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| Year : 2013 | Volume
: 6
| Issue : 6 | Page : 653-657 |
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| LRSA suspect, taken to molecular court, PCR trialled, proven guilty |
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Baishali Chakraborty1, Banya Chakraborty1, Yeshi Palden Dopthap1, Dibyendu Banerjee2
1 Department of Microbiology, Calcutta Medical College, Kolkata, West Bengal, India
2 Department of Microbiology, Calcutta National Medical College, Kolkata, West Bengal, India
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| Date of Web Publication | 6-Sep-2014 |
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 Abstract | | |
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
How to cite this article:
Chakraborty B, Chakraborty B, Dopthap YP, Banerjee D. LRSA suspect, taken to molecular court, PCR trialled, proven guilty. Ann Trop Med Public Health 2013;6:653-7 |
How to cite this URL:
Chakraborty B, Chakraborty B, Dopthap YP, Banerjee D. LRSA suspect, taken to molecular court, PCR trialled, proven guilty. Ann Trop Med Public Health [serial online] 2013 [cited 2021 Mar 4];6:653-7. Available from: https://www.atmph.org/text.asp?2013/6/6/653/140243 |
| Introduction | |  |
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:
- Mutations the domain V region of one or more of the 5 copies of 23S rRNA gene.
- Acquisition of plasmid-mediated ribosomal methyltransferase cfr gene.
- Deletion/mutation in the ribosomal protein L3 of PTC.
Among these three mechanisms, LZD resistance has been largely attributed to the first one. [3]
| Materials and Methods | | |
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]. | Figure 1: PCR with Taq polymerase showing primer set for 5 copies of 23S rRNA gene
Click here to view |
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.
| Discussion | | |
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.
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| 7. | Farrell DJ, Mendes RE, Ross JE, Sader HS, Jones RN. LEADER Program Results for 2009: An activity and spectrum analysis of linezolid using 6,414 Clinical isolates from 56 medical centers in the United States. Antimicrob Agents Chemother 2011;55:3684-90.
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Correspondence Address:
Dibyendu Banerjee
27-H, Rajkrishna Street, Uttarpara - 712 258, West Bengal
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/1755-6783.140243
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