extfieldsofstudy: Biology Medicine

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Antibiotics, Resistome and Resistance Mechanisms: A Bacterial Perspective

History of mankind is regarded as struggle against infectious diseases. Rather than observing the withering away of bacterial diseases, antibiotic resistance has emerged as a serious global health concern. Medium of antibiotic resistance in bacteria varies greatly and comprises of target protection, target substitution, antibiotic detoxification and block of intracellular antibiotic accumulation. Further aggravation to prevailing situation arose on observing bacteria gradually becoming resistant to different classes of antibiotics through acquisition of resistance genes from same and different genera of bacteria. Attributing bacteria with feature of better adaptability, dispersal of antibiotic resistance genes to minimize effects of antibiotics by various means including horizontal gene transfer (conjugation, transformation, and transduction), Mobile genetic elements (plasmids, transposons, insertion sequences, integrons, and integrative-conjugative elements) and bacterial toxin-antitoxin system led to speedy bloom of antibiotic resistance amongst bacteria. Proficiency of bacteria to obtain resistance genes generated an unpleasant situation; a grave, but a lot unacknowledged, feature of resistance gene transfer.

INTRODUCTION
Antibiotics, representing both naturally as well as chemically synthesized entities, emerged as a powerful tool in counteracting infectious diseases, following serendipitous discovery of penicillin from Penicillium notatum by Alexander Fleming in 1928. Widespread usage of antibiotics that imposes strong selection pressure for resistance development (ability to withstand effects of antibiotics) took a strong grip over the health care system globally as concerns regarding resistance to available drug regime restrict therapeutic options available to treat the disease. Emergence of resistance at rapid pace made the pathogens well-fit and well-adapted, resulting in causing serious life threatening complications as we lack robust drugs to curb the menace of multidrug resistance. Growing menace of antibiotic resistance is inevitable fallout of the introduction of new antibiotics aimed at long-term efficacy in the treatment of infectious diseases. Deteriorating public health ensuing emergence among pathogenic and commensal bacteria of resistance, illustrates a grave predicament globally (Bennett, 2008). Steadily increase in the development of resistance among bacteria thwarts current treatment regimes in hospitals and community settings. Through each passing day, treatments of infectious diseases require administration of high doses of antibiotics and longer stay in hospital. Widening gap between lean productions of drugs increases need of either to rejuvenate the drying antibacterial pipelines or design innovative strategies to combat bacterial antibiotic resistance. The present review analyses development of resistance and focusing on the factors that regulate acquisition of resistant determinants.

Chloramphenicol
It acts as a broad spectrum antibiotic resistance mechanism for chloramphenicol involves enzymatic inactivation via acetylation mediated by chloramphenicol acetyltransferases (CATs) (Schwarz et al., 2004;Wright, 2005). Apart from enzyme inactivation chloramphenicol resistance mechanisms, also involves inactivation by phosphotransferases, target site mutation, permeability barriers and efflux pumps (Schwarz et al., 2004).

Glycopeptide
The vancomycin resistance originated from the production of modified peptidoglycan precursor, d-Ala-d-Lac (VanA, VanB, and VanD) or d-Ala-d-Ser (VanC, VanE, and VanG), to which glycopeptides display diminished binding affinities. The vanA and vanB operons are positioned on plasmids as well as on chromosome; whereas the vanC1, vanC2/3, vanD, vanE, and vanG solely show their presence on chromosomes (Klare et al., 2003;Depardieu et al., 2007).

Quinolone
Though resistance mechanism for quinolone was found restricted to chromosomes, three plasmid-mediated resistance mechanisms have also been reported (Courvalin, 2008;Martinez-Martinez et al., 2008). The chromosome-encoded resistance produce a declined outer-membrane permeability linked with porin loss, while over expression of the naturally existing efflux pumps create mutations in the molecular targets, DNA gyrase and topoisomerase IV (Hooper, 2000;Jacoby, 2005). Mutations were found occurring at quinolone resistance determining regions (QRDR) in the genes gyrA, gyrB, parC, and parE; which program the subunits of DNA gyrase and topoisomerase IV. Despite the fact that qnr determinant is the first recognized plasmid-mediated quinolone resistance gene, five new lineage of qnr genes have been accounted: qnrA, qnrB, qnrC, qnrD, and qnrS. Second kind of plasmid positioned quinolone resistant gene is a cr variant of aac(6)-Ib, that is aac(6)-Ib-cr, encoding aminoglycoside acetyl transferase (Park et al., 2006;Strahilevitz et al., 2009). The third means of resistance involves qepA, a plasmid-mediated efflux pump along with its E. coli derivative QepA2 (Cattoir et al., 2008), is able to expel hydrophilic fluoroquinolones, e.g., ciprofloxacin (Perichon et al., 2007).

Chloromphenicol
It is made of nitrobenzene ring consisting of nitro and dichloroacetyl group. Efflux mediated resistance AcrF efflux pumps have been known to cause resistance. Reynolds, 1989Kluytmans et al., 1997Lina et al., 1999 5.

Quinolone
Quinolone antibiotics possess a quinolone core that typically has a N linked cyclic moiety and various substituents at the C(6) and/or C(7) positions.
The most critical changes to the quinolone skeleton were the introduction of a fluorine at position C6 and a major ring substituent (piperazine or methyl-piperazine) at Polymyxins, which are polycationic, displace stabilizing magnesium and calcium ions to electrostatically interact with the anionic lipopolysaccharide (LPS) outer layer of gram negative cell membranes. This disrupting interaction leads to increased cell membrane permeability, cell leakage, and rapid cell death. (Yu et al., 2015) Biswas et al., 2012Velkov et al., 2010Liu et al., 2016 Frontiers in Microbiology | www.frontiersin.org and sul2 were described after 1980s (Swedberg and Sköld, 1983;Rådström and Swedberg, 1988). In addition a third plamid mediated gene sul3 has also been recognized (Perreten and Boerlin, 2003).

Colistin
Modifications in the lpxA, lpxC, and lpxD genes of A. baumannii result in neutralization of lipid A biosynthesis, causing total loss of LPS leading to a loss of the polymyxin target (Moffatt et al., 2010). Polymyxin resistance is controlled by twocomponent systems PhoP/PhoQ and PmrA/PmrB (Olaitan et al., 2014), which react to cation (calcium, iron, and magnesium) concentrations and pH variations. These systems are concerned in the alterations of LPS resulting in polymyxin resistance. Several molecular mechanisms have been associated with colistin resistance in Gram-negative bacteria, like modifications with PmrA/PmrB, PhoP/PhoQ, ParR/ParS, ColR/ColS, and CprR/CprS two-component systems and alterations in the mgrB gene, that codes for negative regulator of PhoPQ. Addition of cationic groups on lipid A due to mutations creat less anionic lipid A ultimately causing less fixation of polymyxins. The polymyxins remains one of the last classes of antibiotics in which resistance is not known to spread from cell to cell via plasmid mediated. There is a current report of plasmid mediated colistin resistance in china designated as mcr-1 gene (Liu et al., 2016) which is also reported closely in five continents viz, Asia, Europe, Africa, North America, and South America (Schwarz and Johnson, 2016). Antibiotics do not, in themselves, cause resistance but frequent and high exposure of antibiotics to bacteria creates a selection pressure which triggers resistance strategies of bacteria. Acquirement of resistance genes has been viewed as main donor in favor of the extensive dispersal and increase in antimicrobial resistance through horizontal transfer involving MGEs (Xu et al., 2011). Their presence on mobile genetic elements facilitate transfer to un-related bacteria in a process referred to as horizontal gene transfer (HGT) via, conjugation, transduction, or transformation (Aminov and Mackie, 2007;Martinez, 2008). Transformation involves movement of cellular DNA among closely linked bacteria, imparted by chromosomal set of proteins that occur in naturally transformable bacteria. Conjugation needs autonomously replicating genetic elements known as conjugative plasmids that cause movement of plasmid from the donor cell to a recipient cell that is devoid of it. Transduction involves transfer of DNA facilitated by bacteriophages, constituted host DNA in their capsid and insert this DNA into a new host, where it combines with cellular chromosome and is inherited (Frost et al., 2005). Movement of genes confers new metabolic capabilities to the recipient, thereby helps them in their adaptation to new ecological niches. Resistance to antibiotics conferred by chromosomal or mobile genetic elements, is achieved by following strategies: (i) reduction of membrane permeability to antibiotics either by decreasing uptake or increasing efflux, (ii) drug inactivation either by hydrolysis or by modification, (iii) alteration in drug target and decreased binding permeability, and (iv) mutation (Walsh, 2000; Figure 1).

GENETIC BASIS OF ANTIBIOTIC RESISTANCE
Bacteria appeared on this planet billion of years ago, so have their skills sharpened due to genomic flexibility at shielding themselves from toxic chemicals. Bacteria are well-known potent originators for the dissemination of antimicrobial-resistant genetic apparatus (Woodford et al., 2011). They are competent to offer secured platform for the upholding and transmission of genes accountable for antimicrobial resistance as part of mobile genetic elements (MGE; plasmids, transposons and integrons). The transposons and integrons, owing to their genomic plasticity have contributed a great deal to the fitness quotient and robustness of bacteria to survive in varying environments. Integrons, typically transported by plasmids or enclosed in transposons, performing the task of resistance gene dissemination plays an important role in the revealing of Super Bugs (Xu et al., 2011). Since its earliest assessment in 1989 (Stokes and Hall, 1989) molecular mechanisms involved in the mobility of integrons, their excision and integration for gene cassettes, is currently being scrutinized (Hall et al., 1999;Mazel, 2006). Establishing role of MGEs in genomic evolution justifies the predictions of Barbara McClintok that transposons play a major role in the genomic diversity and evolution. Owing to their capacity to relocate between host genomes, MGEs play a vital function of acting as vehicles for resistance gene acquisition and their successive propagation.

Resistance Mediated by Plasmids
Plasmids that mediate horizontal movement of plasmidborne genes are accountable for global spread of resistance (Carattoli, 2013). Resistance plasmids (attributing resistance to commonly used antibiotics) are mostly conjugative; additional are mobilizable. Conjugative plasmids display both broad (no host restriction within the division) and narrow (shifting limited to small number of related bacterial groups) host range. Genes acquired through homologous recombination, integration and excision from the host chromosome relocate from donor to recipient cells by conjugation. These type of plasmid-encoded complexes help the contributor by attaching to promissing recipient that lead to the generation of secured association, required prior to the relocation of DNA. Plasmids that fails to get reloctaed by this approach are transferred by conjugative elements subsequent to the development of transitory or steady fusions called cointegrates. Plasmids also encourage cell contact development through production of pheromone influenced microfibrillar exterior covering substances. Mobilizable plasmids carry DNA transfer genes essential for structure of all or element of the relaxasome, but are deficient of genes essential for mating pore formation. They have a capacity to use conjugative plasmids FIGURE 1 | Various ways of resistance mechanisms to counteract effect of antibiotics. Horizontal Gene transfer facilitates transfer and exchange of genetic material among bacterial cells. Transformation involves direct uptake of genetic material from the surrounding by competent recipient having chromosomal set of proteins. Transduction involves DNA insertion into chromosome as prophage which then replicates, packages host DNA alone or in combination with the host cell chromosome. Conjugative plasmids utilize a protein structure pilus to make a link with the recipient cell so as to move them into the recipient cell that ultimately transfers the copy of entire bacterial chromosome, multicopy plasmid or a small portion to a recipient cell, where these genetic elements insert into the chromosome or replicate independently if compatible with the inhabitant plasmids. Integrons use site specific recombination mechanism where it provides a promoter for gene cassettes to exchange and disseminate. Transposons and insertion sequences insert into new sites on the chromosome or plasmids by non-homologous recombination and increase the copy number of transferred genes giving rise to chromosomal mutations, deletions and rearrangements.
for horizontal spread; these are immobile in cells which are short of mobile elements carrying compatible mating-pore genes. The majority of known mobilizable plasmids utilize conjugative element mating-pores by expressing their own relaxase (Mob) which works on the plasmid's cognate oriT (Joshua et al., 2016). Current studies have revealed that plasmid transfer can also take place even when the mobilizing plasmid and the plasmid being mobilized are in two diverse bacterial cells (Andersen and Sandaa, 1994;Sia et al., 1996). This type of recruitment, in which a contributor strain possessing a self-transmissible plasmid is getting a second plasmid from a receiver strain, is known as retrotransfer. Retrotransfer take place through two stages; (1) self-transmissible plasmid move from the contributor to the receiver, and (2) mobilization of plasmid from receiver back to the contributor (Ankenbauer, 1997). As the capacity of a self-transmissible plasmid to promote the acquirement of novel plasmids by its bacterial host possibly grant a benefit to the contributor bacterium, it can be said retrotransfer may play a significant job in the progression of plasmid transfer system.
Transphylum mobilization events that incorporate elements from entirely diverse phylogenetic group of bacteria, underscores broad range of interactive capacity originated in gene relocation elements. Besides being recognized through PCR amplification of identified incompatibility groups (Götz et al., 1996), mobilizing plasmids are identified by conducting a triparental mating among E. coli having a mobilizable IncQ plasmid, a recipient lacking plasmid and amalgam of local soil or marine bacteria (Top et al., 1994). Endurance of plasmids in natural isolates and their perceptible firmness in absence of antibiotics, opposes a common thought that in non-existence of selection pressure plasmids additional gene transport factors are easily lost. The multitude of antibiotic-resistant strains in environmental milieu where bacteria apparently do not appear to be in touch with antibiotics, suggest that resistance genes can also be firmly retained even in the paucity of antibiotic selection (Andersen and Sandaa, 1994;McKeon et al., 1995;Calva et al., 1996). As plasmids exhibit remarkable property of crossing species borders effortlessly (Hatch and Michael, 2011) co-mobilization of resistance genes aggravates furthermore clinical crisis.

Resistance Mediated by Transposons
Transposable elements (TE) are the DNA sequences that provide flexibility to the genome (Archana et al., 2013). Being proficient to alter their position, they are able to alter their genetic background along with that they change the genetic setting of the locus, where they get inserted (Wicker et al., 2007;Shapiro, 2010). Based on their role in identification and recombination of particular sequences, TEs are categorized into two classes; composite transposons (Class I; holding a range of resistance genes which possess identical structural and functional characteristics, but small DNA homology) and complex transposons (class II; constituting three dissimilar but interrelated families; Tn3, Tn21 and Tn2501; Schmitt, 1986;Wiedemann et al., 1986;Lafond et al., 1989). Some of the composite transposons in gram negative bacteria are Tn5, Tn9, Tn10, Tn903, Tn1525, and Tn2350 and among gram positive bacteria are Tn4001 and Tn4003. Compared to Tn1, Tn3, Tn21, Tn501, Tn1721, and Tn3926 found among gram negative bacteria, gram positive bacteria encompass Tn551, Tn917 and Tn4451 complex resistance transposons. These components are possessed with the capability to progress both intra and inter-molecularly which means they can jump within a DNA molecule or from one DNA molecule to another (Bennett, 2008). Tn21 being majorly studied, bear OXA (a carbapenems, possess oxacillinase activity) and PSE (β-lactam gene Pseudomonas specific enzyme) determinants that makes them resistant to aminoglycoside antibiotics. Tn21 also show resistance toward mercury compounds (Brown et al., 1986) and trimethoprim imparted by dhfr II and V (Sundström et al., 1988). Class I or retro-transposons work by copying RNA from DNA by transcription and RNA to DNA by reverse transcription; thereby get inserted into the genome at a diverse location (Kapitonov and Jurka, 2008). Acting in cut and paste manner, class II transposons does not involve RNA intermediate (Wicker et al., 2007). These, transposases create staggered cut at specific site, creating sticky ends; following its transposition to the aimed site, generally followed by target site duplication and construction of short direct repeat at insertion sites (Madigan et al., 2006). Though transposons provide antibiotic resistance due to the existence of an extra gene on a plasmid, there are chances that transposons can jump from chromosomal DNA to plasmid DNA and vice versa for development of resistance (Wagner, 2006). Insertion Sequences (ISs; size < 2.5 kb) are basic form of mobile genetic elements disseminated in bacteria. ISs are contemplated as non-complex bacterial mobile DNA taking into account their structure (Allaaeddin El et al., 2013). They include more than 19 families, having dissimilar size (Wagner et al., 2007). ISs include an open reading frame that codes for a transposase enzyme, surrounded by inverted repeat sequences of 10-40 base pairs at both ends. The transposase enzyme cuts target DNA and inserts the IS due to possible association with the inverted repeat sequences. Exhibiting fondness toward ATrich region of DNA, higher chances of undergoing homologous recombination, creates variety of possibilities such as deletions, inversions and duplications. There are evidences that when two identical IS elements surround a region of DNA, a composite transposon is produced, and the total interceded DNA flanked by the terminal inverted repeats get mobilized by one or both of the IS coded transposases (Ochi et al., 2009;Gyles and Boerlin, 2014).

Resistance Mediated by Integrons
Integrons attribute a great deal to the fitness quotient and robustness of bacteria to survive in varying environments. Harboring resistance determinants such as antibiotic resistance genes, their mobilization as part of chromosomes and plasmids and integration far off from their origin confer resistance to antimicrobials. Their categorization is based on amino acid sequences of integrase IntI; those carrying IntI1 are referred as class 1, IntI2 as class 2, IntI3 as class 3 and so on. Integrase IntI1, IntI2, and IntI3 were found associated with mobile genetic elements, while IntI4 was found linked with chromosomal integrons.

Class 4 Integrons
Class 4 integrons are distinguished from other Resistance Integrons (RIs) by two key features; incorporation of hundreds of cassettes (V. cholerae, 216 unidentified genes in an array of 179 cassettes, holding roughly 3% of the genome) and the high similarity between the attC sites of those assembled cassettes (Poirel et al., 2010). Class 4 integrons carry gene cassettes for antibiotics chloramphenicol and fosfomycin (Fluit and Schmitz, 2004). Inspite of huge array of cassettes, identification of class 4 integron has been limited within members of Pseudomonas, Xanthomonas, Shewanella, Vibrionaceae, and other proteobacteria (Clark et al., 2000;Rowe-Magnus et al., 2001).

Integrative and Conjugative Elements
Integrative and conjugative elements (ICEs) were first of all anticipated by Burrus et al. (2002) are different mobile elements found in both Gram-positive and Gram-negative bacteria. These are self transmissible integrative elements that code for complete match of conjugation apparatus. ICEs can integrate into and excise from a host chromosome. These versatile entities support their own mobilization facilitating horizontal transfer of antibiotic-resistant genes, virulence factors and various bacterial traits. ICEs possess three genetic modules: (i) integration and excision module; (ii) conjugation module; and (iii) regulation module. These modules contain different array of genes that code for proteins operating by distinct mechanisms. ICEs contain a gene encoding an integrase (Int) that promotes site-specific integration and excision of the element, frequently into a unique site on the chromosome of the host organism (Boyd et al., 2009). Some ICEs bear maintenance modules such as toxin-antitoxin systems (Wozniak and Waldor, 2009) and additional partition systems that guarantee thriving vertical inheritance of these elements. In contrast to plasmids, ICEs, are not found in extrachromosomal state, because they lack autonomous replication, the first known MGEs with ICE-like properties were Tn916 in Enterococcus faecalis and CTnDOT in Bacteriodes thetaiotaomicron; Franke and Clewell, 1981;Shoemaker et al., 1989). Bacteroides CTnDOT promote dissemination of antibiotic-resistant genes (Whittle et al., 2002). ICEs are distinguished by elementspecific properties although they possess a general life cycle and modular structure. Apart from resistance to antibiotics (Böltner et al., 2002;Whittle et al., 2002) ICEs show a extensive collection of phenotypes on their hosts, including resistance for heavy metals (Böltner et al., 2002;Davies et al., 2009) and the power to degrade aromatic compounds (Ravatn et al., 1998). In addition, complex traits such as the capacity to inhabit a eukaryotic host (Sullivan and Ronson, 1998) fix nitrogen (Sullivan and Ronson, 1998) or encourage virulence and biofilm development have been recognized (Drenkard and Ausubel, 2002;He et al., 2004;Davies et al., 2009) the connection between ICEs and the propagation of antibiotic resistance genes in some pathogens show that these mobile elements have considerable clinical significance (Hochhut et al., 2001;Whittle et al., 2002;Mohd-Zain et al., 2004). ICEberg (http://db-mml.sjtu.edu.cn/ICEberg/) is an integrated database that provides comprehensive information about integrative and conjugative elements (ICEs) found in bacteria (Bi et al., 2012).

Bacterial Toxin Anti-toxin Systems
Toxin-antitoxin (TA) systems, initially identified as plasmid addiction modules, are plentiful in the chromosomes of most free-living bacteria (Xie et al., 2018). TA systems provide endurance to bacterial populations in conditions of stress like nutrient deprivation or antibiotic pressure (Harms et al., 2016). Generally TA systems are made of a stable toxin and a labile antitoxin coded by a bicistronic locus (Lee and Lee, 2016). Toxin genes encode for proteins, while the antitoxin genes code for RNAs or antitoxin proteins, classifying them as type I-VI TA loci (Gerdes and Maisonneuve, 2012;Chan et al., 2016;Page and Peti, 2016) categorized due to mechanisms applied by the antitoxins to counteract the actions of the toxins. In TA systems I-VI product of the toxin gene is typically a protein, whereas the antitoxin gene is either a non-coding RNA among TA I and III or a low-molecular-weight protein in TA systems II, IV, V and VI. Toxins work on diverse targets to distress various cellular processes such as DNA replication, cell wall synthesis or protein synthesis. Amongst six types of TA system, type II is broadly studied, due to great quantity and high quality of freely accessible data. Currently two openaccess bioinformatics resources in the field of type II TA loci, the online tool RASTA (Sevin and Barloy-Hubler, 2007) and the web-based database TADB (Shao et al., 2011) are available.
Presently, a new toxin is reported that contains a Gcn5related N-acetyltransferase (GNAT) domain that transfers the acetyl group from acetyl coenzyme A (Acetyl Co∼A) to the amine group of tRNAs (Jurenas et al., 2017b;Van Melderen and Wood, 2017) resulting in acetylation of tRNAs following inhibition of translation in bacterial cells. Similarly TacT of Salmonella enterica Typhimurium (Cheverton et al., 2016) and AtaT of Escherichia coli O157:H7 (Jurenas et al., 2017a), also transfer the acetyl group from acetyl coenzyme A to the amine group of the tRNAs, In stress environment, the intensity of the alarmone molecule, (p)ppGpp, is amplified, which activates a particular proteinase that degrades the antitoxin by proteolytic cleavage, thus permitting the toxin to stop cell expansion. The  Hassan et al., 2012Gordya et al., 2017Maria-Neto et al., 2015Kościuczuk et al., 2012Kaur et al., 2016 Defensins They are a group of AMPs containing α-helix/β-sheet elements coordinated by three disulfide bridges.
They are effective against Gram positive bacteria. Lehrer et al., 1989;de Leeuw et al., 2010 Cecropins They are linear amphipathic α-helical AMPs They act selectively active against Gram-negative bacteria. Cirioni et al., 2008 Diptericins They are members of glycine-rich AMP family Their selective toxicity against Gram-negative Enterobacteria like E. coli occurs via, disruption of cell wall.

Cathelicidins
They are small, cationic, antimicrobial peptides, varying in amino acid sequence, structure and size. They are stored in the secretory granules of neutrophils and macrophages, released extracellular upon leukocyte activation.
They exhibit broad spectrum of activity against bacteria, enveloped viruses and fungi. Main target is bacterial cytoplasmic membrane. Zanetti, 2004;Kaneider et al., 2007 Microcins It is a low-molecular weight antimicrobial peptide produced by Gram negative Enterobacteria as host defense peptides. They are <10 kDa in size, much smaller than other antimicrobial peptides.
They display strong antimicrobial activity against Gram-negative bacteria, such as E. coli O157:H7, Salmonella enteritidis and S. typhimurium. Inhibit DNA replication by targeting DNA gyrase. Nocek et al., 2012;Rebuffat, 2012 Auranofin's Its ability to suppress bacterial protein synthesis leads to significant reduction in the production of key methicillin-resistant Staphylococcus aureus (MRSA) toxins Inhibition of multiple biosynthetic pathways including cell wall, DNA, and bacterial protein synthesis. Thangamani et al., 2016 Buforin II 21 amino acid cationic and linear molecule peptide. Crosses cell membrane without permeabilizing it.
Inhibition of DNA replication and protein synthesis Cho et al., 2009;Xiea et al., 2011 2. Phage therapy OMKO1, wksl3 and Φ1 A new approach to therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics.
Ability of phages to kill antibiotic-resistant bacteria allied with their ubiquitous nature, high specificity (minimal disruption of normal flora), self-replication ability at the infection site, and more importantly low inherent toxicity qualifies them as "safe" and "green" technology. Chan et al., 2016 3.

Combination therapy Antibioticantibiotic
Colistin in association with tigecycline, aminoglycoside, meropenem, imipenem These antibiotic combination showed a decrease of 2.6-to 2.8-fold in MIC Soudeiha et al., 2017Bae et al., 2016 Antibiotic inhibitor Combination of inhibitor and antibiotic such as Augmention i.e., combination of clavulanate and amoxycillin) Frontiers in Microbiology | www.frontiersin.org Demonstrated the improvised antibacterial activity. Since nanoparticles do not enter the bacterial cell, and its mechanism of killing bacteria is fundamentally done via direct contact with the bacterial cell wall. Beyth et al., 2015;Franci et al., 2015;Wang et al., 2017 5. Liposomes as drug delivery vehicles

Drug loaded liposomes
Liposomes are spherical vesicles, with particle sizes ranging from 30 nm to several micrometers, consisting of one or more lipid bilayers surrounding aqueous spaces used as targeted drug delivery systems.  (Gerdes and Maisonneuve, 2012). Persisters are a fraction of bacterial cells in the culture that survive through a prolonged antibiotic treatment. Many studies have shown that toxins of diverse chromosomal TA systems encourage the development of persister cells. GNAT-RHH TA system is a newly exposed approach of bacterial cells to support persister cell formation by disturbing tRNA functions (Jurenas et al., 2017b), in S. enterica over expression of the TacT causes drug tolerance (Cheverton et al., 2016). Chromosomal type II TA loci have been reported to be activated by environmental stress (Li et al., 2016). To assess if antibiotic stress would stimulate the transcription of kacAT operon, exponential phase HS11286-RR2 cells were checked with different antibiotics at the minimum inhibitory concentration (MIC). Transcript levels of the toxin gene, kacT, were quantified by RT-qPCR at various time points after antibiotic challenge. RT-qPCR results showed that the exposure to meropenem or tigecycline antibiotics caused 10fold or 40-fold increase in kacT transcription levels (Qian et al., 2018). Klebsiella pneumoniae faces a wide diversity of environmental conditions, including antibiotic stress. Fifteen pairs of putative type II TA loci are detected on the K. pneumoniae HS11286 chromosome. Activation of the toxin plays an important role in bacterial multidrug tolerance (Harms et al., 2016). The chromosomally encoded kacAT bicistronic operon of K. pneumoniae HS11286 is a functional GNAT-RHH TA locus with kacA encoding the cognate antitoxin to the toxic product of kacT. (Hall et al., 2017). Over expression of KacT inhibited K. pneumoniae cell growth and resulted in dormant cell formation. Crystal structure analyses show that the KacT toxin adapts a typical GNAT-fold, which may confer the same catalytic mechanism as the one revealed for TacT of S. enterica (Cheverton et al., 2016). It may bind cellular tRNAs via its positive groove and transfer the acetyl group from AcCoA to tRNAs halting translation leading to cell arrest.

STRATEGIES TO COMBAT BACTERIAL ANTIBIOTIC RESISTANCE
Emerging antibiotic resistance is a problem of global magnitude. Confronted by increasing amounts of antibiotics use, emergence and dissemination of antibiotic resistant strains has compromised therapeutic potential of antibiotics. Of the different strategies adopted, techniques that materialize ideally include; (1) Designing antimicrobial peptides (AMPs; Bacteriocins, Cathelicidins, Microcins, etc.) with broader spectrum of targets (Gordya et al., 2017), (2) Phage therapy (exploiting phages such as OMKO1, wksl3 and Φ1 to kill antibiotic-resistant bacteria; Chan et al., 2016), (3) combination therapy (using combination of antibiotics e.g., colistin in association with tigecycline, aminoglycoside, meropenem, etc or combination of inhibitor and antibiotic such as Augmention i.e., combination of clavulanate and amoxicillin; Soudeiha et al., 2017), (4) Delivery of drugs, AMPs and essential oils as nanoparticles (NPs) for sustained and controlled release (AgNPs of penicillin G, amoxicillin, erythromycin, and vancomycin; Wang et al., 2017), (5) Liposomes as drug targeting vehicles (Poerio et al., 2017), (6) Use of natural compounds such as Flavonoids, Alkaloids, Coumarins, etc. (Chandra et al., 2017), and (7) Modification of antimicrobials e.g., Plazomicin (ACHN-490); derivative of sisomicin produced by addition of a hydroxy-aminobutyric acid substituent at position 1 and a hydroxyethyl substituent at position 6 (Lopez-Diaz et al., 2017; Table 2). Other approaches include use of genomics to find out new bacterial targets and optimization of newer approaches that target bacterial pathogens while exerting selection for reduced pathogenesis, if bacteria evolve resistance to therapeutic intervention. Additionally, strategies such as designing molecules that can block bacterial attachment to surfaces and target bacterial virulence factors along with contribution to protect through production of inactivating antibodies, seems other suitable options to over the menace of drug resistance.

CONCLUSION
Bacterial infections continue to be one of the leading causes of morbidity and mortality worldwide. Fallout of excessive and imprudent antibiotic use, widespread dissemination of resistant determinants as part of MGEs has increased the rate of resistance development. Being capable to relocate between host genomes, they act as vehicles for resistance gene acquisition and their successive propagation. Thorough molecular studies have identified several mechanisms in microbes to attain the antimicrobial resistance. Among these mechanisms, plasmids, transposons, insertion sequences, integrons, ICEs and bacterial Toxin Anti-toxin systems have exposed how and why resistance has attained alarming stage. The possibility for recombination of genes from different bacterial populations is huge and it seems that it doesn't take bacteria much time to acquire the genetic resources to flourish in surroundings that would have otherwise hindered it's growth. Occurring with increasing frequency, resistance limits therapeutic option, resulting in the cases where certain human infections cannot be treated. Pertinently, where there is stiff resistance on the implementation of evidence-based clinical practice, scientists of the health care organizations are still searching as how to keep pace with the demand of actionable knowledge. This adverse condition of antimicrobial resistance demands the rejuvenation of dried pipeline for the development of new and efficient drugs to treat the deadly infection. With a goal to get hold of the menace of antibiotic resistance, it seems essential for everybody to have some basic knowledge about the systems in order to ensure optimal use of antibiotics from the surrounding milieu, to slow down the development of antibiotic-resistant superbugs.

AUTHOR CONTRIBUTIONS
QH and AJ conceived the idea. IS, SR and AJ contributed to writing of the manuscript. MS and AM contributed to reference and graphics section.

ACKNOWLEDGMENTS
Author extends thanks to colleagues from Department of Biosciences, Jamia Millia Islamia and School of Biosciences and Biotechnology, Baba Ghulam Shah Badshah University, for criticism that helped to improve the quality of contents in the perspective of broader audience. Authors also acknowledge help from Dr. Waseem Ahmad Wani, who helped with the designing of antibiotic structures. AJ would like to thank University Grants Commission (UGC), New Delhi for startup.


extfieldsofstudy: Medicine Biology

text:
Large-Scale Structural Variation Detection in Subterranean Clover Subtypes Using Optical Mapping

We selected two genetically diverse subspecies of the Trifolium model species, subterranean clover cvs. Daliak and Yarloop. The structural variations (SVs) discovered by Bionano optical mapping (BOM) were validated using Illumina short reads. In the analysis, BOM identified 12 large-scale regions containing deletions and 19 regions containing insertions in Yarloop. The 12 large-scale regions contained 71 small deletions when validated by Illumina short reads. The results suggest that BOM could detect the total size of deletions and insertions, but it could not precisely report the location and actual quantity of SVs in the genome. Nucleotide-level validation is crucial to confirm and characterize SVs reported by optical mapping. The accuracy of SV detection by BOM is highly dependent on the quality of reference genomes and the density of selected nickases.

INTRODUCTION
Subterranean clover is the key forage legume in Australia, producing valued feed for livestock on a sown area of more than 29 million hectares (Nichols et al., 2013). As with other legumes, symbiotic nitrogen fixation in subterranean clover contributes to soil improvement. Subterranean clover is diploid (2n = 2x = 16) with a genome size around 556 Mb/1C. Its inbreeding nature, annual habit, and well-assembled reference genome (subterraneum) have established it as a model for Trifolium (Nichols et al., 2013). Based on morphology, genetic, and cytogenetic data, subterranean clover is classified into three subspecies: subterraneum, yanninicum, and brachycalycinum (Katznelson and Morley, 1965a,b). The subspecies differ morphologically, enabling them to adapt to different soil environments, e.g., ssp. subterraneum and ssp. yanninicum are adapted to moderately acidic soils, with ssp. subterraneum found on well-drained soils and ssp. yanninicum adapted to waterenriched soils (Francis and Devitt, 1969). In contrast, ssp. brachycalycinum is adapted to dry and neutral-to-alkaline soils that contain cracks or stones facilitating burr development. In this study, we examined the sympatric subspecies subterraneum and yanninicum to check the performance Abbreviations: BOM, Bionano optical mapping; DAPI, 4 ,6-diamidino-2-phenylindole; DNA, deoxyribonucleic acid; MQR, molecule quality report; N/A, not available; NGS, next generation sequencing; SNP, single-nucleotide polymorphism; SV, structural variation. of optical mapping in structural variation (SV) detection and validate the findings using short read sequencing.
Structural variations are genomic alterations in sequence size, copy number, orientation or chromosomal location between individuals (Feuk et al., 2006). They are important genetic features that enrich genetic diversity and lead to important phenotypes (Escaramis et al., 2015).
We selected the Trifolium model species, subterranean clover (Trifolium subterraneum L.), with a high-quality reference and high-resolution Bionano optical maps (BOM) for two genetically diverse subspecies. These BOM findings were then validated by high coverage Illumina short read data generated for the two subtypes.

Purification of Cell Nuclei
Suspensions of intact cell nuclei were prepared following Vrána et al. (2016). Approximately 20 g each of mature dry seeds of ssp. subterraneum cultivar Daliak and ssp. yanninicum cultivar Yarloop were germinated at 25 • C on moist paper towels in a dark environment. When the roots reached 2-3 cm in length, they were excised about 1 cm from the root tip, fixed in (2% v/v) formaldehyde at 5 • C for 20 min, and subsequently washed three times with Tris buffer (5 min each time). The root tips (∼40/sample) were excised and transferred to 1 ml IB buffer (Šimková et al., 2003), in which cell nuclei were isolated using a homogenizer at 13,000 rpm for 18 s. Large debris was removed by filtering through 50-µm nylon mesh, and the nuclei in suspension were stained with DAPI (2 µg ml −1 ).

Preparation of High Molecular Weight (HMW) DNA
High molecular weight (HMW) DNA was prepared according to Šimková et al. (2003) with modifications. Four batches of 700,000 G1-phase nuclei each were sorted into 660 µl IB buffer in 1.5 ml polystyrene tubes using a FACSAria II SORP flow cytometer and sorter (BD Biosciences, San Jose, CA, United States). One 20 µL agarose miniplug was prepared from each batch of nuclei. The miniplugs were treated by proteinase K (Roche, Basel, Switzerland), washed in wash buffer (10 mM Tris, 50 mM EDTA, pH 8.0) four times, and subsequently five times in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). After the plugs had been melted for 5 min at 70 • C and solubilized with GELase (Epicentre, Madison, WI, United States) for 45 min, DNA was purified by drop dialysis against TE buffer (Merck Millipore, Billerica, MA, United States) for 90 min.

Construction of Bionano Optical Map
The latest genome assembly of T. subterraneum (cv. Daliak) (Kaur et al., 2017) was used as a reference and digested in silico using Knickers (v1.5.5). Four available nickases (Nt.BspQI: GCTCTTC, Nb.BbvCI: CCTCAGC, Nb.BsmI: GAATGC, Nb.BsrDI: GCAATG) were used to check the frequency of enzyme restriction sites in the reference genome with Nt.BspQI, being the most appropriate enzyme to nick the HMW DNA with the expected frequency of 7.1 sites per 100 kb. In all Bionano experiments, Nt.BspQI was used. The DNA was labeled and stained following the manufacturer's NLRS protocol as described in Kaur et al. (2017). Four runs on the Bionano Irys R instrument (30 cycles/run) were carried for subspecices yannicum (cv. Yarloop) to achieve sufficient genome coverage (∼425 ×).

Structural Variation Detection by BOM Validated Using Illumina Short Reads
After de novo assembly, runBNG was used for SV calling with default parameter settings (medium configration settings from Bionano Genomics). To check the accuracy of the SVs detected, we followed the pipeline from Shelton et al. (2015) using the default, strict and relaxed parameters in optical mapping and then performed SV calling. To further confirm the findings, we selected short paired-end reads for validation. The plants were grown in the field at Shenton Park, Western Australia (31 • 57 S, 115 • 50 E) and the genomic DNA was extracted from a single plant of cv. Yarloop and cv. Daliak -representatives of two subterranean clover subspecies. Truseq Illumina libraries were prepared with an insert size of approximately 550 b and the short paired-end reads were generated using Illumina HiSeq 2000 at coverage of 48 × in cv. Yarloop and 56 × in cv. Daliak [the same dataset used in Kaur et al. (2017)]. Reads from both cultivars were aligned to the latest nucleotide reference (cv. Daliak) respectively (Kaur et al., 2017) using Speedseq (v0.1.0) (Chiang et al., 2015). Results were visualized using the integrative genomics viewer (IGV) (v2.3.91) (Robinson et al., 2011). Nucleotide-level SV calling was performed using Lumpy (v0.2.11) (Layer et al., 2014) by analyzing the mapping result from Speedseq (v0.1.0) (Chiang et al., 2015). The settings of Speedseq reads mapping were the deafult. The program used from Lumpy was 'lumpyexpress'. The nucleotide reference was the same one used in the Speedseq reads mapping. The short sequence reads of cv. Yarloop were the same as used in reads mapping. When large-scale regions containing SVs from BOM were identified, we checked the corresponding regions to see if these regions contain SVs from the results of Lumpy and the visualization by IGV.

De Novo Assembly of cv. Yarloop Optical Map
A total of 1,083,671 single molecule maps (directly from the Irys platform without filtering) were generated with a total length of 235.5 Gb (∼48 × genome coverage), of which the molecule N50 was 212.7 kb, and the average label density was 7.5 per 100 kb ( Table 1). After filtering maps < 150 kb, 958,136 single molecule maps remained with a total length of 212.7 Gb (∼385 × genome coverage), of which the molecule N50 was 218.6 kb, and the average label density was 8.3 per 100 kb. Using the filtered single molecule maps, 375,975 single molecule maps were finally de novo assembled to generate 377 consensus maps. The total length of the generated consensus maps was 475 Mb (∼89% of the total length of the reference genome) with a map N50 of 1.8 Mb.

SVs Assessment With BOM Validated by Illumina Short Reads
The mapping rate between cv. Yarloop Bionano consensus maps and the cv. Daliak NGS reference is 7.4%. Bionano SV calling between cv. Yarloop Bionano molecule maps and the cv. Daliak reference genome identified 12 regions (tens of kb regions) containing deletions and 19 containing insertions in cv. Yarloop compared to cv. Daliak (Supplementary Figure S1). The average deletoin length was 6.2 kb (Supplementary Table S1) and in these regions, 9.7% of the sequences were assembly gaps (Ns). The average length of the insertions was 8.04 kb, and the percentage of unknown sequences in these regions was 3.6%. Compared to the average coverage of all cv. Yarloop Bionano consensus maps (95.64 ± 23.52), the average coverge of the deletion and insertion regions in BioNano consensus maps is 101.12 ± 6.75. When checking the SVs called from the pipeline presented by Shelton et al. (2015) the results are consistent with our findings. Speedseq/Lumpy SV calling detected 20,887 deletions, 115 inversions, and 1,331 duplications in cv. Yarloop. Among those 20,887 deletions there are 71 (on average 2,533 b) that supported the 12 regions (on average 6,196 b) implied by BOM in cv. Daliak (Supplementary Figure S2 and Supplementary  Table S2). Lumpy did not detect any insertions. Coverage 425 385 0.89 † In the set of molecules, N50 represents the length of the shortest molecule whose length is greater than half of the total sum of lengths of all molecules; it is the point of half of the mass of the distribution.

DISCUSSION
Although whole genome sequencing has been widely used to detect SVs, it is challenging to characterize large-scale SVs in a genome, particular for large-scale insertions. Current sequencing technologies can produce sequencing reads from a hundred to tens of thousands base pairs (Yuan et al., 2017a). However, they are still not long enough to span some variant regions (Li et al., 2011). Optical mapping, as an alternative, has promised the accuracy in SV detection (Li et al., 2017).
In the de novo assembly of cv. Yarloop Bionano single molecule maps, the total length of the consensus maps accounted for ∼89% of the estimated T. subterraneum genome size contrary to our expectation of ∼100%. This incomplete assembly could be caused by the low-quality single molecule map filtering step or the single map fragmentation due to the close proximity of Nt.BspQI restriction sites leading to DNA double-strand breaks in some DNA regions (Hastie et al., 2013). If the fragmented Bionano maps are repeats, they may collapse due to sharing the same nicking site partten. If there is no restriction sites in the fragmented maps, in the de novo assembly, they could be excluded leading to a smaller consensus map size.
The mapping rate between cv. Yarloop Bionano optical maps to the Daliak NGS reference indicates that the genomes of cv. Yarloop and cv. Daliak are diverged and most of the genome cannot be aligned. This could be an explanation that only 12 deletions and 19 insertions were detected. When checking the avergae coverage of the detected SV regions and the average coverage of the Yarloop Bionano consenus maps, the results suggeste that our findings are reliable. To get rid of the bias from one paramter settings in the de novo assembly step, we followed the pipeline presented by Shelton et al. (2015). Our results have also been supported.
In the Lumpy SV calling, we identified 71 small deletions in the 12 large-scale regions reported by BOM (Figure 1). While, the total length of the 71 deleted genomic regions reported by Lumpy was close to the total length reported by BOM (71.7 kb vs. 74.4 kb respectively), some length differences remained, probably due to the incorrect gap size or misassemblies in the reference genome, or also could be due to the incomplete SV calling in Lumpy. Interestingly, the gaps in the detected SV regions which were highly likely caused by collapse in the repetitive regions, were complemented by the Bionano super-scaffolding process for the generation of the advanced reference assembly (Kaur et al., 2017).
No insertions were reported by Lumpy in the SV calling, it is probable that those sequences being novel in cv. Yarloop compared to the reference assembly based on the cv. Daliak. When nucleotide level alignments were carried out using short sequence reads from the cv. Yarloop with the cv. Daliak, Yarloop reads from genomic regions not present in the reference assembly (either Yarloop-specific or unassembled in the reference) could not be mapped. As such, SV could not be called in these regions. Such novel sequences were grouped as unmapped sequences, earlier abandoned by Lumpy in SV calling. This issue has also been reported previously by (Xia et al., 2016) for most reference based SV calling methods, which cannot efficiently report large-scale insertions if there are many novel sequences in the examined individuals.

CONCLUSION
Based on the physical location of nicking sites, optical mapping provides an attractive method to detect SVs. Single molecule maps produced by optical mapping are long enough to span most of the large and complex genome regions that traditional sequencing technologies are unable to achieve. However, optical mapping has some limitations in discovering the precise location and actual number of SVs owing to enzyme physical locations. NGS is useful to characterize SVs identified by optical mapping.
Although optical mapping provides the total size of SVs in a detected region, the total size of those SVs can be misreported due to the inaccurate gap size in the reference genome and/or absent enzyme restriction site information in the gap regions. To improve SV detection and characterization, a high-quality reference genome is crucial. In the absence of a high-quality reference genome, possible nucleotide-level validation of those identified SV regions is recommended to assess the accuracy of SV calling in optical mapping.

AVAILABILITY OF DATA
All raw nucleotide data and Bionano data are under BioProject PRJNA404013.

AUTHOR CONTRIBUTIONS
PK, DE, PB, and YY conceived and designed the research. ZM, JV, and JD performed the Bionano Irys R System genome mapping experiments. YY performed the bioinformatics analysis, prepared the figures and wrote the manuscript with contributions from PK, PB, ZM, WE, DE, JD, and JV. All authors read and approved this manuscript.

FUNDING
This study was conducted by the Centre for Plant Genetics and Breeding (PGB) at The University of Western Australia (UWA) in close collaboration with Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Czech Republic. This project was also supported by grant award LO1204 from the National Program of Sustainability I and by the Czech Science Foundation (Award No. P501/12/G090).

ACKNOWLEDGMENTS
YY was supported by the China Scholarship Council for his Ph.D. studies at the University of Western Australia. We thank Zdeňka Dubská for assistance with nuclei flow sorting, Helena Staňková for help with Bionano mapping, and Hana Šimková for advice on Bionano mapping. We acknowledge the supercomputing resources provided by the Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia.


extfieldsofstudy: Medicine Chemistry

text:
Hypolipidemic activities of partially deacetylated α-chitin nanofibers/nanowhiskers in mice

Partially deacetylated α-chitin nanofibers/nanowhiskers mixtures (DEChNs) were prepared by 35% sodium hydroxide (NaOH) treatment followed by disintegration in water at pH 3–4. The aim of this study was to investigate the hypolipidemic effects of DEChNs at different dosage levels in male Kunming mice. The male mice were randomly separated into five groups, that is, a normal diet group, a high-fat diet group, and three DEChN groups that were treated with different doses of DEChN dispersions (L: low dose, M: medium dose, H: high dose). Primarily, the DEChNs significantly decreased body weight (BW) gain and adipose tissue weight (ATW) gain of mice. Meanwhile, the decreasing extent of weight ratios between ATW and BW was dependent on the dose of DEChNs. Moreover, the DEChNs prevented an increase in plasma lipids (cholesterol and triacylglycerol) in mice when they were fed a high-fat diet. Histopathological examination of hepatocytes revealed that the DEChNs were effective in decreasing the accumulation of lipids in the liver and preventing the development of a fatty liver. The results suggested that the DEChNs reduced the absorption of dietary fat and cholesterol in vivo and could effectively reduce hypercholesterolemia in mice.

H yperlipidemia is a major cause of coronary atherosclerosis and subsequent related cardiovascular disease, which has always been related to obesity to some extent (1). In recent years, most studies have examined how to decrease plasma lipid concentrations and the absorption of fat in the intestinal tract to reduce diet-related chronic disease (2). The conventional therapeutic modalities for hyperlipidemia are lipid-lowering drugs such as atorvastatin, lovastatin and fibrates (3). These synthetic drugs are available and effective but may cause adverse effects at the same time (4). Therefore, the antihyperlipidemic activity of many biologically active components from natural materials, such as polysaccharides and dietary fiber, has been explored. Polysaccharides, such as Ulva pertusa, Ulva lactuca, chitosan, and chitosan derivatives, are novel potential hyperlipidemic agents (3). Other dietary fibers, such as pectin and psyllium, also exhibit potent hypolipidemic effects (5).
Studies have shown that chitosan has a beneficial lowering effect on plasma cholesterol, which may play an important role in the prevention and treatment of cardiovascular disease (6,7). Some previous research work has revealed the hypolipidemic mechanism of chitosan hypolipidemic activity of low molecular weight chitosan was better than high molecular weight chitosan (8,9). The positively charged amino groups of chitosan may have the ability to bind negatively charged molecules, such as lipids and bile acids, and subsequently be excreted in the feces (10). The adsorption of chitosan to oil droplets affects the digestibility of oil. Access to the oil by digestive enzymes is decreased, thereby decreasing oil digestion.
Chitosan is a derivative of chitin. Chitosan is a cationic polysaccharide produced by the deacetylation of chitin under alkaline conditions, normally with a degree of deacetylation (DDA) greater than 0.7. Chitosan is, consequently, a copolymer of N-acetyl-D-glucosamine and D-glucosamine, which is similar to chitin structurally (11). Chitin, the source of chitosan, is the second most abundant renewable natural polysaccharide after cellulose. Chitin is the main component of the exoskeleton of arthropods, such as crabs, prawns, and shrimp, as well as the cuticles of insects and the cell walls of certain fungi, coexisting with various types of proteins and certain minerals (12). Isolated chitin is composed of β-(1-4)-linked 2-acetamido-2-deoxy-β-D-glucose units and a small amount of D-glucosamine. Because a large amount of crab and shrimp shell-derived chitins is discarded every year, utilizing chitin as a functional material or commodity is highly beneficial (13). In addition to converting chitin to its highly deacetylated derivative, chitosan, which may be used in a variety of applications, chitin itself has the potential to be converted into individual nanofibers through some downsizing processes.
One of the methods, partial deacetylation with 30-35 wt% NaOH treatment followed by disintegration in water at a pH of 3-4, was used to prepare partially deacetylated α-chitin nanofiber/nanowhisker mixtures (DEChNs) from α-chitins. The as-prepared DEChNs had DDA values of 0.25-0.35 and a high density of cationic charged C2-amino groups on the surface (14). The DEChNs had lower DDA values (0.25-0.35) than chitosan (normally >0.70) and did not dissolve at a molecular level but dispersed at a nanofibril level, maintaining high crystallinity (14). Our previous studies showed that partially deacetylated α-chitin can be converted to nanofiber/nanowhisker mixtures by mechanical pretreatment under acidic conditions (15). The DEChNs have been studied as nanostructured materials for further applications, such as gels and films (16). Other research work has been conducted to study the biochemical properties of DEChNs. Kazuo Azuma et al. evaluated the effects of oral administration of surface-deacetylated chitin nanofibers, chitosan, and cellulose nanofibers on hypercholesterolemia in rats and indicated that chitin nanofibers suppressed an increase in serum total cholesterol (TC), chylomicron, very-low-density lipoprotein (VLDL), and phospholipid (PL) levels while increasing alanine transaminase levels, vacuolar degeneration, and accumulation of lipid droplets in liver tissue (17). Recently, Anraku et al. proposed that surface-deacetylated chitin nanofibers are more effective in decreasing renal injury and oxidative stress than deacetylated chitin powder in 5/6 nephrectomized rats (18). The result of this study suggested that ingestion of chitin nanofibers, not chitin powder, resulted in significantly reduced levels of pro-oxidants, such as uremic toxins, in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation. All of these studies stated the potential of chitin in the biotechnology field, emphasizing the importance of nanofibrillation of chitin. However, research has rarely reported the mechanism and the effects of chitin nanofibers/nanowhiskers on hypolipidemic function. Although the DEChNs and chitosan have similar macromolecular structures, DEChNs are characterized by nanofibril morphology, lower amino groups, and higher crystallinity. Thus, it is worth further examination of the relationship between the biochemical properties of DEChNs and their hypolipidemic effects.
In the present study, the hypolipidemic effects of the DEChN dispersions were investigated in male Kunming mice at different dosage levels. The effects of the DEChNs on the weight of the body, organ tissue, and adipose tissue were examined in this study. Serum blood chemistry analysis and histopathological examination of the liver were also performed.

Materials
The α-chitin was purified from swimming crab (Portunus trituberculatus) shells collected from Nantong, a seaside city in Jiangsu Province, China, as follows (11). Discarded crab shells were soaked in 1 M HCl for 12 h to remove the mineral salts. The sample was then washed thoroughly with distilled water, followed by treatment with 1 M NaOH for 12 h to remove the proteins. These two steps were repeated three times. The obtained residual solid was decolored by immersing it in 0.5% (w/w) NaClO 2 , and the pH was adjusted to 5 using acetic acid. The suspension was heated for 2 h at 70°C. The purified α-chitin solid residues were obtained by filtration, rinsed with distilled water, and stored at 4°C for further use. TC, triacylglycerol (TG), high-density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) kits were purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. All other reagents and solvents were of analytical grade.

Preparation of deacetylated a-chitin nanofiber/nanowhisker mixtures
The α-chitin (1 g) was suspended in 35% (w/w) NaOH solution (25 mL) at 90°C for 4 h with continuous stirring. The final partially deacetylated chitin product with a DDA of 0.29 was collected and thoroughly washed with deionized water until a neutral pH was obtained and then stored at 4°C (14). The DDA of the original and partially deacetylated chitins were determined by the electrical conductivity titration method (19). In this case, a dried sample (0.1 g) was added to water (60 mL), and a small amount of 0.5 M NaOH was used to adjust the pH to 9. The slurry was stirred for 30 min for adequate distribution. Afterward, 0.1 M HCl was added to adjust the pH to 2.5-3.0, and a 0.05 M NaOH solution was added at a rate of 0.1 mL/min to adjust the pH to 11 using a pH-stat titration system. The conductivity and pH curves obtained reflect the amount of C2-amino groups in the chitins.
The partially deacetylated chitin was suspended in deionized water at a concentration of 0.1% (w/v), and the pH of the suspension was adjusted from the original pH of 6-7 to 3-4 by adding acetic acid with constant stirring. Next, this suspension was homogenized and treated with ultrasonication, the DEChNs were obtained after centrifugation, and the dispersions were successfully prepared (16).

Animals and diets
Kunming mice (males, Specific pathogen free (SPF), 18-20 g) were used as the experimental animals. The mice were procured from the Animal Center for Disease Prevention and Control in Hubei Province, China. All animal protocols were approved by the institutional animal care and use hospital of Huazhong Agricultural University (Wuhan, China). The animal study was performed under strict adherence to the international rules for animal experiments and the internationally accepted ethical principles for laboratory animal use and care. The mice were housed in cages in a controlled environment (25 ± 2°C, 50-70% relative humidity, 12±1 h light-dark cycle). There were five mice in each cage, and these mice were allowed free access to food and water for 5 days. Then, the mice were randomly divided into five groups as follows (10 mice per group). The normal fat control group (blank group, n = 10) received a normal fat diet and saline solution. The normal fat diet consisted of 40% corn starch, 22% wheat, 20% soybean, 7% peptone (fish), 4% casein, 4.2% mineral mixture, 0.8% amino acid, 0.3% vitamin mixture, 0.2% choline chloride, and 1.5% fat. The high-fat control group (control group, n = 10) received a high-fat diet and dilute acetic acid solution. Finally, there were three DEChN groups, namely, the L-DEChN (n = 10), M-DEChN (n = 10), and H-DEChN groups (n = 10), which received a high-fat diet and low, medium, or high doses of DEChN dispersions, respectively, dispersed in dilute acetic acid at a pH of 3-4. The high-fat diet consisted of 10% lard, 10% egg yolk powder, 2% cholesterol, 0.5% sodium deoxycholate, and 77.5% normal fat diet.

Experimental design
All mice were fed under experimental conditions for 21 days. The male mice were randomly separated into five groups: a normal diet group, a high-fat diet group, and three DEChN groups that had been treated with different doses of DEChN dispersions (low, medium, and high). During the experimental period, the method described by Qi et al. (20) was employed in this study. The mice were allowed free access to food and water for 5 days to acclimate to the animal facility and were weighed and randomly divided into five groups as described above. Each day the body weights (BW) were recorded, and in the DEChN groups, the mice were intragastrically administered DEChN dispersions at dosages of 18.75 mg/kg·bw·d (L-DEChN group), 37.5 mg/kg·bw·d (M-DEChN group), and 75 mg/kg·bw·d (H-DEChN group). Correspondingly, each day the mice in the blank group were given saline solution, and the mice in the control group were given dilute acetic acid solution. At the end of the third week, mice fasted for 18 h prior to blood withdrawal. Necropsies were performed on all mice after blood withdrawal, and organ and adipose tissues were collected and measured.

Collection of blood and bioassay
All blood samples for the bioassay were obtained by retro-orbital blood collection from mice that fasted overnight (18 h). Blood samples were centrifuged at 3,000 g for 10 min to separate the serum for lipid profile estimation and stored at -4°C. The serum TC, TG, HDL-C, and LDL-C concentrations were measured by an enzymatic colorimetric method using commercially available kits. Meanwhile, liver, kidney, and spleen samples were also collected and weighed.

Statistical analysis
Statistical analyses were performed using Student's t-test and compared to the control group (21). The data are presented as the mean ± standard deviation; p < 0.05 was considered a statistically significant difference.

Results and discussion
Characterizations of deacetylated a-chitin nanofiber/nanowhisker mixtures The atomic force microscopy image of the DEChNs is shown in Fig. 1a. Complete individualization of DEChNs was achieved by partial deacetylation and the following downsizing process. The as-prepared individual DEChNs had widths that ranged consistently between 10 and 30 nm and lengths that ranged between 150 and 500 nm. The DDA of the DEChNs was 0.29.
Photographs of the DEChNs in water at pH 1.5-6 are shown in Fig. 1b. The DEChNs were well dispersed in water at pH 1.5-4.5; however, at pH 6.0 there was severe turbidity with white flocculent particles, indicating that DEChNs flocculate in water at higher pH values. The well dispersion of DEChNs at pH 1.5-4.5 was in accordance with its high zeta-potential (+30-70 mV), which indicated the high cationic surface charges of DEChNs. Thus, the addition of acetic acid cationized the C2-amino groups on the DEChNs, promoting the fibrillation of the DEChNs into nanofibrils by electrostatic repulsion and stable dispersion in water. However, DEChNs lost a certain amount of positive charge when the zeta-potential decreased to +24.1 mV at pH 6.0 and therefore became flocculent and precipitated.
While pH is normally neutral in most parts of the body, it is acidic in the gastric environment. The changes in the zeta-potential and dispersion-flocculation that occurred in relation to the pH value of the DEChNs reflect the potential of DEChNs to bind molecules such as lipids, cholesterol, and bile acid in the stomach at pH < 4.5 (acidic gastric juice) and to flocculate with the binding molecules at a higher pH in the intestine for excretion outside of the body.

Body and tissue weight of mice
The BW and BW gain of experimental mice are shown in Fig. 2a and b, respectively. The DEChN dispersions were divided into the following three dosage groups: L-DEChNs, M-DEChNs, and H-DEChNs, in which the mice were fed dosages of 18.75, 37.5, and 75 mg/kg·bw·d of DEChN dispersions, respectively. The mice were fed the DEChNs along with a high-fat diet to estimate their effect on the resulting weight change of the mice. As a control, because the DEChNs were dispersed under acidic conditions mediated by acetic acid, the dilute acetic acid solution was given to the mice along with the high-fat diet.
As shown in Fig. 2a and b, the mice fed the high-fat diet plus the dilute acetic acid solution (control group) showed decreases in BW during the experimental period, compared with those mice fed the normal-fat diet plus a saline solution (blank group). This indicates that the dilute acetic acid could reduce the weight gain of the mice even though they were fed the high-fat diet. Until Day 14, there were some differences in the BW of mice fed the L-DEChNs and mice in the control group. However, after 14 days, the BW of these two groups showed little difference. The results suggested that the L-DEChN (18.75 mg/ kg·bw·d) dispersion dosage slightly mitigated increases in the BW of mice fed a high-fat diet after the initial 2 weeks, when mice had acclimated to the feeds. Compared with the control group and the L-DEChN group, there was significantly less BW gain in mice that had been fed the M-DEChN and H-DEChN doses. Especially for the mice that received H-DEChNs along with a high-fat diet, a minimal increase in the BW was observed.
Overall, the BW of the mice obviously changed; both acetic acid and DEChNs were able to prevent the weight gain of mice, even those fed a high-fat diet, and the degree of prevention was correlated with the dosage of DEChNs.
Necropsies were performed on the experimental mice after 21 days. The tissue weights (TW) of the mice (including liver weight, kidney weight, and spleen weight) are shown in Table 1. As shown in Table 1, compared with the control group, the TW of the mice fed DEChN dispersions were significant lower along with a lower overall BW. The results suggest that DEChNs reduced both the BW and TW gain of mice. In contrast, the ratios of liver, spleen, and kidney weights to BW (TW/BW) showed no significant differences among all groups.

Adipose tissue and serum lipids
The adipose tissues (including renal, epididymal, and mesenteric adipose tissue) of experimental mice were collected and measured. The adipose tissue weight (ATW) and the ATW/BW ratios are shown in Table 2. Compared with the mice fed the normal-fat diet plus a saline solution (blank group), the mice fed the high-fat diet plus a dilute acetic acid solution (control group) showed an increase in ATW at the end of the experimental period. This indicates that feeding the mice a high-fat diet resulted in a high accumulation of lipids.

Hypolipidemic activities
Compared with the control group, a decrease in the weights of the renal, epididymal, and mesenteric adipose tissues were observed in the H-DEChN and L-DEChN groups. The H-DEChN group showed a greater decrease than the L-DEChN group. The weights of the renal ATW of mice in the L-DEChN group and the H-DEChN group were 0.235 ± 0.068 and 0.146 ± 0.041 g, respectively, compared with 0.418 ± 0.148 g in the control group and 0.234 ± 0.116 g in the blank group. The weights of the epididymal adipose tissue of mice in the L-DEChN and H-DEChN groups were 0.621 ± 0.16 and 0.412 ± 0.098 g, respectively, compared with 0.819 ± 0.238 g in the control group and 0.695 ± 0.107 g in the blank group. The weights of the mesenteric adipose tissue of mice in the L-DEChN and H-DEChN groups were 0.476 ± 0.187 and 0.471 ± 0.097 g, respectively, compared with 0.728 ± 0.085 g in the control group and 0.616 ± 0.17 g in the blank group. The weights of the renal, epididymal, and mesenteric adipose tissues of mice in the M-DEChN group were 0.268 ± 0.075, 0.700 ± 0.159, and 0.594 ± 0.115 g, respectively.
There was an obvious decrease in the weights of the collected adipose tissue compared with the control group, while there was no significant difference in the renal, epididymal, and mesenteric ATW/BW ratios of the M-DEChN group and the blank group. The results indicated that DEChNs could interfere with the absorption of dietary fat and cholesterol in vivo. This finding was likely a result of the unique ability of chitin to bind lipids and bile acids. The H-DEChN group (75 mg/kg·bw·d DEChNs) was more effective than the L-DEChN group (18.75 mg/kg·bw·d DEChNs) or the M-DEChN group (37.5 mg/kg·bw·d DEChNs) in decreasing the accumulation of lipids in tissue and preventing the development of obese mice.
The serum lipid concentrations of mice were measured, including TC, TG, HDL-C, and LDL-C. The results are shown in Table 3. After 21 days of experimental diets, the serum TC concentration of the mice fed the high-fat diet was higher than that of the mice fed a normal-fat diet. However, the serum TC and HDL-C concentrations of   which is approximately a 2.44-fold decrease, indicating that while feeding the mice a high-fat diet normally resulted in a higher accumulation of lipids in the blood, DEChNs can suppress hyperlipidemia in mice that are fed high-fat diets.

Liver histological examination
The livers from the mice were subjected to histopathological examination, and the results are shown in Fig. 3. At the end of the experiment, the livers of the mice from the control group had become swollen, oily, and orange. By comparison, the livers from the blank and DEChN groups were normal and dull red in color. As shown in Fig. 3, there were no evident fat vacuoles in the cytoplasm from the blank group, while hepatocytes from mice in the control group had accumulated a large number of fat vacuoles. The results indicated that feeding the mice a high-fat diet resulted in a higher accumulation of lipids in the livers of mice, thereby leading to a severe fatty liver. Histopathological examination of hepatocytes from mice in the DEChN groups revealed that livers had a small number of droplet accumulation compared with those in the control group. In addition, there was no evidence of fat vacuoles in the cytoplasm among the DEChN groups and the livers of the DEChN groups appeared normal, that is, similar to the blank group. These results suggested that a small dosage of DEChN dispersions was effective in decreasing the accumulation of lipids in the liver, thus preventing the development of a fatty liver. The histopathological examination of the liver was consistent with the data collected for BW gain, ATW gain, and serum lipid levels, thereby proving that DEChNs possesses effective hypolipidemic activity.
Optimum dosage of deacetylated a -chitin nanofiber/nanowhisker mixtures Azuma et al. recently proposed that an oral administration of surface-deacetylated chitin nanofibers (SDCNFs) decreased the diet-induced increase in serum TC, chylomicron, very-low-density lipoprotein, phospholipid levels, and suppressed vacuolar degeneration and accumulation of lipid droplets in liver tissue (17). They also suggested that administration of SDCNFs did not affect BW change or organ weights. However, our study showed that DEChNs significantly reduced the BW and ATW gains in mice when fed a high-fat diet. The difference may be attributed to the dosage of DEChN dispersions. In the present study, the dosage was under precise control and the DEChNs were intragastrically administered.  The present study suggests that the BW and ATW gains of the mice in the DEChN groups were significantly lower than those of the control group and changed dramatically in the H-DEChN group. On the contrary, the changes of the weight ratio between tissue and body were observed to be slight. For the H-DEChN group, the suppression of BW gain and the reduction of adipose tissue accumulation were the most obvious. The drastic changes may indicate that mice fed excessive DEChNs exhibited a pathological growth state. Therefore, in consideration of the efficacy of ATW loss and ensuring healthy growth of mice as mentioned above, a dosage of 37.5 mg/kg·bw·d DEChNs (the M-DEChN dose), in the medium dosage range, was recommended and proposed in the present study.
At the recommended optimum dosage, DEChNs can facilitate weight loss and reduce accumulation of fat in diet-induced obese mice and significantly reduce the rise of liver lipid levels in mice when fed a high-fat diet. Meantime, at this dosage, the DEChNs reduced the plasma lipid levels, the serum TC, and TG concentration as well. These values were lower than those of mice fed the high-fat diet and dilute acetic acid solution, indicating that DEChNs can effectively prevent hypercholesterolemia when consuming a high-fat diet. The most probable explanation for this is the binding of lipids to the DEChNs, thereby reducing the absorption of dietary fat and cholesterol in vivo.
Chitosan, with an average molecular weight of 650 kDa or greater, was approved by the Ministry of Health, Labour and Welfare (Japan) in 1997 as a specific healthy functional food, and the approved oral dosage of chitosan was 0.5-3 g per day in humans (22). In particular, doses between 2 and 5 g/kg were used to evaluate the hypolipidemic effects of chitosan. In this study, the best dosage of DEChNs for optimal hypolipidemic effects was 37.5 mg/kg·bw·d, which is much lower than the recommended dosage of chitosan. Thus, we conclude that the hypolipidemic effects of DEChNs are more efficient than those of chitosan.
Possible hypolipidemic mechanism of deacetylated a -chitin nanofiber/nanowhisker mixtures According to previous reports, chitosan, a derivative of chitin, can reduce BW and ATW gains in mice that are fed high-fat diets (22). The hypolipidemic mechanism of chitosan was investigated in male Sprague-Dawley rats and the results suggested that chitosan reduced the absorption of dietary fat and cholesterol in vivo, thereby effectively reducing the occurrence of hypercholesterolemia in rats (23). There have been many studies that have attempted to explain the mechanisms of chitosan in inhibiting hyperlipidemia. A classical perspective, which is widely accepted, is that chitosan binds lipids in the gastrointestinal tract, which are then excreted in the feces (23,24). DEChNs have a similar molecular structure to chitosan but can be identified by their nanofibrillar morphology, lower amino groups, and increased crystallinity. However, there have not been many research studies that have examined the use of DEChNs in supporting the hypolipidemic activities of mice, as well as the underlying mechanisms of how the DEChNs inhibit hyperlipidemia.
Our zeta-potential detection revealed that even though the DEChNs possess lower amino groups (DDA values approximately 0.25-0.35) than chitosan (DDA values normally >0.70), they still possess high cationic surface charges under acidic conditions. That is to say, chitin, as well as chitosan, may have the unique ability to bind lipids and bile acids. Moreover, the nanofibrillar morphology and high crystallinity might be the other advantages. In detail, the C2-amino groups on the surface of the DEChNs were cationized in gastric juice (pH 1-2), which gave the DEChNs a high zeta-potential of +30-70 mV in the stomach. Positively charging the DEChNs allowed for the attraction and accumulation of anions, such as large amounts of lipids, cholesterol, and bile acid, to the DEChNs via electrostatic attraction. It is noteworthy to mention that the DEChNs do not dissolve at a molecular level but disperse at a nanofibril level in the stomach. Therefore, the DEChNs adsorb to the surface of an oil droplet, decreasing its accessibility to digestive enzymes, thereby decreasing its digestion. Moreover, the DEChNs could act as an emulsifier, affecting the emulsification and adsorption of lipids because DEChNs and oil could contribute to a large accumulation through hydrophobic interaction and nano-effects. Therefore, the binding of lipids and the emulsification could be the main cause for the hypolipidemic action of DEChNs. When the protonated DEChNs enter the intestine (pH 7-7.5), they lose the positive charge then flocculate and precipitate along with the lipids, cholesterol, and bile acid. Thereafter, these precipitates are trapped in the intestine and eventually excreted.

Conclusions
The present study examined the following effects of DEChNs: 1) reducing accumulation of adipose tissues, 2) preventing increases in plasma in mice that are fed a longterm high-fat diet, and 3) guarding against the symptoms of hypercholesterolemia and fatty liver in mice. Further studies are needed to examine the pharmacokinetics and biodegradability of DEChNs. Finally, the TC and lipid levels of the liver, serum, and liver enzymes and receptors regulating the lipid metabolism were not examined in this study. Therefore, these areas also need further examination.