High Prevalence of Metal Resistant Genes in Salmonella enterica MDR Plasmids Correlates Severe Toxicities of Water with higher Typhoid AMR

AMR and drug void have caused huge panic today with few thousand death per year. MDR Typhoid was a serious old disease and caused serious health hazard in humen and animals demanding an update on molecular biology of the status on transferable genetic elements. Rplasmids combined in F’-plasmid and the new MDR conjugative plasmids were shown abundant in Sanmonella ranging 70-440kb with similarities. BlaTEM, blaCTX-M, blaOXA, blaNDM mdr genes were abundant in >50 plasmids analyzed and metal resistant gene clusters are predominant in most large plasmids. Among the acetyltransferase all catA1, aacA1 and aac-1b-cr genes were located. Abundant streptomycin phosphotransferases (StrAB) and rarely colistin resistant Mcr-5/9 phosphoethanolamine–lipid A transferase were detected. Altered isomeric dihydropterote synthases (Sul1/2/3) were present giving sulfamethoxazole resistance and dhfr gene frequently associated giving trimethoprim resistance. Metal resistant gene clusters like SilABC (CusABC), PcoAB, RcnA, terABC, and merABCXT etc were found in many Salmonella enterica plasmids. Toxin genes like HipA and virulence genes like spvABD were located in few plasmids increasing virulence and pathogenesis. Drug efflux genes tetA or tetB and OqxB, floR, CmlA were frequent where as QepA and EamA genes were rarely seen. Thus, Salmonella metal resistant genes combined with antibiotic resistant genes has tried to overcome the both toxic antibiotics and metalions causing Typhoid AMR. Such acquisition spreads salmoniasis in the live stocks (pig, cow, chicken) where toxic soil and water dominate increasing chance of MDR typhoid in human.

capsule formulation for those over six years of age but injectable Vi polysaccharide vaccine also available. Diagnosis is by either culturing the bacteria or detecting their DNA in the blood, stool, or bone marrow using PCR technique. During prognosis serum AST and ALT may be very high (200-400U/L). The disease was treated with antibiotics such as azithromycin, fluoroquinolones, or third-generation cephalosporins. Salmonella enterica plasmids harbour a composite transposon that can carry multiple resistance genes, including blaTEM-1 (ampicillin resistance), dfrA7 (trimethoprim resistance), sul1+sul2 (sulfamethoxazole resistance), catA1 (chloramphenicol resistance), and strAB (streptomycin resistance) genes. This composite transposon has also been found integrated into the chromosome in some H58 S. Typhi lineages (Klemm et al. 2018). Many drug resistant determinants are abundant in Salmonella emterica plasmids isolated from different animal sources as well as water and thus AMR is a problem increasing salmoniasis in animal and typhoid in human (Rasheed et al. 2019). Typhoid fever, the causative agent of Salmonella enterica serovar Typhi is spreading in the Asian countries due to acquisition of MDR plasmids from multidrug resistant Escherichia coli and Klebsiella pneumonia (Mandal et al. 2012). However, non-typhoidal MDR Salmonella enterica Serovar Typhimurium were isolated in meat foods (chicken, pork and beef) as well as milk and egg, Such MDR bacteria cause serious diarrhoea and bacteraemia and need hospitalization as happening in the Asia as well as United States due to widespread contamination in livestocks (Sadhanthirakodi et al. 2016). Other than Serovar Typhimurium, Serovar Kentucky, Serovar Idican and Serovar Entiritidis are predominant. Interestingly, we found few very small plasmids those have ether mdr genes or virulence genes or metal resistant genes suggesting toxicities of different kind prevail first generating such plasmids but now such small plasmids combned with F'plasmids and then such Salmonella enterica plasmids further recombined residing in the polluted water resources. There are many mdr genes located in Enterobacteriaceae plasmid since 1950s as shown in Figure-1  . First, amp and tet genes were sequenced in pBR322 in 1965 and since the application of colour di-deoxy DNA sequencing, millions plasmid sequences were deposited in GenBank. Amp gene was renamed as bla or beta-lactamse and now 20 different beta-lactamases classes were reported with million of mutated isomers and most importantly ESBL and MBL multiple isomers were located in MDR single conjugative plasmid with size >100kb (Chakraborty, 2017). Similarly, tetAB, acrAB, mexAB/CD/EF, bcr, mcr types MFS and RND drug efflux genes were reported in E. coli, P. aeruginosa, K. pneumonia as well as S. enterica plasmids. Many metal efflux genes (silABC, merB, rcnA) and metal binding genes (telC, silz) were reported in MDR plasmids. Abundance of metal resistant gene cluster in association of mdr genes suggested that metal toxicity in water might be precede the antibiotic toxicity. We will describe here the different types of those genes in Salmonella plasmids causing recent outbreaks of salmoniasis in animals and typhoid in human.

Material & Methods
We got the plasmid sequences from NCBI Nucleotide GenBank Database by typing "Salmonella" and "plasmid". We retrieve the each sequence and searched for mdr genes, drug efflux genes, toxin genes, and virulence genes. BLAST search was performed to get relation among the peers (www.ncbi.nlm.nih.gov/blast). Plasmids were divided into small (3-15kb), medium (15-49kb), large (50-100kb) and very large (>100kb) and plasmid may have mdr gene or no madr gene but virulence genes. Similarly, few plasmids have both metal resistant gene and mdr genes but mdr gene may not be found in few plasmid. Interestingly, such review was absent in the pubmed.

Result
Table-1 showed the overall description of the plasmids describing mdr genes , drug efflux genes, metal resistant genes as well as other genes involved in the Salmonella pathogenesis.
Abundance of metal binding proteins and metal efflux proteins in Salmonella plasmids indicated that such MDR bacteria suffered in the environmental water and had forced to accumulate multiple metal resistant locus where as in the human host such bacteria may lost few metal resistant genes acquiring many antibiotic resistant genes . A similar huge accumulation of silver, copper, mercury and tellurium resistant genes were seen in large plasmid pRH-R27 (accession no. LN555650) where very rare nical-cobult specific efflux protein rcnA was also present (protein id. CED95467) in association with blaVIM, aac6'-1b, sul1 and strAB mdr genes (Kaldhone et al.2019). Plasmid pCFSA300-1 appeared very similar in plasmid pHK0653 with respect to mdr genes (dhfr, sul, blaOXA1, aac3', ANT3") but citrate lyase, Adenine-Guanine phosphoribosyl transferase and carbomoyl phosphate synthase were inserted at tellurium resistant locus. Interestingly, a very small 14kb Salmonella typhimurium plasmid pMG101 (accession no. AF067954) had all silver resistant genes but no other antibiotic resistant genes indicating metal resistance was primitive and likely occurred during European industry development between 1760-1850s where as mdr genes were created after 1940 (Gupta et al. 1999;. We also found a medium plasmid pSA20044414 (accession no. CP030210) with many arsenic and copper resistant genes in association of Tra conjugative proteins but no mdr gene was detected indicating F'-plasmid may be combined with small metal resistant plasmids like pMG101 with silver resistant locus and then small R-plasmids like pSc101 and pMB were combined to originate modern day large MDR conjugative plasmids like p87912 (accession no. CP041180) which contained sixteen mdr genes and two drug efflux genes like oqxA/B . Never the less, WGS of Salmonella (accession nos. CP000026, AE014613) indicated that metal resistant locus were also frequently associated with Salmonella genome (Calva et al. 2015). Mcr-9 enzyme (protein id. ANV19589) was detected in plasmid p09-036813-1A_261 (261kb; accession no. CP016526) in association of aph6-Id, aph3", dhfr, aac3", aacA4 and tellurium, mercury and arsenic metal resistant genes. Such mutant mcr-1 was detected in many S. enterica isolates (Lozano-Leon et al. 2019) giving colistin resistance and more deadly blaNDM-1 also was detected giving imipenem resistance (Banerjee et al. 2018).Salmonella enterica serovar Seftenberg pNDM-SAL plasmid (accession no. KP742988) has both cephamycinase and carbapenemase and thus highly resistant to all beta-lactams and similar Salmonella plasmids pHS36-NDM (accession no. KU726616) and pRH-1238(accession no. KR091911) ware sequenced (Huang et al. 2013;Villa et al. 2015).  Table-1 Note: BlaTEM is similar to amp gene of pBR322 and it lyses benzyl penicillin and ampicillin but not cefotaxime and oxacillin. TetA and TetB enzymes are ~400 aa transmembrane protein and remove tetracycline from bacterial cytoplasm giving tetracycline resistance. Such gene (tetC) was discovered first in plasmid pBR322. StrA and StrB phosphorylates streptomycin and phosphorylated streptomycin could not able to bind ribosome giving resistance. Other phosphotransferases (aph) are known to give gentamycin and kanamycin resistance. Cat enzyme acetylates chloramphenicol and acetylated chloramphenicol did not bind ribosome. AacC1 and aacA1 types acetyltransferases are abundant in plasmids causing aminoglycosode resistance. Hpt is hygromycin phosphotransferase and Arr3 is refampicin phosphotransferase. Dhfr enzyme gave resistance to trimethoprim and sul1/2/3 are altered dihydropterote synthase enzyme giving sulphonamide resistance. spvB is Actin ADP ribosyl transferase and inactivates muscle function. Dcm is cytosine MTase and Dam is adenine methyltransferase where as rmtB is 16S rRNA methyltransferase giving drug resistance altering rRNA structure in the ribosome. HipA is a serine-threonine protein kinase that likely phosphorylates tRNA(Glu) synthetase (GltX). CmlA is chloramphenicol efflux membrane protein and acrAB is RND-MFS drug efflux proteins and similar to OqxAB. Colicin resistance is due to a colcin1b transporter and colicin drug bind to cell membrane inhibiting mureib biosynthesis in bacteria. QepA drug efflux protein located in Salmonella plasmid pGDD25-16 gives floroquinolone resistance as also possible for the presence of aac6'-1b-cr protein in plasmid pHXY0908 due to N-acetylation of ciprofloxacin. Mercuric reductase (merA) and multi-copper oxidase (PcoA) were abundant and many mutations were observed in PcoA but merA was conserved among the plasmids (Figure-7).       Salmonella typhi (genotype 4.3.1) may be a threat to South Asian population (Rahman et al.2020). However, blaTEM, catA1, dhfrA7, sul1, sul2, strAB and gyrase A subunit mutations were detected where as our review demonstrated the presence of blaOXA1, blaCMY and blaNDM like deadly beta-lactamases (  (Hill et al. 1983;Bradley, 1985).

RcnA Ni++/Co++ transporters of
Abundance of tellurium resistance genes is obscure as it is not an essential element like zinc but its applications in electronics, optics, batteries and mining industries have expanded during the last few years, leading to an increase in environmental contamination. Gold ores containing Tellurium are calaverite (AuTe2), sylvanite (AgAuTe4), and nagyagite [AuPb(Sb, Bi)Te2-3S6] and thus gold use increase may correlates its abundance in water. TeO3( -2 ) may cause garlic like smell of dimethyltellurite on ingestion of bismuth salt contaminated with tellurite (Cairnes, 1911) whereas, in another mechanism of detoxification, TeO3( -2 ) was reduced to Te( 0 ) causing precipitation because TeO3( -2 ) was very toxic to bacteria at <1µg/L concentration. TeO3( -2 ) could also be reduced chemically to lower oxidation states by glutathione or by other reduced thiol-containing proteins (metallothionine) with drastic decrease in the concentration of antioxidant molecules such as glutathione and cysteine causing a phenotype of higher TeO3( -2 ) tolerance. In this context, mutants of cysteine biosynthetic pathway have shown highly sensitive to telleurite (Dyllick-Brenzinger et al. 2000;Fuentes et al.2007). Prevalence of mdr genes and metal resistant genes were also demonstrated in many Salmonella sp isolated from food animals (Na et al. 2020;Anwar et al. 2020). Surprisingly, ampC beta-lactamse producing plasmids were not detected in the database but many papers had detected such gene in Salmonella sp (Roschanski et al. 2014). The genetic exchange and acquisition of mdr genes were happed in the gut and thus gut microbiome plaed a central role in shaping both mdr and metal resistant genes (Jain et al. 2018). WGS of Salmonella has showed the existence of MDR-islands in Salmonella genome and thus virulence and multi-resistance will be more prominence in Salmonella infections (Saroj et al. 2008;Liu et al. 2009;Sudhanthirakodi et al. 2016;Parkhill et al. 2018;Luo et al. 2020) Conclusion We explained the recent salmoniasis outbreaks in India as well as abroad due to over expression of plasmid-mediated mdr genes, drug efflux genes as well as metal resistant genes which have acquired when Salmonella spends its life in the contaminated water originated due to huge expansion of metal industry, coal industry as well electronics industry. We presented small plasmids with only metal resistant genes or drug resistant genes or toxin genes. However, combination of such plasmids with 62.5kb F' conjugative plasmids created large mdr conjugative plasmids accumulating different genes that might not necessary for drug resistance. The localization of complete metal resistant operons like sil, cus, mer and ter with 5-15 metal resistant genes in large plasmids indicated that live stocks (pig, chicken, goat) grew in the metal contaminated soil and water with poor hygienic condition. Such report thus confirmed the spread of animal salmoniasis and Salmonella enterica could be located in cow milk and chicken meat. Salmonella typhi plasmids also analyzed to dictate same notion indicating the passage of the organisms in zoonotic reservoirs have to be carefully studied. Never the less we have authenticated the metal resistant proteins as well as their relation to transposons with mdr genes like blaTEM1, blaNDM1, blaCTX-M15, strAB, mcr5/9, dhfr, sul1/2 and drug efflux genes like tetA, tetB, floR, and oqxB. This report is thusa valuable source of drug resistant and metal resistant proteins and their symbiotic relation with respect to co-passage of Salmonella enterica to intestine (to make gut microbiome) and water resources. We are studying the metal resistant bacteria in lakes near Midnapore City where clusters of metal and steel industries are accumulating at the side of the Bombay Road and Kangsabati River of West Bengal, India.