates of Acinetobacter species were biofilm producers especially from device in ICU samples and they are multidrug resistant.All biofilm producing Acinetobacter species were found to be resistant to clinically achievable levels of most commonly used antibiotics such as penicillin, cephalosporin, aminoglycosides, quinolone, carbapenem and monobactam group of drugs.Polymyxins remain the only agent that may be consistently active in vitro against Acinetobacter species.However, polymixin resistant Acinetobacter isolates are slowly emerging.This is very alerming for us that biofilm forming multidrug resistant Acinetobacter species represents a severe threat in the treatment of hospitalized patients.Combination therapy may be the only therapeutic option to preserve the clinical utility of the polymixins against Acinetobacter.So, antibiotic policy and guidelines are essential to eliminate major outbreak in future.
. No growth of Acinetobacter species were detected in other samples namely ascitic fluid, sputum, aural swab, oral swab, burn samples, cerebrospinal fluid and catheter tips.From 32 ICU and 20 Non ICU Acinetobacter isolates 28 (87.5%) and 11 (55.0%) were biofilm producers.Biofilm forming capacity of Acinetobacter species was significantly (p<0.008)higher in ICU than Non ICU isolates.The rate of biofilm production by isolated Acinetobacter species from different clinical samples is shown in Table-1.From ICU and Non ICU samples all the biofilm forming Acinetobacter isolates were 100% resistant to amoxicillin, ceftriaxone ceftazidime, cefotaxime, cefuroxime and aztreonam.Higher level of resistance was also recorded in Table-2.Only colistin showed 7.1% resistance in biofilm forming Acinetobacter isolates in ICU and 100% sensitivity in Non ICU isolates.