Studies on the mechanism of multidrug resistance of Acinetobacter baumannii by proteomic analysis of the outer membrane vesicles of the bacterium

Acinetobacter baumannii, an opportunistic pathogen is responsible for pneumonia, meningitis and a wide range of other infections. It is typically involved in nosocomial infections. Outer membrane vesicles (OMVs) released from the bacteria play an important role in the bacterial physiology including pathogenesis. In this study, OMVs were isolated and characterized form sensitive and multidrug-resistant clinical strains of A. baumannii. A comparative proteomics analysis of the OMVs from the A. baumannii sensitive and MDR strain revealed the increased content of carbapenemase and a host of other related antibiotic resistant enzymes in the OMVs of the A. baumannii MDR strain. The OMVs of antibiotic-sensitive strain contained only 8 antibiotic resistance-conferring proteins, whereas the OMVs of multidrug resistance contained 24 proteins. Growth studies on the sensitive and MDR strain in the presence of polymyxin B and OMVs prepared from the respective strains revealed that the OMVs protected the bacteria against the antibiotic. Bacteria were grown in the presence of antibiotic and an indole derivative has shown that the minimum inhibitory concentration was reduced in the presence of derivative indicating that the derivative regulated the production of OMVs. Interestingly, it was also found that the degradosome complex involved in RNA processing was associated with the OMVs. These studies reveal that OMVs may play an important role in antibiotic resistance/persistence.


Introduction
Acinetobacter baumannii is an opportunistic nosocomial pathogen in humans and infects people with the compromised immune system. It is associated with a range of clinical syndromes like pneumonia, meningitis, bacteremia, urinary tract infections and others (Fournier et al. 2006). A. baumannii has a remarkable capacity to acquire resistance to most of the antibiotics used in the clinical practice and is a cause of major concern (Vila et al. 2007;Bou et al. 2000). It has acquired resistance against carbapenems which are a drug of choice for treating A. baumannii infections, and polymyxin the drug of last resort (Lean et al. 2016). This bacterium represents most common bacterial strains acquiring multidrug resistance that are grouped with the acronym "ESKAPE" indicating Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa and 1 3 infectious nature of the A. baumannii is able to acquire multi-drug resistance and methods involved in the clinical management were reviewed earlier (Howard et al. 2012). The epidemiology and multidrug resistance of A. baumannii are well studied, but pathogenesis and virulence warrant further in-depth investigations (Long et al. 2013). Several secreted molecules are known to play an important role in the pathogenesis of this bacterium. Production of outer membrane vesicles (OMVs) is a unique mechanism through which bacteria release virulence factors to the host. Recent studies from our laboratory and several other reports suggest that outer membrane vesicles of bacteria contain virulence factors are playing an important role in antibiotic resistance (Chowdhury and Jagannadham 2013;Kulkarni et al. 2014;Chattopadhyay and Jagannadham 2015;Devos et al. 2015;Manning and Kuehn 2011).
OMVs are released form bacteria are spherical in structure ranging from 20 to 250 nm and composed of lipids, proteins and in some cases nucleic acids. OMVs possess several biological functions such as transporting virulence factors to host. Therefore, several studies are directed to identify the virulence factors associated with OMVs using proteomic approaches. In a recent study, OMVs isolated from two clinical strains of A. baumnnii revealed contents of virulence factors varying from one more virulence associated factors are found in the OMVs of the MDR strain (Li et al. 2015).
The extracellular secretary proteins and the proteins released through OMVs were identified from a highly virulent multidrug-resistant strain and OMVs carries 39 proteins with virulence and pathogenesis (Mendez et al. 2012). In another study, it was shown that outer membrane protein A (OmpA) possess cytotoxic activity and plays an important role in the biogenesis of OMVs from A. baumannii (Jin et al. 2011;Moon et al. 2012). The proteins present on the surface of the OMVs prepared from A. baumannii were shown to elicit a potent immune response both in vivo and in vitro (Jun et al. 2013). OMVs were shown to transfer the carbapenem resistance genes between different strains of A. baumannii Fulsundar et al. 2014). Some studies on proteomics analysis were carried out from the OMVs prepared from different A. baumannii strains. The OMVs were shown to contain 132 proteins including the cytotoxic outer membrane protein A (Kwon et al. 2009). Quantitative proteomics studies were carried out from the cell wall and plasma membrane fractions prepared from A. baumanni to identify the proteins involved in the antibiotic resistance (Yun et al. 2011). The current status of quantitative proteomics from the carbapenem resistance strains was reviewed suggesting that different studies should be combined to understand the mechanism of MDR (Tiwari and Tiwari 2014). Several studies need to be carried out form different clinical strains for a better understanding of proteins involved in the antibiotic resistance in A. baumannii.
In the present study, the proteomic analysis of the OMVs prepared from sensitive and multidrug-resistant strain was carried out. In addition, we have observed OMVs released from A. baumannii protects the bacterium against the membrane active antibiotic polymyxin B in a concentration dependent manner. As far as we aware this is the first report demonstrated that OMVs from the MDR strain carry the proteins of the degradosome complex.

Bacterial strains and growth condition
Seventy one strains of A. baumannii collected from a tertiary care hospital in Guwahati, Assam, India from Aug 2012 to Nov 2013. Two strains of A. baumannii (one sensitive and other most resistant) were selected for the isolation of OMVs. The source of the samples was isolated from the suction catheter tube of the patients. The species level identification was carried out by 16s rRNA gene sequencing. The bacterium was grown in Luria-Bertani broth (LB) (HiMedia, India) at 37 °C with shaking at 250 rpm. The disc diffusion by Kirby-Bauer method (Bora and Ahmed 2012) was used to determine the sensitive and resistance phenotype according to the Clinical and Laboratory Standards Institute (CLSI; M100-S22, 2012).

Determining the minimum inhibitory concentration
The antibiotic polymyxin B used in the study is a cationic cyclic polypeptide; it acts on the bacteria by binding membrane phospholipids and disrupting the cytoplasmic membrane including pore formation in bacterial wall. The stock of the peptide was prepared in a concentration of 1 mg/ml. The minimum inhibiting concentrations of polymyxin B was determined by the broth dilution method in Bioscreen C (Kulkarni et al. 2014). LB medium inoculated with the bacterial strains was distributed in different wells. Different concentrations of antibacterial molecules were added to the bacterial cultures. The minimum concentration of the antibiotic molecule where the bacteria failed to grow was defined as MIC. The growth inhibiting concentrations (above MIC) for polymyxin was used to study the effect of OMVs from both the resistant and sensitive strains of A. baumannii.

Preparation of OMVs
The OMVs were prepared from both sensitive and resistant strains using the protocol (Kulkarni et al. 2014). In brief, the A. baumanniii culture was grown till reaches the stationary phase at OD 600nm 1.0 in LB broth and incubated at 37 °C with constant shaking (250 rpm). The cell pellet was 1 3 separated from the culture by centrifugation at 10,000 × g for 30 min at 4 °C. (Beckman Colter Avanti 100, rotor JLA.10.500). The supernatant was filtered using 0.45 µm (Millipore). The OMVs were prepared by ultracentrifugation at approximately 150,000 × g (35,000 rpm, rotor type Ti 45, Beckman coulter Optima max 100 XL) for 2 h at 4 °C. The pellet containing OMVs were re-suspended in 10 mM phosphate buffer (pH 7.4) and further purified by sucrose density gradient method. Sucrose solutions of 70, 60, and 20% were added from the bottom to top in polyallomer tubes and the suspension of OMVs was layered on the top of it. The tubes were subjected to ultracentrifugation at 35,000 rpm (160,000 × g for 'SW 60 Ti' rotor, Beckman) at 4 °C for 6 h. A pellet hanging in between 70 and 60% sucrose solution was collected and diluted in 20 times in 10 mM phosphate buffer (pH 7.4) and further subjected to ultracentrifuge at 160,000 × g (45,000 rpm, Beckman Coulter Optima max ultracentrifuge XL, rotor TLA.300) for 2 h. The purified OMVs thus obtained were resuspended in 10 mM phosphate buffer (pH 7.4) and stored at − 20 °C for further experiments.

Preparation of outer membrane, inner membrane, and total cell lysate proteins from Acinetobacter baumannii and quantification
The bacterial cells harvested via centrifugation were used to prepare the cell extract. The cell pellet was transferred to lysis buffer (tris HCl 50 mM, EDTA 8 mM Nacl 1 M), after addition of lysozyme (60 μg/ml), 1 M Dithiotritol, and 100 mM PMSF. Then the mixture was vortexed and sonicated for 2 min in a Branson sonifier (pulses 5 s on 5 s off at 30% ampere). The remaining cells and cell debris were separated by centrifugation at 8000 rpm for 10 min the supernatant was collected in a fresh tube. This supernatant is used as whole-cell protein (WCP) extract. Using WCP, inner membrane (IM) and outer membrane (OM) was separated by the triton X method. WCP was then mixed with 2% triton X-100 and ultracentrifuge at 160,000 × g (45,000 rpm, Beckman Coulter Optima max ultracentrifuge XL, rotor TLA.300) for 2 h. The pellet was collected and the supernatant was removed. The pellet was again dissolved in 2% triton X-100 and again centrifuged for 2 h. Now the supernatant contains inner membrane (IM) proteins and the pellet comprise of outer membrane (OM) proteins. The pellet was resuspended in 10 mM phosphate buffer (pH 7.4) and stored at − 20 °C until used for further experiments. The protein concentration of OMVs was determined by the Folin Coucalteau method.

Dynamic light scattering
Dynamic light scattering instrument DLS (HORIBA SZ-100 particle size analyzer) at 90° angle at 25 °C with a refractive index of the medium as 1.334 was used to estimate the size distribution of these OMVs. SZ-100 software was used to analyze the data. The OMVs were diluted to 0.24 µg/mL in 10 mM phosphate buffer pH 7.4. Triplicate values were taken in nano analysis mode and the mean values were calculated to determine the diameter of the vesicles.

Transmission electron microscope (TEM) analysis
TEM was carried out for OMVs isolated from sensitive and MDR A. baumannii strains using a Jeol transmission electron microscope (JEM 2100, Tokyo, Japan) at 200 kV. The samples were loaded onto a carbon coated copper grid, negatively stained with 0.2% uranyl acetate and images were captured.

Growth curve assay
The role of OMVs on the growth inhibiting concentration of antibacterial peptide polymyxin B of sensitive and MDR A. baumannii in the presence of their respective OMVs and the growth pattern of sensitive strain in presence of OMVs isolated from MDR strain was observed. The study was carried out in a Bioscreen C using an equal amount of inoculum and equal conc. of antibiotics with varying conc. of OMVs were observed. An inoculum having a cell density of 2 × 10 5 CFU/mL was added in each well of honey comb plates. The growth inhibiting concentrations (above MIC) for polymyxin is 2.5 μg for sensitive strain and 4.5 μg for MDR strain was used. The concentration of OMV used for both the sensitive and MDR strains were 1, 2, 4, 6, 8, 12, and 16 μg. Whereas the concentration of MDR OMVs used for sensitive strain were 1, 2, 4, 6, and 8 μg, respectively. In each well, the bacterial cells, polymyxin, and OMVs in different concentrations were mixed and incubated for 2 h. After 2 h incubation, they were inoculated with the LB broth. The growth inhibitory concentrations of the antibacterial for each strain are 2.5 μg for sensitive and 4.5 µg for MDR. Control for the experiments includes media with bacterial inoculam for both the strains, only growth media and OMVs with media. The plate was kept inside the growth chamber of the Bioscreen C growth monitoring system. The growth temperature was maintained at 37 °C with constant shaking on "low" mode (120 rpm) with a wavelength of 600 λ and duration was for 24 h. The qualitative assays on agar plates were also carried out to examine the effect of OMVs on the growth of A. baumannii in the presence of antibiotics. For qualitative studies, the constituents of the wells were streaked on a 90 mm LB agar plate segmented into sections and labeled. The plates were incubated at 37 °C for 24 h.

Efficacy of OMVs in restoring the growth of A. baumanii in presence of antibiotics
After 2 h of incubation, 20 μL of each well was spread plated onto a 90 mm LB agar plate for a viable count. The qualitative plate assays were performed to estimate the extent of protection provided by OMVs to the bacterial cells in the presence of antibiotics in inhibitory concentration. The colonies grown on the plates were counted following incubation at 37 °C for 24 h. The experiment was repeated three times, and an average of the colony count was obtained for each concentration of the vesicles.

Evaluation of growth of A. baumanii strains with the presence of indole derivatives and polymyxin B
A. baumannii Ab69 and Ab71 were treated with various concentration of an indole derivative (Ethyl-2 (3-indolyl)-4-methyl-thizole-5-carboxylate, indole-3-carboxymidoxime) along with the polymyxin B. Briefly, A cell density 2 × 10 5 CFU/mL was added and treated with various concentrations of polymyxin B and indole compound. In each well, the bacterial cells, indole derivatives and polymyxin B were mixed and incubated 24 h with regular interval. Control for the experiments includes media with bacterial inoculum and growth inhibitory concentration of polymyxin B for both the strains. The plate was incubated inside the growth chamber Bioscreen C growth monitoring system. The growth temperature was maintained at 37 °C with constant shaking on "low" mode (120 rpm) with a wavelength of 600 λ and duration was for 24 h.

SDS PAGE
The whole-cell extract, outer membrane (OM), inner membrane (IM) and OMVs proteins were separated on 10% SDS-PAGE gel according to the Laemmli protocol. About 150 µg of all the protein were dissolved in SDS-PAGE loading buffer, (250 mM Tris-HCl (pH 6.8), 25% beta mercaptoethanol, 10% SDS, 0.05% Bromophenol blue, 50% glycerol) denatured at 95 O C for 5 min and loaded in different wells in the gel of a thickness 1.5 mm. The gel was stained with coomassie brilliant blue dye (R250) staining solution overnight and de-stained.

Trypsin digestion
The lanes containing the OMV proteins from sensitive and resistant strains subjected to in-gel digestion. First, the lanes were cut into small pieces and washed with water. Secondly, the pieces were treated for 15 min each in acetonitrile, 50 mM ammonium bicarbonate (ABC), in 1:1 ratio to remove excess coomassie dye. The gel pieces were dehydrated with acitonitrile for 5 min and dried using a speed vac concentrator. They were then reduced with dithiothreitol (DTT) at 60 °C for 60 min and irreversibly alkylated with iodoacetamide (IAA) for 30 min in dark. The reduction alkylation was followed by the addition of 50 Mm ABC solutions, 1:1 ratio of 20 mM ABC/50% ACN (15 min each). Finally, an ACN wash was done for 5 min and then excess ACN dried with a speed vac concentrator. To the dehydrated gel pieces trypsin (20 µg in 25 mM ABC) 25-30 μl (Promega trypsin gold mass spectrometry grade) was added and incubated at 37 °C for 18 h. The peptides were extracted twice from the gel pieces with 100 μl of 50% ACN, 45% Milli Qwater and 5% Trifloroacetic acid (TFA) solution by vortexing for 45 min each. These peptides were concentrated and dried them in a Speed Vacconcentrator and stored at -30 O C until further use.

LC/MS/MS analysis
The tryptic peptides were subjected to mass spectrometry using an LC-ESI-MS/MS Orbitrap velos instrument obtained from Thermo Scientific (San Jose, CA). The peptides were separated using Proxeon LC system on a Biobasic C18 (100 mm × 0.18 mm) reverse phase column, with a pore size of 300 Å and particle size of 5 μm (New Objective, MA, USA) using a 90 min gradient. The LC system was connected to ESI-MS/MS which recorded the collision induced dissociation (CID) MS of the peptides. The flow rate was set at 300 nL/min. The mobile phases used in the separation of peptides are Solvent A (0.2% formic acid in water) and Solvent B (0.2% formic acid in 95% acetonitrile). The gradient was started at 5% B and increased to 15% B in 45 min, and at 68 min % B reached up to 35%, at 78 min % B reached to 70% and kept up to 81 min. Later % B decreased to 5% by 90 min and equilibrated the column. The MS (m/z 400-2000) and MS/MS spectra were obtained using activation type HCD at a heated capillary temperature of 200 °C, and the electrospray ionization (ESI) voltage was set at 1.6 kV. The peptides were fragmented using normalized collision energy of 35%. The MS/MS spectrum of the top 10 peptides with a signal threshold of 1000 counts was acquired with 10 ms activation time and a repeat duration of 30 s.

Identification of proteins
The database of A. baumannii was downloaded from NCBI in fasta format and used it for the identification of proteins. The MS/MS spectra of the doubly charged peptides were searched against this database of A. baumannii. The LC-ESI MS/MS spectra were analyzed using the Proteome Discoverer Version 1.4 supplied by the manufacturer. All the MS/ 1 3 MS spectra were analyzed using SEQUEST (Thermo Fisher Scientific) selecting the enzyme trypsin and applying the search parameters of precursor tolerance of 10 ppm and a fragment tolerance of 0.6 Da. The increase in mass due to the oxidation of methionine (15.99 Da) and carboxyamidomethylation of cysteine (57.02 Da) was set as the variable and fixed modification, respectively. Maximum equal modifications per peptide were set as three; maximum dynamic modifications per peptide are four for the identification of proteins. Only peptides identified with high confidence were included in the list. All the proteins were identified by at least two unique peptides.

Bioinformatic analysis of the proteome of A. baumannii (Ab69) and A. baumannii (Ab71)
The subcellular localization of a protein and the pathway analysis was predicted by PSORTb ( Yu et al. 2010) and CELLO2GO (Yu et al. 2014), respectively. Antibiotic resistance proteins associated with OMVs of both strains were predicted by RGI software (Jia et al. 2017).

Results and discussion
The main aim of the study is to compare the proteins from the OMVs present in sensitive and resistant strain to understand the functional significance of OMVs. The effect of OMVs on growth of both sensitive and resistant strains in the presence of growth inhibiting concentrations of polymyxin B, an antibacterial agent was also studied.

Physical characterization of OMVs of both the sensitive and MDR strains
Using the disc diffusion method, we screened A. baumannii (Ab 69) and A. baumannii (Ab71) (sensitive and resistant strain, respectively) with different groups of antibiotics. Ab 69 was found to be sensitive to most of the antibiotics and Ab71 was found to be resistant to all the classes of antibiotics. The two A. baumannii strains were grown to the early stationary phase in LB broth (OD 600 = 1.0), and the OMVs were harvested. The size of OMVs from both the strain was estimated using Transmission electron microscopy (TEM) and Dynamic light scattering (DLS). The diameter of both the strains was estimated to be in the range of 50-100 nm (Fig. 1a, c) in Transmission electron microscopy. In DLS, the diameter of the vesicles was found to be the 40-210 nm (Fig. 1b) for the sensitive strain and 80-300 nm (Fig. 1d) for the MDR strain. The diameter obtained by DLS method depends on the concentration of the particles, the diffusion coefficient, the viscosity of the solvent, surface structure, and the type of ions in the medium. Hence, the size measured by this technique can be larger than the value obtained by electron microscopy.

Separation, identification, and analysis of OMV proteins
A comparative profile of whole-cell extract (WC), inner membrane(IM), and outer membrane (OM) vesicles of both sensitive and MDR on SDS PAGE shows that the proteins present in OMVs were also present in WC extract and OM (Fig. 2). The protein profiles clearly indicates a selective sorting of proteins in the vesicles of both the strains. The comparative protein profile picture of OMVs shows that there are more bands in OMVs of MDR as compared to that of sensitive strain.
The protein bands form the OMVs were cut and subjected to in-gel digestion using trypsin. The peptides were extracted using standard protocols and subjected to LC coupled ESI MS/MS studies. The proteins were identified by SEQUEST. A total of 320 proteins (Supplementary table S1) were identified in sensitive OMVs, and 560 proteins (Supplementary table S2) were identified in MDR OMVs. An overlap of 15 proteins was observed, whereas 545 proteins were exclusively present in MDR OMVS and 305 proteins were specific to sensitive OMVs. It is possible that the OMVs of MDR strain might have released more proteins under stress conditions.

Functional analysis of the identified proteins related to antibiotic resistance
The proteins related to antibiotic resistant present in the OMVs of MDR strain and their functional annotation was predicted with the help of Uniprot KB and literature review. The proteins that confer resistance to aminoglycosides, ampicillin penicillin-binding proteins, carbapenamase enzymes and the metallo-beta-lactamase enzymes and other proteins were identified from the OMVs of the MDR strain. These important enzymes for imparting antibiotic resistance were found to be present in OMVs of multidrug-resistant strain. The list of proteins identified along with the predicted function is shown in Table 1. We have found 24 drug resistance proteins in Ab71 strain whereas Ab69 contains only 8 proteins as shown in Table 2. These results clearly demonstrate that most of the drug resistance proteins are enriched in OMVs of the resistant strain than normal. This observation suggests that OMVs play an essential role in drug resistance mechanism of A. baumanii. This finding in accordance with previous reports suggests that dissemination of carbapenem resistance gene via OMVs of A. baumanii ).

Outer membrane vesicles mediate protection against antimicrobial peptides
Secreted OMVs might help to defend cells against outer membrane-acting antibiotics based on the nearly identical surface constituents of the OMVs and the bacterial outer membrane. In the present study, it was observed that OMVs protect the producer bacterium against growth inhibitory effects of membrane active antimicrobial peptide polymyxin B. The extent of protection provided by OMVs was studied in the both sensitive and MDR strain. The minimum inhibitory concentration of polymyxin B for sensitive strain was 2 μg of polymyxin and for resistant strain it was found to be 4 μg of polymyxin B. To study the effect of OMVs on the growth of bacteria, polymyxin B was added above its growth inhibitory concentration, i.e., 2.5 μg for sensitive strain and 4.5 μg for resistant strain. And OMVs were added in increasing concentration. The concentration of OMV used for both the sensitive and MDR strains was 1, 2, 4, 6, 8, 12, and 16 μg and for the OMVs of MDR supplemented in sensitive strain was 1, 2, 4, 6, and 8 μg. From this study, it was observed that OMVs protected both the stains against the antimicrobial effect polymyxin B. The concentration OMVs required in both the stain was different. In Ab69, it required the higher concentration, i.e., 12 μg OMVs as compared to MDR strain which required lower concentration, i.e., 4 μg OMVs (Fig. 3a, b). The corresponding viable counts were calculated. No growth was observed in the antibioticcontaining plate and plate containing only purified OMVs. The viable count observed in the absence of antibiotics was taken as 100%. The percentage of survivors in the presence different concentrations of OMVs was calculated by counting the CFUs. When the sensitive strain was supplemented with OMVs of MDR strain it was seen that it protected the bacterium at lower concentration (4 μg) as shown in Fig. 3. These results show that OMVs played a role in protecting the bacteria from membrane active antimicrobial peptide polymyxin B.
The growth inhibition effect of the indole derivative was also studied. Earlier studies demonstrated that the production of OMVs is regulated in the presence of some indole derivatives (Tashiro et al. 2010). Studies from our laboratory and elsewhere (Kulkarni et al. 2014(Kulkarni et al. , 2015Manning and Kuehn 2011) revealed that OMV protects bacteria against some antibacterial molecule. Combining these observations, it appears rational to postulate that the activity of antibacterial might be increased in the presence of antibacterial molecule polymyxin B. To test this prediction we have grown the A. baumanni normal and MDR strains in the presence of ethyl-2-(3-indolyl)-4-methyl-thiazole-5-corboxylate and polymyxin B. It is observed that the normal strain, the growth is inhibited at higher concentrations of the indole derivative, whereas in the MDR strain the effect is less as compared to normal strain as shown in Fig. 4. These observations suggest that the usage of indole derivative may be reducing the production of OMVs coupled with the sensitizing the antibacterial molecule activity and exhibits the activity. In the case of MDR srtain, the activity of indole derivative is not as pronounced as in normal strain. This may be due to the presence of more drug resistant protein in the OMVs of MDR strain. These studies suggest that using antibiotics in combination with appropriate indole derivatives may help in reducing the quantity of antibiotic usage. However, several studies are needed to evaluate the effect of Indole derivatives, before any clinical applications.

Presence of degradosome complex enzymes in the OMVs of the MDR strain
In bacteria RNA maturation takes place with the help of several ribonucleases and some of them organize as protein complexes. These complexes were identified in several bacterial species including Escherichia coli, Pseudomonas syringae Lz4W, Rhodobacter capsulates (Liou et al. 2001;Purusharth et al. 2005;Jager et al. 2001). In this complex is organized by the endoribonuclease RNase E. This is tightly associated with an exoribonulcaese polynucleotide phosphorylase, the DEAD-box helicase PhlB, and enolase. Other proteins Polynucleotide kinase, Poly (A) polymerase, the cheperons DnaK and GroEL were also implicated to be present in the complex (Purusharth et al. 2005). Interestingly the proteins RNase E, Dna K and other proteins present in the degradosome complex of several bacteria are found to be segregated in to the OMV of the MDR strain of A. baumanni. These observations indicate the OMVs carrying the degradome complex proteins can be transported in the host systems and elicit the reactivity of regulating/controlling the host RNA maturation. Thus, this is the first time it was shown such a complex proteins were carried by the OMVs.  Ramos et al. (2005) 1 3 OMVs are known to interact with host cells in different ways and transfer their contents to host (O'Donoghue and Krachler 2016). The interaction of OMVs cargo with human cell line like small intestine epithelial cell lines, colon epithelial cell lines and airway epithelial cell lines were studied (Lynch and Alegedo 2017). OMVs are capable to act at the interface of bacteria and host and mediate interorganismal interactions. The role of OMVs in cell proliferation, apoptosis and autophagy are being studied in detail. The cell type and organism plays a crucial role in this interaction. There is an urgent requirement to understand the functions of OMVs with multicellular organisms.

Conclusion
A. baumannii is one of the most nosocomial MDR and most pathogenic organism. The OMVs released from MDR strain contains more proteins related to antibiotic resistant. They are found to contain carbapenemase, and metallo-beta lactamase and other enzymes which are threatening mechanism of antibiotic resistance. OMVs released from the strains of A. baumannii protected the bacterium against the membrane active antibiotic polymyxin B. Even the OMVs of MDR strain protected the sensitive strain from polymyxin B at lower concentration as compared to the OMVs of sensitive strain. The OMV isolated from the MDR strain contains more drug resistant proteins as compared to the OMVs from the normal strain. So OMVs are playing a role in providing antibiotic resistant to the bacteria. Moreover, the OMVs are also found to carry degradosome complex proteins which control the host RNA maturation. The role of degradosome complexes present in OMVs warrants further studies.
Control Ab69 strain alone. b A. baumannii 71 growth in the medium with presence of different concentration of indole derivative (3, 4, 5, 6 μg) and polymyxin is 5 μg. Control Ab71 strain alone