Introduction

Acinetobacter baumannii, is one of the most challenging non fermenter, a gram negative pathogen for health care providers across the world. The organism is an opportunistic pathogen responsible for several genitourinary, neurological, respiratory and surface infections (Karlowsky et al., 2003). A. baumannii topped the list of (World Health Organization) bacteria with an imminent need for newer drugs and regimen (Willyard, 2017). Recent times have witnessed antibiotic resistance in A. baumannii species due to indiscriminate prescription practices and irrational drug use in both hospital and environmental settings. Studies have shown a rapid surge in the prevalence of MDRAB over the past two decades (Sukanya, Lakshmi, & Padmaja, 2014).

Recently, multidrug resistant A. baumannii isolates have been reported and this is of a significant threat to the health care system across the world (Dijkshoorn, Nemec, & Seifert, 2007). The mechanisms of resistance of Acinetobacter baumannii are at various levels, involving the production of enzymes involved in antibiotic degradation/modification, changes in permeability patterns and functioning of efflux pumps, and most importantly, the formation of biofilm (Gurung, Khyriem, & Banik, 2013).One of the uniqueness of A.baumanii which contributes to its extensive survival in the environment is its ability to bioflim formation, or communities of bacterial cells associated with a surface and encase in an extracellular matrix of carbohydrates, nucleic acids, proteins, and other macromolecules (Gaddy & Actis, 2009).

In addition to the above-mentioned infections, since A. baumannii is associated with infections of medical implants and devices like catheters and shunts, the susceptibility of biofilm formation is also higher with this organism. Biofilms on abiotic surfaces may facilitate their survival in the hospital environment. The drawback with these biofilm producing organisms is not only the increased risk of antibiotic resistance but also the synthesis of exopolysaccharide (EPS) in large quantities which can create a protective environment which in turn could result in poor antibiotic penetration and development of resistance (Kaliterna & Goic-Barisic, 2013).

The formation of biofilm by A.baumannii is attributed to the factors which promote subsistence of the pathogen in the hospital environment. These include specific resistance to disinfectants and other antimicrobial drugs, and, the capability of biofilm formation on various biotic and abiotic surfaces (Tomaras, Dorsey, Edelmann, & Actis, 2003). It has been established that the presence of biofilm is associated with antibiotic resistance and poses a significant challenge in the eradication of biofilm (Patel, 2005).

Therefore, it is essential that a causal relationship between biofilm formation and antibiotic resistance is established among the clinical isolates of A. baumannii. The present study was carried out to estimate the frequency of biofilm formation using different methods and evaluate the linkages between biofilm formation and antibiotic resistance.

Materials and Methods

Ethical clearance was obtained from the institutional ethics committee.

Bacterial isolation and identification

The prospective type of study was carried from January 2019 to August 2019 in Microbiology, in a tertiary care hospital in Kancheepuram district. The study was done on 73 isolates of A. baumannii from clinical samples such as urine, pus, sputum and miscellaneous (body fluids excluding blood).

The study samples were processed using phenotypic methods (Gram stain, culture, and biochemical tests: I-Indole, MR-Methyl Red, VP- Voges Proskauer, C-Citrate, MMM-Mannitol Motility Test Medium, TSI- Triple Sugar Iron, U-Urease) as per standard methodology (Gerner-Smidt, Tjernberg, & Ursing, 1991) and confirmed by conventional microbiological methods

Antimicrobial Susceptibility Testing

All 73 isolates were tested for antimicrobial susceptibility with Ampicillin (10 μg), Amikacin (10 μg), Ciprofloxacin (5 μg), Norfloxacin (30 𝜇g), Levofloxacin (5 𝜇g), Gentamicin (10 μg), Amoxyclav (30 μg), Ceftazidime (30 μg), Cefepime(30 𝜇g), Imipenem (10 μg), Meropenem (10 μg), Piperacillin-Tazobactam(100 𝜇g/10 𝜇g) by Kirby Bauer disc diffusion method.

Antimicrobial susceptibility testing was done by disc diffusion test and the findings were interpreted as per Clinical Laboratory and Standard Institute (CLSI) Guidelines. Isolates resistant to at least three classes of antibiotics were defined as Multidrug Resistant A. baumannii (MDRAB). A. baumannii ATCC 19606 was used as control (CLSI, 2017). After complete identification, isolates were subcultured and preserved in Trypticase soy broth (TSB) with 20% glycerol at -20ºC.

Biofilm formation

Two different assays were used to evaluate biofilm production by all 73 A.baumannii isolates using two conventional phenotypic methods.

Tube Method (TM) (Qualitative Assay)

A.baumannii isolates for biofilm formation were tested by the glass test tubes adherence test. Ten milliliters of Trypticase soy broth (TSB) with 1% glucose was inoculated with single colonies of the test bacterial strains on nutrient agar individually and the cultures were incubated for 48 hrs at 37ºC. The experiment was performed in triplicate. After incubation, the contents were decanted and Phosphate Buffer Saline (PBS) (pH 7.3) was used for washing. The contents were then dried. Crystal violet (0.1%) was used for staining the tubes for 7 min, gentle rotation of each of the tubes was done so that uniform staining of any adherent material on the inner surface took place. The contents were then gently decanted. Distilled water was used to remove and wash the excess stain. Tubes were then dried by placing them in an inverted position. The tubes were then observed for biofilm formation (Freeman, Woods, Welsby, Percival, & Cochrane, 2010).

Microtitre Plate Method (MTPM) (Quantitative assay)

Trypticase soy broth with 0.25% glucose at 37 ^oC was used to achieve overnight growth of each isolate and this was diluted in the ratio of 1:40 in TSB-0.25% glucose. Sterile 96 well polystyrene microtiter plates were used for inoculation of 200μL of cell suspension and incubated for 24 hours. Following this step, 200μL of phosphate buffered saline was used for washing the wells gently three times after which it was inverted for drying. Further to this, staining was done with 1% crystal violet for 15 minutes. On further rinsing the well with 200μL of ethanol-acetone (80:20 v/v) to solubilize crystal violet, the optical density was measured at 620 nm (OD 620) using ELISA reader. Each assay was performed in triplicate and the average optical density was recorded (Christensen et al., 1985).

The following values were assigned for biofilm determination (Table 1).

Controls for biofilm forming property

Biofilm producing reference strains of Acinetobacter baumannii(ATCC 19606) and Pseudomonas aeruginosa (ATCC 27853) were used (Deighton & Balkau, 1990).

Statistical Analysis

Statistical analysis of MDR and biofilm formation

The prevalence of biofilm formation was expressed as percentages. Chi-square test was used to evaluate the association between MDR and biofilm producing capacity of the isolates. The observed difference between biofilm formation and MDR was found to be statistically significant at 95% C.I. A p value < 0.05 was considered as statistically significant.

Statistical analysis of different degrees of biofilm forming ability among 73 Acinetobacter baumannii isolates by TM, MTPM

The degree of biofilm formation among 73 Acinetobacter baumannii was very significantly associated with Tube method and Microtiter plate method. A p value of 0.001 was analyzed statistically by Chi-square tests.

Statistical evaluation of TM and MTPM for detection of biofilm formation

The comparative statistical analysis for TM and MTPM was done by using 2x2 tables by Greenhalgh (Knobloch, Horstkotte, Rohde, & Mack, 2002). Data obtained from MTPM was compared with data from TM. Sensitivity of TM and MTPM were found to be (78.78%) and (90.74%). Sensitivity of MTPM was more than TM, which is supported by higher Positive Predictive Value of 87.5%. Therefore MTPM is a better method than TM and can be used as a screening method in detection of biofilm among Acinetobacter baumannii isolates.

Results and Discussion

During the 8 months of study from January 2019 to August 2019, 73 clinical isolates of A. baumannii were collected from various clinical samples. The phenotypic identification of the A.baumannii isolates was performed by Bacteriological methods (Grams staining, colony morphology and biochemical tests) using standard methodology (Figure 1).

Overall, 26 A. baumannii isolates (36%) were detected in urine, followed by 19 (26%) from pus, 17 (23%) from sputum and 11 (15%) from other miscellaneous (body fluids excluding blood) (Table 2).

In the present study, A. baumannii demonstrated resistance against most of the routinely used antibiotics. Out of 73 isolates, 81% (59/73) of A. baumannii isolates were identified as MDR and 19% (14/73) of isolates were susceptible (Table 3).

In the present study, Of 73 Acinetobacter baumannii, 59 isolates were MDR and qualitative (TM) and quantitative method(MTPM) revealed 45(76%), 57(97%) as biofilm producers respectively. Among all 73 isolates, Microtiter plate method demonstrated maximum positivity with MDRAB. The relationship between biofilm production and Multi Drug Resistance was statistically significant (p=0.05) (Table 4). In addition, biofilm formation was verified for 14 antibiotic susceptible isolates and the results of TM and MTPM showed 1(7%), 4(21%) respectively as weak biofilm producers (Table 5).

Data from TM and MTPM were compared. The potential for Biofilm formation were categorized by OD values as strong, moderate, and weak and non-biofilm producers. Qualitative TM of biofilm screening revealed 63% isolates positive for biofilm production of which 26(35%) of isolates displayed strong, 13(17%) indicated moderate, 7(9%) weak biofilm formation and 27(39%)were non biofilm producers (Figure 2).

Quantitative MTPM assay for biofilm screening showed 84% positive for biofilm formation, among which 49(67%) isolates were strongly positive, remaining isolates were moderate7(10%), weak 5(7%) and non-biofilm producers12(16%) (Figure 3).

The degree of biofilm formation among 73 Acinetobacter baumannii isolates was very significantly (p=0.001) associated with Tube method and Microtiter plate method (Table 7; Table 6).

Sensitivity, specificity, PPV and NPV were evaluated. Tube method showed sensitivity (78.78 %), specificity (67.5 %), PPV (66.66%) and NPV (79.41%) parameters and MTPM: sensitivity (90.74 %), specificity (63.15 %), PPV (87.5%), NPV (70.58%). Sensitivity (90.74 %) of MTPM was more than TM (78.78 %), which is supported by higher PPV (87.5%). Therefore MTPM is a better method than TM and can be used as a screening method in detection of biofilm among Acinetobacter baumannii isolates (Table 8).

In this study, 73 Acinetobacter baumannii were isolated from various clinical samples. 26 (36%) isolates were from the urine samples, followed by pus 19 (26%), sputum 17 (23%) and 11(15%) from other miscellaneous (body fluids excluding blood) samples. Similarly, in another study by (Lahiri, Mani, & Purai, 2004) majority of isolates were from urine samples (51.3%). In contrast, (Pattanaik & Banashankari, 2019) reported maximum isolation of Acinetobacter baumannii from pus samples 54 (30.5%) and 47 (27.6%) were from the endotracheal (ET) secretions, 5(2.9%) were from urine, and 3(1.7%) were from sputum.

Globally, there is an increased prevalence of nosocomial infections pertaining to various hospital settings due to the persistence and survival of MDR A. baumannii (Ghasemi et al., 2018). This situation further complicates the treatment of MDRAB infections, especially in Turkey, India and Iran (Shirmohammadlou, Zeighami, Haghi, & Kashefieh, 2018). In this study, out of 73 isolates, 14 (19%) were sensitive to all antibiotics tested and 59 (81%) isolates were MDR. Consistant with our study, some researchers (Gurung et al., 2013) and (Abdi-Ali, Hendiani, Mohammadi, & Gharavi, 2014) demonstrated that the biofilms were stronger with MDR strains compared to the sensitive strains.

Formation of biofilm is an essential factor in determining the virulence in the pathogenesis of A. baumannii (Vijayakumar et al., 2016). In this study, out of 59 MDR isolates, 45(76%) and 57(97%) isolates produced biofilm in varying degrees when tested by Tube method and Microtitre plate method respectively. This study results showed that there is a statistically significant association between Multiple Drug Resistance and biofilm formation. This result is in accordance with previous studies by (Chaturvedi, Chandra, & Mittal, 2019) and (Avila-Novoa, Solís-Velazquez, & Rangel-Lopez, 2019) which showed 90% and 100% of bacteria with the ability to form biofilm were MDR.

The present study demonstrated that out of 73 isolates, 46(63%) and 61(84%) isolates were able to form biofilm by Tube and Microtiter plate method. Among 73 isolates tested for biofilm production, TM showed 39% non biofilm producers, 9% weak, 17% moderate, and 35% strong, whereas MTPM showed 16%non biofilm producers, 7% weak, 10% moderate, and 67% strong. In a similar study, (Abdi-Ali et al., 2014) have assessed the biofilm formation using modified Microtiter Plate and test tube methods. Microtiter method showed 18% strong, followed by 41% weak and 25%non biofilm producers. Tube method showed 22% strong, 42% weak and 18%non biofilm producers.

Variations in biofilm production could be due to different methods. The results of the present study showed a higher rate than studies conducted by (Gurung et al., 2013) which stated that 50% were biofilm formers by Tube method. (Kailasbadave & Dhananjay, 2015) reported 62.5% isolates produced biofilm by Microtitre plate method. In contrast, the percentage was lower in our study compared to studies conducted by (Bardbari et al., 2017) who showed 100% of isolates were capable of forming biofilms and in a similar study by (Vijayakumar et al., 2016), all isolates formed biofilms.

There was a strong correlation between TM method and MTPM in identifying biofilm producers. However, it was challenging to distinguish between moderate, weak and non biofilm producers due to the observer variations. Sensitivity of MTPM was more than TM, which is supported by higher PPV. Thus Microtitre Plate assay method showed better detection of biofilm formation. Hence findings of this study suggest that MTPM quantitative is a better method than TM and can be used as a screening method in detection of biofilm among Acinetobacter baumannii isolates.

Conclusions

Most of the A. baumannii isolates showed resistance to commonly used antibiotics. The majority of them were biofilm producers in various degrees. This study elucidated the importance of biofilm formation in MDRAB infections. Persistence of MDRAB could result in prolonged hospitalization and increased mortality. TM could be used to detect biofilm producing A.baumannii, but when compared with the MTPM, indicates that the MTPM method should be the first choice because this method is a more sensitive and reproducible method. It can be used for screening and is a quantitative tool for determining biofilm formation by Acinetobacter baumannii. This study indicates the need for further molecular support to find out biofilm producing virulence genes of MDRAB.

Acknowledgement

We would like to thank Dr R. Vijayaraghavan, SIMATS and G. Poornima(Technician), SMMCH and RI.

Conflicts of Interest

The authors declare that they have no conflict of interest for this study.

Funding Support

The authors declare that they had no funding support for this study.