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A Method of Universal Primer Design for The Detection of Diverged CTX-M Beta-Lactamases in Multi-Drug Resistant Superbugs
Abstract
Complexity of many diverged β-lactamases in common multi-drug resistant bacteria has reached very high level. We found >40% of common bacteria in Kolkata water bodies were ampicillin resistant and reported clinical isolates were >95% ampicillin resistant worldwide. More than twenty unique sequence classes of β-lactamases were known to cause MDR inactivating penicillin, cephalosporin and carbapenem drugs. Further, there are other diverged MDR genes like drug efflux genes (acrAB, mexAB-EF, tetA), drug modifying genes (aacA1/C1, strA/B, cat3B, sul1/2) and drug transporter genes (mexAB, MdtABC) were present in single bacterial plasmid suggesting too many PCR reactions to be performed to understand the drug resistomes. Thus, we devised a method of reduction in PCR assays using seq-2 and forced multi-align analysis of ~195 blaCTX-M protein sequences for universal primer design. The universal primer set recognized major type-1, type-2 and type-9 blaCTX-M mutants including clinically relevant blaCTX-M-15. However, such universal primers were detected mostly CTX-M-1 type β-lactamase gene and a TEM-SHV another universal primer were identified blaTEM-1 type gene in Kolkata superbugs whereas SHV-isomers were infrequent. BlaTEM-1 β-lactamase 132 alanine amino acid polymorphism (GCT-GCG) was detected signifying the amp gene of pBR322 plasmid origin. This study was supported the WHO recommendations to adopt a uniform policy for AMR detection and antibiotic therapy worldwide.
Keywords: Universal primers, Diverged CTX-M β-lactamases, PCR diagnosis, MDR conjugative plasmids
Cite this Article
Asit Kumar Chakraborty, Sourav Kumar Nandi. A Method of Universal Primer Design for The Detection of Diverged CTX-M Beta-Lactamases in Multi-Drug Resistant Superbugs. Research & Reviews: A Journal of Biotechnology. 2019; 9(2): 1–10p.
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References
Laxminarayan R, Duse A, Wattal C et al (2013) Antibiotic resistance. The Lancet Infect Dis 13:1057-1098.
Chakraborty AK (2016) In silico analysis of hotspot mutations in the bacterial NDM-1 and KPC-1 carbapenemase. Biochem Biotechnol Res 4(1):17-26.
Chakraborty AK (2015) High mode contamination of multi-drug resistant bacteria in Kolkata. Ind J Biotechnol 14:149-159.
McKenna M (2013) Antibiotic resistance. Nature (Lond) 499:394-396.
Mataseje LF, Peirano G, Church DL et al (2016) Colistin non-susceptible Pseudomonas aeruginosa ST654 with blaNDM-1 arrives in North America. Antimicrob Agents Chemother Jan 11. pii: AAC.02591-15.
Li X-Z, Plésiat P, Nikaido H (2015) The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin Microbiol Rev 28:337–418. doi:10.1128/CMR.00117-14.
Chakraborty AK (2016) Complexity, heterogeneity, 3-D structures and transcriptional activation of multi-drug resistant clinically relevant bacterial beta-lactamases. Trends Biotechnol-open access 2(1):1-001.
Ambler RP (1980) The structure of β-lactamases. Phil Trans Royal Soc. London 289:321–331.
Bush K, Jacoby GA, Medeiros AA (1995) Functional scheme for β-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 39(6):1211-1233.
Poirel L, Kampfer P, Nordmann P (2002) Chromosome-encoded Ambler class A beta-lactamase of Kluyvera georgiana, a probable progenitor of a subgroup of CTX-M extended-spectrum beta-lactamases. Antimicrob Agents Chemother 46(12):4038-4040.
Wang H, Kelkar S, Wu W et al (2003) Clinical isolates of Enterobacteriaceae producing extended-spectrum beta-lactamases. Antimicrob Agents Chemother 47:790–793.
Yamasaki Y, Komatsu M, Yamashita T (2003) Production of CTX-M-3 extended-spectrum beta-lactamases and IMP-1 metallo- beta-lactamase by five Gram-negative bacilli. Antimicrob Agents Chemother 51:631–638.
Ensor VM, Shahid M, Evans JT et al (2006) Occurrence, prevalence and genetic environment of CTX-M β-lactamases in Enterobacteriaceae from Indian hospitals. J Antimicrob Chemother 58:1260–1263.
Saladin M Cao VT, Lambert T et al (2002) Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol Lett 20(2):161-168.
Li H, Li JB (2005) Detection of Five Novel CTX-M-Type Extended Spectrum Beta-Lactamases with One to Three CTX-M-14 Point Mutations in Isolates from Hefei, Anhui Province, China. J Clini Microbiol 43(8):4301–4302.
Park YJ, Park SY, Oh EJ et al (2005) Occurrence of extended-spectrum beta-lactamases among chromosomal AmpC-producing Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens in Korea and investigation of screening criteria. Diagn Microbiol Infect Dis 51(4):265-269.
Lee S, Park Y-J, Kim M et al (2005) Prevalence of Ambler class A and D β-lactamases among clinical isolates of Pseudomonas aeruginosa in Korea. J Antimicrob Chemother 56:122–127.
Dallenne C, Da Costa A, Decré D et al (2010) Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J Antimicrob Chemother 65(3):490-495.
Parveen RM, Subha Manivannan S, Harish BN et al (2012) Study of CTX-M Type of Extended Spectrum β-Lactamase among nosocomial Isolates of Escherichia coli and Klebsiella pneumoniae in South India. Ind J Microbiol 52(1):35-40.
Guo YF, Zhang WH, Ren SQ et al (2014) IncA/C plasmid-mediated spread of CMY-2 in multidrug-resistant Escherichia coli from food animals in China. PLoS One 9(5):e96738.
Bedenic B, Sardelic S, Luxner J et al (2016) Molecular characterization of class B carbapenemases in advanced stage of dissemination and emergence of class D carbapenemaes in Enterobacteriaceae Infect Genet Evol May 9, pii: S1567-1348(16)30183-6.
Wang S, Zhao SY, Xiao SZ et al (2016) Antimicrobial resistance and molecular epidemiology of Escherichia coli causing bloodstream infections in three hospitals in Shanghai, China. PLoS One 11(1):e0147740.
Rocha FR, Pinto VP, Barbosa FC (2016) The Spread of CTX-M-Type extended-spectrum β-lactamases in Brazil: A Systematic Review. Microb Drug Resist. 22(4):301-311.
Bora A, Hazarika NK, Shukla SK et al (2014) Prevalence of blaTEM, blaSHV and blaCTX-M genes in clinical isolates of Escherichia coli and Klebsiella pneumoniae from Northeast India. Ind J Pathol Microbiol 57:249-254.
George EA, Sankar S, Jesudasan MV et. al (2015) Molecular characterization of CTX-M type extended spectrum β-lactamase producing E. coli isolated from humans and the environment. Indian J Med Microbiol 33 (Suppl S1):73-79.
O’Neill J (2016) Review on antimicrobial resistance. Tackling a crisis for the health and wealth of nations. London. Review on antimicrobial resistance 2016. https://amr-review.org/sites/default/files/.
Chakraborty AK (2016) Multi-drug resistant genes in bacteria and 21st Century problems associated with antibiotic therapy. Biotechnol Ind J 12(12):114.
Blair JMA,Webber MA, Baylay AJ et al (2015) Molecular mechanisms of antibiotic resistance. Nat Rev Microbiol 13:42–51. doi:10.1038/nrmicro3380
.
World Health Organization (2014) Antimicrobial resistance: global report on surveillance. World Health Organization, Geneva, Switzerland: http://apps.who.int/iris/bitstream/10665/112642/1/ 9789241564748_eng.pdf.
Laxinarayan R, Chaudhury RR (2016) Antibiotic resistance in India. Drivers and opportunities for action. PLoS Med 13: e1001974.
Chakraborty AK (2017) Multi-drug resistant bacteria from Kolkata Ganga River with heterogeneous MDR genes have four hallmarks of cancer cells but could be controlled by organic phyto-extracts. Biochem Biotechnol Res 5(1):11-23.
Chakraborty AK (2017) MDR genes are created and transmitted in plasmids and chromosomes to keep normal intestinal microbiota alive against high dose antibiotics- a hypothesis. J Mol Med Clin Appl 2(1):109. DOI: http://dx.doi. org/10.16966/2575-0305.109.
Chakraborty AK (2019) Current status and unusual mechanism of multi-resistance in Mycobacterium tuberculosis. J Health Med Informatics. 10 (1): 328. Doi: 10.4172/2157-420.1000328.
Chakraborty AK, Pradhan S, Das S, Maity M, Sahoo S, Poria K. (2019) Complexity of OXA Beta-Lactamases involved in Multi-Resistance. British J Bio-Medical Res. 3(1): 772-798. Doi: 10.24942/bjbmr.2019.424.
Chakraborty AK, Poira K, Saha D, Halder C, Das S. (2018) Multidrug- Resistant Bacteria with activated and diversified MDR Genes in Kolkata Water: Ganga Action Plan and Heterogeneous Phyto-Antibiotics tackling superbug spread in India. Ame J Drug Deli Ther. 5 (1), Pp.1-9.
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