Background: A 31-year-old patient was admitted in our Medical College and Hospital following a road traffic accident, and with a history of Linezolid treatment for 10 days. Materials and Methods: Culture from wound swab revealed multiple pathogens. Among them, Staphylococcus aureus turned out to be Methicillin resistant (MRSA), and also showed resistance to Linezolid disc diffusion. Then PCR of the resistant strain as well as a sensitive strain followed by DNA sequencing was done. Results: Sequencing of the domain V region of the 23S rRNA gene revealed the presence of a G2576U mutation in LRSA (LZD-resistant S. aureus) in two 23s rRNA copy, rrn1 and rrn5. Conclusions: The patient’s wound was infected with an MRSA strain, which was LRSA as well. LRSA is a rare occurrence, and rarely reported from Indian subcontinent. A constant vigil is necessary to detect this resistance, if possible with tools of molecular epidemiology. Keywords: Gene sequencing, LRSA, MRSA
Staphylococcus aureus , a well-known pathogen, is notorious for causing pyogenic infections, both in the community setup, as well as in hospital environment. It is also remarkable for developing multidrug resistance day by day-especially becoming methicillin-resistant Staphylococcus aureus, otherwise known as MRSA. Linezolid, an oxazolidinone, has shown potent activity against Gram-positive organisms like MRSA, methicillin-resistant coagulase-negative Staphylococci (MR-CoNS), vancomycin-resistant Enterococci (VRE), and multidrug-resistant Streptococcus pneumoniae, and has thus become the clinicians’ favorite blue-eyed boy. [1] It uniquely inhibits protein synthesis by binding to the peptidyltransferase center (PTC) of the 50S ribosomal subunit. [2],[3] Linezolid resistance in S. aureus is extremely uncommon, and the surveys have revealed that >99% isolates are susceptible. [4],[5] There are three main mechanisms for developing LZD resistance:
Among these three mechanisms, LZD resistance has been largely attributed to the first one. [3]
We, hereby, present a case where a 31-year-old male was admitted to our tertiary care Medical College and hospital on 2 nd November, 2012. He presented with non-healing ulcer following road traffic accident around 2 months before the date of admission. There was no history of diabetes mellitus or hypertension. He was treated with linezolid from 01.10.2012 for about 10 days-as advised by a quack. No other drug history or prescription was available. Culture report from wound swab revealed S. aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa as well. Susceptibility testing was performed by Kirby-Bauer disc diffusion method following CLSI guideline. [6] The S. aureus isolate was found to be sensitive to vancomycin, cotrimoxazole, amikacin and ofloxacin, and resistant to penicillin, erythromycin, cefoxitin (hence MRSA), and linezolid. Growth upto the edge of 30 mg linezolid disc was noted. Linezolid susceptibility was then repeated by Stokes method with S. aureus ATCC strain as control, and again the test strain showed resistance. The isolate was preserved by stabbing it in a semisolid media. At the same time a linezolid sensitive isolate of S. aureus was also preserved. DNA extraction was done from both isolates and PCR was done with Taq DNA polymerase [Figure 1].
Then, following Agarose gel electrophoresis of the amplified product, DNA sequencing was done from lanes 2, 3, 4 and 6 of gel (lane 2 and 6 representing LNZ rrn1 and LNZ rrn5, respectively). After sequencing we got the following three sequences of two different copy of 23s rDNA (LNZr1, LNZr5) and as control we used pcr product of LSSA (LZD-sensitive S. aureus) DNA with rDNA1 primer set. >LNZr1 TGGACGCCCTCAGTCCTCAGAATGGTTGGAA TCATTCATAGAG TGTAAAGGCAT AAGGGAGCT TGACTGCGAGACCTACA AGTCGAGCAGGGTCGAAAGACGGACTTAGTGA TCCGGTGGTTCCGCATGGAAGGGCCATCGCTC AACGGATAAAAGCTAC CCCGGGGATAACAGGCTTATCTCCCCCAAGA GTTCACATCGACGGGGAGGTTTGGCACCTCG ATGTCGGCTCATCGCATC CTGGGGCTGTAGTCGGTCCCAAGGGTTGGG CTGTTCGCCCATTAAAGCGGTACGCTAGCTG GGTTCAGAACGTCGTGAGA CAGTTCGGTCCCTATCCGTCGTGGGCGTAGGA AATTTGAGAGGAGCTGTCCTTAGTACGAGAGG ACCGGGATGGACATAC CTCTGGTGTACCAGTTGTCGTGCCAACGGCA TAGCTGGGTAGCTATGTATGGACGGGATAAGT GCTGAAAGCATCTAAGC ATGAAGCCCCCCTCAAGATGAGATTTCCCAAC TTCGGTTATAAGATCCCTCAAAGATGATGAGGT TAATAGGTTCGAGGT GGAAGCGTGGTGACACGTGGAGCTGACGAATA CTAATCGATCGAAGACTTAATCAATTTATTTCA ATGTTTTGCGAAGCA AAATCATTTACTTACTATCTAGTTTTGAATGTA TAATCATTCTCTTGTCTGGTGACAATGGCAAG GAGGTCACACCTGTT CCCATGCCGAACACAGAAGTTAAGCTCCTTA GCGCCGATGGTAGTTGGATTTACGTTCCGCT AGAGTAGGACGTTGCCAG CCAAAAAAATAGAGATGGGAA (821nt) >LNZr5 CCTAAAAAAAAAAGAGGAGCTAAGTTCCTC AGAATGGTTGGAAATCATTCATAGAGTGTAAA GGCATAAGGGAGCTTGAC TGCGAGACCTACAAGTCGAGCAGGGTCGA AAGACGGACTTAGTGATCCGGTGGTTCCGCAT GGAAGGGCCATCGCTCAAC GGATAAAAGCTACCCCGGGGATAACAGGCTT ATCTCCCCCAAGAGTTCACATCGACGGGGAGG TTTGGCACCTCGATGTC GGCTCATCGCATCCTGGGGCTGTAGTCGGT CCCAAGGGTTGGGCTGTTCGCCCATTAAAGC GGTACGCTAGCTGGGTTCA GAACGTCGTGAGACAGTTCGGTCCCTATCCG TCGTGGGCGTAGGAAATTTGAGAGGAGCTGT CCTTAGTACGAGAGGACC GGGATGGACATACCTCTGGTGTACCAGTTG TCGTGCCAACGGCATAGCTGGGTAGCTATGTAT GGACGGGATAAGTGCTG AAAGCATCTAAGCATGAAGCCCCCCTCAAG ATGAGATTTCCCAACTTCGGTTATAAGATCCC TCAAAGATGATGAGGTTA ATAGGTTCGAGGTGGAAGCGTGGTGACACG TGGAGCTGACGAATACTAATCGATCGAAGACT TAATCAATTTATTTCAAT GTTTTGCGAAGCAAAATCATTTACTTACTATC TAGTTTTGAATGTATAATCATTCTCTTGTCTGG TGACAATGGCAAGGA GGTCACACCTGTTCCCATGCCGAACACAGAA GTTAAGCTCCTTAGCGCCGATGGTAGTTGGAT TTACGTTCCGCTAGAGT AGGACGTTG CCAGGCAAAT TAAATTATTCCACA GTAGTCTATTGTGGTTTAGGAAAGAAAA (861nt) >LNZcontrl CGAGAGCG CTAAGGGTTTCCCTCA GGAATGGT TGGAATCATTCATAGAGTGTAAAGGCATAAG GGAGCTTGACTGCGAGA CCTACAAGTCGAGCAGGGTCGAAAGACGG ACTTAGTGATC CGGTGGTTCCGC ATGGAAGGG CCATCGCTCAACGGATAAA AGCTACCCCGGGGATAACAGGCTTATCTCCCC CAAGAGTTCACATCGACGGGGAGGTTTGGC ACCTCGATGTCGGCTCAT CGCATCCTGGGGCTGTAGTCGGTCCCAAGG GTTGGGCTGTTCGCCCATTAAAGCGGTAC GCGAGCTGGGTTCAGAACGTC GTGAGACAGTTCGGTCCCTATCCGTCGT GGGCGTAGGAAATTTGAGAGGAGCTGTC CTTAGTACGAGAGGACCGGGATGG ACATACCTCTGGTGTACCAGTTGTCGTGCC AACGGCATAGCTGGGTAGCTATGTGTGGACG GGATAAGTGCTGAAAGCAT CTAAGCATGAAGCCCCCCTCAAGATGAGAT TTCCCAACTTCGGTTATAAGATCCCTCAAA GATGATGAGGTTAATAGGTT CGAGGTGGAAGCATGGTGACATGTGGAG CTGACGAATACTAATCGATCGAAGACTTAAT CAAAATAAAT GTTTTGCGAAG CAAAATCACTTTTACTTACTATCTAGTTTT GAATGTATAATTTACATTCATATGTCTGGTGA CTATAGC AAGGAGGTCAC ACCTGTTCCCATGCCGAACACAGAAGTTAA GCTCCTTAGCGTCGATGGTAGTCGAACT TACGTTCCGCTAGAGTAGAACG TTGCCAGCCAAAAAAATGAAATGGAAA (827nt) Alignment of the above three sequences revealed LNZr1 TGGACGCCCTCAGT-CCTCAG-AATGGTTGGAA TCATTCATAGA 42 LNZr5CCTAAAAAAAAAAGAGGAGCTAAG-TT CCTCAG-AATGGTTGGAAATCATTCATAGA 55 LNZcontrl CGAGAGCGCTAAGGGTTTCCCTCAGGAAT GGTTGGAA-TCATTCATAGA 48 ********************************** LNZr1 GTGTAAAGGCAT AAGGGAGCTTGAC TGCGAGA CCTACAAGTCGAG CAGGGTC GAAAGACG 102 LNZr5 GTGTAAAGGCATAAGGGAGCTTGACTGCGAG ACCTACAAGTCGAGCAGGGTCGAAAGACG 115 LNZcontrl GTGTAAAGGCATAAGGGAGCTTGACTGCGA GACCTACAAGTCGAGCAGGGTCGAAAGACG 108 *************************************** ********************* LNZr1 GACTTAGTGATCCGGTGGTTCCGCATGGAA GGGCCATCGCTCAACGGATAAAAGCTACCC162 LNZr5 GACTTAGTGATCCGGTGGTTCCGCATGGAAG GGCCATCGCTCAACGGATAAAAGCTACCC175 LNZcontrl GACTTAGTGATCCGGTGGTTCCGCATGGAAG GGCCATCGCTCAACGGATAAAAGCTACCC 168 ***************************************** ******************* LNZr1 CGGGGATAACA GGCTTATCT CCCCCA AGAGTT CACATCGACG GGGAGGTT TGGCACCTCG 222 LNZr5 CGGGGATAACAGGCTTAT CT CCCCCAAGAGTT CACATCGACGGGGAGGTTTGGCACCTCG 235 LNZcontrl CGGGGATAACAGGCTTATCTCCCCCAAGAGT TCACATCGACGGGGAGGTTTGGCACCTCG 228 **************************************** ******************** LNZr1 ATGTCGGCTCATCGCATCCTGGGGCTGTAGT CGGTCCCAAGGGTTGGGCTGTTCGCCCAT 282 LNZr5 ATGTCGGCTCATCGCATCCTGGGGCTGTAGT CGGTCCCAAGGGTTGGGCTGTTCGCCCAT 295 LNZcontrl ATGTCGGCTCATCGCATCCTGGGGCTGTAGT CGGTCCCAAG GGTTGGGCT GTTCGCCCAT 288 ***************************************** ******************* LNZr1 TAAAGCGGTACGCTAGCTGGGTTCAGAACGTC GTGAGACAGTTCGGTCCCTATCCGTCGT 342 LNZr5 AAGCGGTACGCTAGCTGGGTTCAGAACGTCGT GAGACAGTTCGGT CCCTATCCGTCGT 355 LNZcontrl TAAAGCGGTACGCGAGCTGGGTTCAGAACGT CGTGAGACAGTTCGGTCCCTATCCGTCGT 348 ***************************************** ****************** LNZr1 GGGCGTAGGAAATTTGAGAGGAGCTGTCCT TAGTACGAGAGGACCGGGATGGACATACCT 402 LNZr5 GGGCGTAGGAAATTTGAGAGGAGCTGTCCT TAGTACGAGAGGACCGGGATGGACATACCT 415 LNZcontrl GGGCGTAGGAAATTTGAGAGGAGCTGTCCTTA GTACGAGAGGACCGGGATGGACATACCT 408 *************************************** ********************* LNZr1 CTGGTGTACCAGTTGTCGTGCCAACGGCATA GCTGGGTAG CTATGTATGG ACGGGATAAG 462 LNZr5 CTGGTGTACCAGTTGTCGTGCCAACGGCATAG CTGGGTAGCTATGTATGGACGGGATAAG 475 LNZcontrl CTGGTGTACCAGTTGTCGTGCCAACGGCAT AGCTGGGTAGCTATGTGTGGACGGGATAAG 468 ********************************************** ************* LNZr1 TGCTGAAAGCATCTAAGCATGAAGCCCCCCTC AAGATGAGATTTCCCAACTTCGGTTATA 522 LNZr5 TGCTGAAAGCATCTAAGCATGAAGCCCCCCT CAAGATGAGATTTCCCAACTTCGGTTATA 535 LNZcontrl TGCTGAAAGCATCTAAGCATGAAGCCCCCC TCAAGATGAGATTTCCCAACTTCGGTTATA 528 ************************************** ********************** LNZr1 AGATCCCTCAAAGATGATGAGGTTAATAGGTT CGAGGTGGAAGCGTGGTGACACGTGGAG 582 LNZr5 AGATCCCTCAAAGATGATGAGGTTAATAGGTT CGAGGTGGAAGCGTGGTGACACGTGGAG 595 LNZcontrl AGATCCCTCAAAGATGATGAGGTTAATAGGT TCGAGGTGG AAGCATGGT GACATGTGGAG 588 ******************************************** ************** LNZr1 CTGACGAATACTAATCGATCGAAGACTTAAT CAATTTATTTCAATGTTTTGCGAAGCAAA 642 LNZr5 CTGACGAATACTAATCGATCGAAGACTTAAT CAATTTATTTCAATGTTTTGCG AAGCAAA 655 LNZcontrl CTGACGAATACTAATCGATCGAAGACTTAAT CAAAATA-AATGTTTTGCGAAGCAAA 644 *************************************** *************** LNZr1 ATCA-TTTACTTACTATCTAGTTTTGAATGTAT AAT-CATTCTCTT GTCTGGTGACA 697 LNZr5 ATCA-TTTACTTACTATCTAGTTTTGAATGT ATAAT CATTCTCTTGTCTGGTGACA 710 LNZcontrl ATCACTTTTACTTACTATCTAGTTTTGAATG TATAATTTACATTCATATGTCTGGTGACT 704 **************************************** *********** LNZr1 ATGGCAAGGAGGTCACACCTGTTCCCATGCC GAACACAGAAGTTAAGCTCCTTAGCGCCG 757 LNZr5 ATGGCAAGGAGGTCACACCTGTTCCCATGCCG AACACAGAAGTTAAGCTCCTTAGCGCCG 770 LNZcontrl ATAGCAAGGAGGTCACACCTGTTCCCAT GCCGAACACAGAAGTTAAGCTCCTTAGCG TCG764 ********************************************************** LNZr1 ATGGTAGTTGGATTTACGTTCCGCTAGAGTAG GACGTT GCCAGCCAAA-AAAATA-811 LNZr5 ATGGTAGTTGGATTTACGTTCCGCTAGAGT AGGACGTTGCCAGGCAAATTAAATTATTCC 830 LNZcontrl ATGGTAGTCGAACTTACGTTCCGCTAGAGTAG AACGTTGCCAGCCAAA-AAAAT- 817 *************************************** LNZr1 GAGATGGGAA 821 LNZr5 ACAGTAGTCTATTGTGGTTTAGGAAAGAAAA 861 LNZcontrl GAAATGGAAA 827 ***** Sequencing of the domain V region of the 23S rRNA gene revealed the presence of a G2576U mutation (Escherichia coli numbering) in LRSA (LZD-resistant S. aureus) in comparison to LSSA (LZD-sensitive S. aureus) in two 23s rRNA copy, rrn1 and rrn5. Although we noticed other mutations, we could not find any other reported mutation like G2447U, G2505A, C2512U, G2513U, and C2610G.
Linezolid resistance, thankfully, is an extremely rare phenomenon. The Zyvox Annual Appraisal of Potency and Spectrum (ZAAPS) study, 2007 for linezolid resistance came up with an overall resistance rate to linezolid in 23 countries to 0.03%. [5] A similar worldwide program, LEADER 2009, which monitors and tracks linezolid resistance in USA since 2004, reported the resistance rate to be 0.34%. [7] Data on linezolid resistance from Asian countries, especially Indian subcontinent is extremely rare and whatever scarce reports are found-DNA sequencing to point out the type of point mutation/mechanism of resistance has not been performed in almost all the cases. The strain of S. aureus found by us was unique in the sense that it was an MRSA strain showing resistance to linezolid, hence compromising almost all the avenues of treatment. It is high time that we wake up, take notice of impending doom, recognize the dangerous outcomes of a great threat, closely monitor the resistance pattern evolving, and track it down with the tools of molecular epidemiology. The authors gratefully acknowledge Dr. Manas Maiti, Senior Scientist, Department of genetics, for PCR and DNA sequencing.
Source of Support: None, Conflict of Interest: None
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