Roles of Vitamin Metabolizing Genes in Multidrug-Resistant Plasmids of Superbugs

Superbug crisis has rocked this world with million deaths due to failure of potent antibiotics. Thousands mdr genes with hundreds of mutant isomers are generated. Small integrons and R-plasmids have combined with F’-plasmids creating a space for >10-20 of mdr genes that inactivate antibiotics in different mechanisms. Mdr genes are created to save bacteria from antibiotics because gut microbiota synthesize >20 vitamins and complex bio-molecules needed for >30000 biochemical reactions of human metabolosome. In other words, mdr gene creation is protected from both sides, intestinal luminal cells and gut bacteria in a tight symbiotic signalling system. We have proposed, to avert the crisis all vitamin metabolizing genes will be acquired in MDR- plasmids if we continue oral antibiotics therapy. Therefore, we have checked the plasmid databases and have detected thiamine, riboflavin, folate, cobalamine and biotin metabolizing enzymes in MDR plasmids. Thus vit genes may mobilise recently into MDR-plasmids and are likely essential for gut microbiota protection. Analysis found that cob and thi genes are abundant and likely very essential than other vit genes.


MDR bacteria contaminated in air dust and rain water
Life creation is due to chemical evolution and Darwin's adaptation theory tells the unique way of changes in cellular structure and metabolism in different eco-system. Microscopic unicellular bacteria, fungi, yeast, protozoa, and algae are regulating water and atmosphere circling plant and animal kingdoms (Pathak et al., 1993;Ahmadjian, 2000). A battle among the creatures is maximum at all points where simple bacteria create crisis in the human life creating toxins and other metabolic effects (Grossart et al., 2013;Mckenna, 2013). Microbes are in symbiotic relation with human and 2x10 12 gut bacteria perform various syntheses of bio-molecules like vitamins, butyrate, unsaturated fatty acids, bile salt and prostaglandin (Le Chatelier et al., 2013). Recently, multidrug-resistant microbes have increased in global water and atmosphere (PM10 particulates increased) posing a threat to human population worldwide (Ibiene et al., 2011;Chakraborty, 2017b). Antibiotics had cured most infections between1940-1990 although gradual increase in drug resistance was detected in many continents as early as 1960 (Reynolds , 1989;Paul et al., 2013). Indeed the drug industry had always run to discover potent derivatives like cefotaxime, ceftrioxane, imipenem and dorripenem in place of old drug like ampicillin and oxacillin (Laxminarayan et al., 2013).
Most harmful changes have happened when 2-15 kb R-plasmids and integrons are combined with 62kb F'-plasmids and such MDR conjugative plasmids donate mdr genes into all bacteria more easily by conjugation (Chakraborty, 2016a). Thus, 40% common bacteria in Ganga river water and Bay of Bengal sea water are ampicillin resistant and most bacteria (>95%) have isolated from human and animal are ampicillin and tetracycline resistant being two early mdr genes (amp and tet that have sequenced in 1965 as plasmid pBR322) and many mdr genes are detected in plasmids of intestinal bacteria (Morten et al., 2009).
Moreover, OXA-2, OXA-23, OXA-48 and OXA-51/58 have no sequence similarity but all hydrolyses carbapenems and some inhibitors. The Klebsiella pneumoniae New Delhi metallo-β-lactamase-1 (NDM-1) was discovered in 2009 in an Indian patient that could hydrolyse all β-lactums (Mataseje et al., 2016). However, NDM1 outbreaks in England and USA as early as 2010 and were found in plasmid as well as chromosome of K. pneumoniae, E. coli and Acinetobacter baumannii as well as to lesser extent in Providentia sp. and Enterobacter sp. (Chakraborty, 2016a).

Drug efflux genes are accumulating in large plasmids
Major drug efflux MDR genes include tet gene isomers which encode a membrane-bound drug efflux protein (~400aa) which kicks out tetracycline from bacterial cell cytoplasm and also has been diversified into tetA/B/C (accession nos. X75761, KC590080) (McMurry et al., 1980). Other potential genes are MFS, RND and MATE types drug efflux genes that could kick out drugs in a proton-pump mechanism (Sun et al., 2014). The MexAB-OprM system in Pseudomonas aeruginosa has the broadest substrate specificity and contributes to resistance to macrolides, aminoglycosides, sulfonamides, fluoroquinolones, tetracyclines and many βlactams. The loss of the outer membrane protein (porin) OprD, is associated with imipenem resistance and reduced susceptibility to meropenem. Similarly, tetracycline resistant protein, tetM (accession no. AY466395, protein id. AAS45561) binds tetracycline increasing drug MIC (Croft et al., 2013;Chakraborty, 2016a).

Target mutations are prominent mechanism for multi-resistance
The majority of rifampicin-resistant clinical isolates of M. tuberculosis harbour mutations in the 507-533 coding region of rpoB gene creating an altered β-subunit of the RNA polymerase that could not able to bind refampicin and altered KatG gene (S315T mutation) and -15C/T mutation in the promoter may be important in isoniazid resistance targeting NADHdependent enoyl-acyl carrier protein (ACP)-reductase that is involved in mycolic acid synthesis. VanA gene cluster are involved in the vancomycin resistance in Enterococcus facium (Merlo et al., 2015; also see, plasmids pIP501 and pAM_1) and ermA/B genes are diverged 23S rRNA methyl transferases that give resistant to macrolides (Harme et al., 2015).
This implies that bacteria do change its target genes if new mdr gene synthesis is delayed or very high dose antibiotic is used. So bacteria need multiple gateways to inactivate drug quickly to save gut bacteria (Chakraborty, 2016c).

MDR Genes move into chromosome to increase gene dose
In skin infection, methicillin resistance gene (mecA) encoding a penicillin-binding protein and activated with mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec) has been reported in Staphylococcus aureus chromosome which also is associated with bla, aac, aad and sul1/2 types MDR genes. This implies that to avert the drug crisis, mdr genes have been mobilized into chromosome to increase in copy number and in association with multiple mdr genes (Chakraborty, 2016b).
We have extensively reviewed the beta-lactamases (Chakraborty, 2016b) and drug acetyl transferases (Chakraborty et al., 2017) in plasmids and integrons and their association in other mdr genes (Chakraborty, 2016a;Chakraborty, 2017c,f). From GenBank data analysis, we have observed an increase in drug efflux genes and IS-elements in MDR conjugative plasmids with reducing the TRA conjugative genes. Which means most of the bacteria have now many types of MDR-plasmids and as we are continuing insult with complex oral antibiotics, other necessary changes are obvious (1/4 genes in MDR-plasmids are unknown function) to avert the serious health crisis of human and bacteria under strong symbiosis (Ame J Drug Deli Ther., 2018, in press). So we have investigated here the nature of vitamin synthesizing genes in large MDR conjugative plasmids to prove that superbug horror and antibiotic horror are synonemous and many changes have occurred in bacterial genomes and plasmids as we have increased multiple complex antibiotics doses since 1960s.

Materials and Methods
Water from Ganga River was collected at the morning from Babu Ghat (Kolkata-700001) and Howrah Station area. About 100µl of water was spread onto 1.5% Luria Bartoni-agar plate containing different concentration of antibiotics at 2-50µg/ml. MDR bacteria were selected in agar-plate containing ampicillin, streptomycin, chloramphenicol, tetracycline or ciprofloxacin at 50, 50, 34, 20 µg/ml respectively. As imipenem and meropenem resistant bacteria were present low (0.08-0.2 cfu/ml water), a modified method was followed. 2 ml 5x LB media was added into 10 ml River/Sea water at 2-10µg/ml concentration and was incubated 24 hrs to get drug resistant bacteria population (Chakraborty, 2015). Meropenem resistant bacteria further selected on ampicillin, tetracycline, chloramphenicol and streptomycin to get the superbugs.
PCR amplification was performed using 1 unit Taq DNA polymerase, 20ng DNA template, 0.25mM dXTPs, 1.5mM MgCl 2 , for 35 cycles at 95 O C/30" (denaturation)-52 O C/50"(annealing)-72 O C/1.5' (synthesis). The product was resolved on a 1% agarose gel in 1X TAE buffer at 50V for 2-4 hrs and visualized under UV light and photograph was taken (Chakraborty et al., 1993). The 16S rRNA gene amplification and mdr genes sequencing are performed by conventional methods (Sanger et al., 1977;Chakraborty et al., 1991  families acetylating enzymes. This data has suggested that accumulation of vitamin synthesizing enzymes into plasmids is not accidental and only is increasing in MDR-plasmid now due to complex antibiotic use.

Folic acid biosynthetic genes in MDR-plasmids
We also found folate and thimine metabolism enzymes in Sinorhizobium meliloti mega plasmid, pSymA (accession no. AE006469; 1354kb) like 5,10 methylene tetrahydrofolate reductase (protein id. AAK65825; nt. 1211022-1211975), formoyl tetrahydrofolate deformylase (protein id. AAK65824) and thiamine pyrophosphate binding enzyme (protein id. AAK65851) but no classical mdr gene but penicillin binding proteins (protein ids. AAK65894, AAK65893) and many ABC or MFS transporters. This supports our hypothesis that bacteria will acquire more enzymes in the vitamin biosynthetic pathway to avert the action of antibiotics and will preserve the symbiotic relation in the intestine. So how and why such genes are now accumulating in MDR-plasmids is a important observation but needs research to elucidate if our hypothesis that all vitamin synthesizing enzymes will be in MDRplasmids to avert antibiotic actions on gut microbiota which are protected by symbiotic phenomenon during evolution (Wang et al., 2017).
AHK47149) as well as acrB (protein id. AHK47037) and ermA/QacA (protein id. AHK47565) drug efflux proteins, Search indicated that the plasmid had biotin synthesizing enzymes (bioB, bioD and bioF) at the nt. 983383-981237 (protein ids. AHK47270, AHK4768 and AHK47269 respectively). Thus multiple vitamin synthesizing genes are accumulating in mdr plasmids due to repeated use of complex antibiotics.

Pantothenate biosynthetic gene in large MDR Plasmids
Enterobacter cloacae plasmid, p22ES-469 (Accession no. CM008897) has acquired enzymes involved in pantothenate metabolism (protein ids. PIA01523, PIA01562) and also in association with thi genes and many mdr genes.

Niacin biosynthetic genes in MDR-plasmids
Rhizobium leguminosarum large plasmids have nicotinamide amidase gene (protein id.

Many biosynthetic genes in large MDR plasmids
As discussed earlier, we detected many large plasmids have multiple vit genes. Enterobacter cloacae large plasmid p22ES-469 has many thi and pan genes (Miranda-Ríos et al., 1997).
Shewanella bicestrii plasmid pSHE-CTX-M has cob and rib genes. Rhizobium leguminosarum very large plasmid also have multiple thi genes as well as bio and cob genes (accession no. CP025013). This implies that gradual selection of vit genes into plasmids have initiated and maintained during antibiotic exposure.

Discussion
Multi Symbiotic bacteria contribute to the functional biodiversity in the aquatic world and influence the fitness of the host organisms and microhabitats ecosystem. It has been suggested that LPS, vitamins and butyrate from intestinal bacteria activate luminal cells to secrete interleukins and cytokines that help to synthesis diversified mdr genes in conjugative plasmids and chromosome to protect gut microbiota against action of high dose of antibiotics (Gibson et al., 2016). We propose that MDR bacteria will be normal resident of intestine until we stop oral antibiotic use. It is thus G-20 Nations in Germany are united for active research on MDR bacteria to stop superbug horror and WHO has warned to use multiple doses of antibiotics to patients and animals. Many microorganisms are also gathering toxin genes in plasmids and likely very threatening to public. Bacillus thuringiensis plasmid pBMB293 (Accession no. CP007615, 294kb) has no mdr gene but genes for enterotoxins (protein id.
AIM34697), dipterans toxin (protein id. AIM34741) and reverse transcriptase, DNA polymerase β, DNA topoisomerase III and type II secretion system. Similarly, Bacillus anthrus plasmid pX01 (accession no. CM002399; 171kb) has toxin gene (protein id. AFL55645, 809aa) and also in pBMB293 plasmid. We need to reduce global toxicity in water and our industries must know the knowledge of contamination of chemicals and heavy metals in water increasing superbugs spread.

Conclusion
WHO has suggested to follow AMR Action Plan and G-20 leaders and Scientists have agreed to reduce antibiotics use in human, animal, and agricultural land as well as to augment research on novel therapeutics alternate to antibiotics (Villa et al., 2015). We have purified organic phyto-extracts (Cassia fistula, Suregada multiflora, Syzygium aromaticum, Cinnamomum zeynalicum, etc) that inhibit Kolkata superbugs and gives a hope for new drug development (Chakraborty, 2015). We slogan, "Come Back to Nature: Save Plants and Use as Medicine". Many technologies like enzybiotics, phage therapy and gene medicines are under development other than herbal therapy (Kutter et al., 2015). Sadly, household water, sea water, rain water and river water are contaminated greatly with superbugs increasing deadly infections where MRSA Staphylococcus aureus, MDR Acinetobacter baumannii and NDM1 Escherichia coli infections are all antibiotic resistant. Thus we lost our win position using ampicillin, streptomycin, tetracycline, ciprofloxacin, azithromycin, and cefotaxime and imipenem drugs against MDR bacterial infections. The GenBank data analysis has proved our hypothesis that vitamin crisis in human due to antibiotic therapy. We think much analysis of unknown genes in superbug plasmids are necessary and likely NIH (NCBI, USA) should act quickly. Perhaps DNA recombination enzymes like tranposases, resolvases, integrases, topoisomerases and plasmids partition enzymes are good target for development of therapeutics against superbugs. Trully, all famous drug companies are in horror to invest in new antibiotic discovery. But phage therapy (bacteriophages T4/θ/ϕ), enzybiotics (lysin, lysozyme), gene medicines (ribozyme, CASPER-CAS, IL gene therapy) and delivery of toxic drugs efficiently using nano-carriers (fullerenes, DNA-Origami) are frontier medical sciences to regulate the 21 st Century drug industry (Dunbar et al., 2018). This work may also through some highlight into combat measure and novel drug design against superbugs and there is no similar report in the Pubmed database (Xu et al., 2014).