microorganisms Review The Role of Bacterial Membrane Vesicles in the Dissemination of Antibiotic Resistance and as Promising Carriers for Therapeutic Agent Delivery 1, 1, 1 2 3, Md Jalal Uddin y , Jirapat Dawan y, Gibeom Jeon , Tao Yu , Xinlong He * and Juhee Ahn 1,* 1 Department of Medical Biomaterials Engineering, College of Biomedical Science, Kangwon National University, Chuncheon, Gangwon 24341, Korea; [email protected] (M.J.U.); [email protected] (J.D.); [email protected] (G.J.) 2 Shandong Institute of Parasitic Diseases, Shandong First Medical University & Shandong Academy of Medical Sciences, Jining 272033, China; [email protected] 3 Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou 225001, China * Correspondence: [email protected] (X.H.); [email protected] (J.A.); Tel.: +82-33-250-6564 (J.A.) These authors contributed equally to this work. y Received: 29 March 2020; Accepted: 2 May 2020; Published: 5 May 2020 Abstract: The rapid emergence and spread of antibiotic-resistant bacteria continues to be an issue difficult to deal with, especially in the clinical, animal husbandry, and food fields. The occurrence of multidrug-resistant bacteria renders treatment with antibiotics ineffective. Therefore, the development of new therapeutic methods is a worthwhile research endeavor in treating infections caused by antibiotic-resistant bacteria. Recently, bacterial membrane vesicles (BMVs) have been investigated as a possible approach to drug delivery and vaccine development. The BMVs are released by both pathogenic and non-pathogenic Gram-positive and Gram-negative bacteria, containing various components originating from the cytoplasm and the cell envelope. The BMVs are able to transform bacteria with genes that encode enzymes such as proteases, glycosidases, and peptidases, resulting in the enhanced antibiotic resistance in bacteria. The BMVs can increase the resistance of bacteria to antibiotics. However, the biogenesis and functions of BMVs are not fully understood in association with the bacterial pathogenesis. Therefore, this review aims to discuss BMV-associated antibiotic resistance and BMV-based therapeutic interventions. Keywords: outer membrane vesicle; antibiotic resistance; virulence; vaccine; therapeutic agent delivery 1. Introduction Over the last few decades, antibiotic resistance in bacteria has been a serious global threat to public health [1]. Antibiotic-resistant bacteria can survive current antibiotic regimens, resulting in frequent therapeutic failure [2]. The emergence of antibiotic-resistant bacteria spurred the necessity of developing new antibiotics [3,4]. The fundamental understanding of antibiotic resistance mechanisms is an important step in the development of effective therapeutic regimens. The intracellular levels of antibiotics are synergistically regulated by efflux pump systems and membrane permeability barriers [3]. Recently, it has been recognized that bacterial membrane vesicles (BMVs) may play a role in antibiotic resistance. Therefore, understanding the roles of BMVs can provide directions for the control of antibiotic-resistant pathogens. The structural features of the bacterial outer membrane play an important role in the rapid adaptation to environmental stresses such as cold, heat, and antibiotic treatments, resulting in the evolution of antibiotic resistance in bacteria [2,5]. Therefore, the structure, biogenesis, function, Microorganisms 2020, 8, 670; doi:10.3390/microorganisms8050670 www.mdpi.com/journal/microorganisms MicroorganismsMicroorganisms2020 2019, 8, ,7 670, x FOR PEER REVIEW 22 of of 23 22 regulation of BMVs could be a new research area in connection with antibiotic resistance [6]. andAlthough regulation the biological of BMVs functions could be aof new BMV research-containing area components in connection have with been antibiotic considered resistance importan [6].t Althoughfor understanding the biological the mechanisms functions of BMV-containingrelated to antibiotic components resistance, have there been is consideredstill a lack of important information for understandingon the biogenesis the mechanismsof BMVs in terms related of to antibiotic antibiotic resistance resistance, [7] there. Therefore, is still a lackthis ofreview information addresses on thethe biogenesispossible roles of BMVs of BMVs in terms in the of control antibiotic and resistanceprevention [7 of]. Therefore,the emergence this reviewof antibiotic addresses-resistant the possiblebacteria. roles of BMVs in the control and prevention of the emergence of antibiotic-resistant bacteria. 2. Terminology and Characteristics of Bacterial Membrane Vesicles 2. Terminology and Characteristics of Bacterial Membrane Vesicles The term BMV has been used to describe various extracellular substances, known as outer The term BMV has been used to describe various extracellular substances, known as outer membrane membrane vesicles (OMVs), which are specifically released from Gram-negative bacteria. Similarly, vesiclesGram-positive (OMVs), bacteria which are and specifically archaea releasedproduce from vesicles, Gram-negative known as bacteria. membrane Similarly, vesicles Gram-positive (MVs), and bacteria and archaea produce vesicles, known as membrane vesicles (MVs), and eukaryotic bacteria eukaryotic bacteria secrete surface and cellular lipids and proteins, named exosomes or microvesicles secrete[8–10]. surfaceTherefore, and the cellular term OMV lipids is and not proteins, inclusive named as there exosomes are many or vesiclemicrovesicles-producing [8–10 Gram]. Therefore,-positive the term OMV is not inclusive as there are many vesicle-producing Gram-positive bacteria. The BMV bacteria. The BMV could be an inclusive term for membrane vesicles released from both Gram- could be an inclusive term for membrane vesicles released from both Gram-negative (BMVGN) and negative (BMVGN) and Gram-positive (BMVGP) bacteria. The BMVs are nano-sized spherical Gram-positivemembrane particles (BMVGP )released bacteria. Thefrom BMVs the arebacterial nano-sized membranes, spherical membraneencapsulat particlesing proteins, released toxins, from thepeptidoglycan, bacterial membranes, lipopolysaccharide encapsulatings (LPSs) proteins,, and nucleic toxins, acids peptidoglycan, [11]. The B lipopolysaccharidesMVs have less than 370 (LPSs), kbp andin DNA nucleic and acids are [1011–].300 The nm BMVs in diameter have less [6,12] than. 370The kbp BMVs in DNA play an and important are 10–300 role nm in in bacterial diameter cell [6,12-to].- Thecell BMVsinteractions play an [13] important. The rolestruc intural bacterial characteristics cell-to-cell interactionsof BMVs (Figure [13]. The 1) structuralcontribute characteristics to bacterial ofresistance BMVs (Figure to different1) contribute types of to environmental bacterial resistance stresses to different [2,5]. types of environmental stresses [2,5]. A B FigureFigure 1. 1.Structural Structural characteristics characteristics ofof Gram-negativeGram-negative( A(A)) and and Gram-positive Gram-positive ( B(B)) bacterial bacterial membrane membrane vesicles.vesicles. 3.3. IsolationIsolation andand PurificationPurification of of Bacterial Bacterial Membrane Membrane Vesicles Vesicles Isolation,Isolation, purification, purification, and and storage storage techniques techniques have have been been developed developed to to collect collect BMVs, BMVs, whichwhich areare essentialessential steps steps for for understanding understanding structural structural and functional and functional characteristics characteristics of BMVs [of14 ].BMVs Those [14] techniques. Those includetechniques conventional include conventional gradient centrifugation, gradient centrifugation, column chromatography,immune column chromatography, affinity-based immune separation, affinity- andbased the separation, proteomic approach and the [14proteomic–16]. The approach amount and [14 content–16]. The of BMVs amount varies and depending content of on B theMVs bacterial varies growthdepending conditions on the and bacterial genetic growth variation conditions [17]. High and purity genetic is essential variation to characterize [17]. High BMVspurity and is essential applies for to deliverycharacterize system BMVs and vaccineand applies development for delivery [18]. Differential system and centrifugation vaccine development is used to remove [18]. D non-BMVsifferential in bacteria by serial centrifugation from 300 to 2000 g, and 10,000 to 100,000 g [19]. However, centrifugation is used to remove non-BMVs in bacteria× by serial centrifugation ×from 300 to 2000× g, theand differential 10,000 to 100,000 centrifugation× g [19] technique. However, provides the differential low yield centrifugation and insufficient technique purity due provides to the repetitivelow yield ultracentrifugationand insufficient [purity20]. Density due gradientto the ultracentrifugationrepetitive ultracentrifugation is applied to[20] increase. Density the separation gradient efficiencyultracentrifugation of BMV particles is applied according to increase to the the unique separation buoyant efficiency densities of BMV [21]. particles In addition, according this method to the increasesunique buoyant the yield densities of BMVs in[21] terms. In addition, of the purity this of method BMV fraction increase ands the the yield quantity of BMVs of BMV in proteinsterms of and the RNAs.purity Hence,of BMV the fraction density and gradient the qua ultracentrifugationntity of BMV proteins method and is consideredRNAs.