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Kolistin direncinin moleküler tespitinde yanlış tanı: kolistine duyarlı Acinetobacter baumannii izolatlarında yanlış mcr-1-PCR pozitifliği

Year 2023, Volume: 48 Issue: 3, 1139 - 1147, 30.09.2023
https://doi.org/10.17826/cumj.1348548

Abstract

Amaç: Bu çalışmanın amacı Türkiye'deki bir araştırma hastanesinde tedavisi zor enfeksiyonlara neden olan karbapenem dirençli Gram negatif bakterilerde kolistin direncinden sorumlu mcr-1 geninin varlığını araştırmaktı.
Gereç ve Yöntem: 75'i Acinetobacter baumannii, 19'u Pseudomonas aeruginosa ve 9'u Klebsiella pneumoniae olmak üzere karbapeneme dirençli 103 izolatta mcr-1 geni PCR kullanılarak incelendi. Mcr-1 pozitifliğini doğrulamak için DNA dizi analizi yöntemi kullanıldı. PCR ile mcr-1 pozitif olarak saptadığımız izolatlarda ve kolistine dirençli izolatlarda diğer antimikrobiyal direnç genleri araştırıldı.
Bulgular: Tamamı kolistine duyarlı olan 4 (karbapeneme dirençli 103 izolatın %3,9’u ve 75 A. baumannii izolatının %5.3’ü) A. baumannii izolatı PCR ile mcr-1 pozitif olarak belirlenirken, biri A. baumannii ve üçü K. pneumoniae olmak üzere kolistine dirençli dört izolatta mcr-1 saptanmadı. DNA dizileme analizi amplifikasyon ürünlerinden hiçbirinin hedeflenen parça olmadığını belirledi, ancak bunlar A. baumannii suşlarının kromozomal DNA parçalarıyla %70'ten fazla eşleşti. Bu nedenle bu sonuçlar yanlış pozitif kabul edildi. Bu yanlış pozitif izolatlar kolistine duyarlı olmalarına rağmen genişlemiş ilaç dirençliydi (XDR). Bunlardan ikisinin blaOXA23 benzeri ve blaTEM genlerini, bir diğerinin blaOXA23 benzeri, blaTEM ve blaOXA48 benzeri genleri, dördüncüsünün ise blaOXA23 benzeri ve blaCTXM genlerini taşıdığı belirlendi.
Sonuç: PCR ile mcr-1 genini saptamak için kullanılan primerlerin özgüllüğü çoğu çalışmada %100 olarak bildirilse de PCR testlerinin hızlı rutin tanıda tek başına veya antibiyotik duyarlılık testleri ile birlikte kullanılmasında henüz yetersiz olduğu sonucuna vardık. En azından PCR pozitif örneklerin DNA dizi analizi kullanılarak doğrulanması belli bir süre için uygun olacaktır.

References

  • Prim N, Turbau M, Rivera A, Navarro JR, Coll P, Mirelis B. Prevalence of colistin resistance in clinical isolates of Enterobacteriaceae: A four-year cross-sectional study. J Infect. 2017;75:493–8.
  • Baron S, Hadjadj L, Rolain JM, Olaitan AO. Molecular mechanisms of polymyxin resistance: knowns and unknowns. Int J Antimicrob Agents. 2016;48:583–91.
  • Bakthavatchalam YD, Pragasam AK, Biswas I, Veeraraghavan B. Polymyxin susceptibility testing, interpretative breakpoints and resistance mechanisms: An update. J Glob Antimicrob Resist. 2018;12:124–36.
  • Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16:161–8.
  • MacNair CR, Stokes JM, Carfrae LA, Fiebig-Comyn AA, Coombes BK, Mulvey MR et al. Overcoming mcr-1 mediated colistin resistance with colistin in combination with other antibiotics. Nat. Commun. 2018;9:458.
  • Caldes C, de Waard JH, Salgado MS, Villacís MJ, Coral-Almeida M, Yamamoto Y et al. Worldwide prevalence of mcr-mediated colistin-resistance escherichia coli in isolates of clinical samples, healthy humans, and livestock—a systematic review and meta-analysis. Pathogens. 2022;11:659.
  • Hussein NH, AL-Kadmy IMS, Taha BM, Hussein JD. Mobilized colistin resistance (mcr) genes from 1 to 10: a comprehensive review. Mol Biol Rep. 2021;48:2897–07.
  • Xiaomin S, Yiming L, Yuying Y, Zhangqi S, Yongning W, Shaolin W. Global impact of mcr-1-positive Enterobacteriaceae bacteria on “one health”. Crit Rev Microbiol. 2020;46:565-77.
  • Pathak A, Singh S, Kumar A, Prasad KN. Emergence of chromosome borne colistin resistance gene, mcr-1 in clinical isolates of Pseudomonas aeruginosa. Int J Infect Dis. 2020;101:22.
  • Chen H, Mai H, Lopes B, Wen F, Patil S. Novel Pseudomonas aeruginosa Strains Co-Harbouring blaNDM-1 Metallo β-Lactamase and mcr-1 Isolated from Immunocompromised Paediatric Patients. Infect Drug Resist. 2022;15:2929-36.
  • Hameed F, Khan MA, Muhammad H, Sarwar T, Bilal H, Rehman TU. Plasmid-mediated mcr-1 gene in Acinetobacter baumannii and Pseudomonas aeruginosa: first report from Pakistan. Rev Soc Bras Med Trop. 2019;5:52.
  • Özkaya E, Buruk CK, Tosun İ, Toraman B, Kaklıkkaya N, Aydın F. Investigation of plasmid mediated mcr colistin resistance gene in clinical Enterobacterales isolates Mikrobiyol Bul. 2020;54:191-202.
  • Li A, Yang Y, Miao M, Chavda KD, Mediavilla JR, Xie X et al. Complete sequences of mcr-1-harboring plasmids from extended-spectrum-lactamase- and carbapenemase-producing Enterobacteriaceae. Antimicrob Agents Chemother. 2016;60:4351–4.
  • Xie J, Liang B, Xu X, Yang L, Li H, Li P et al. Identification of mcr-1-positive multidrug-resistant Escherichia coli isolates from clinical samples in Shanghai, China. J Glob Antimicrob Resist. 2022;29:88-96.
  • Karakonstantis S, Kritsotakis EI, Gikas A. Pandrug-resistant Gram-negative bacteria: a systematic review of current epidemiology, prognosis and treatment options. J Antimicrob Chemother. 2020;75:271-82.
  • Voets GM, Fluit AC, Scharringa J, Cohen Stuart J, Leverstein-van Hall MA. A set of multiplex PCRs for genotypic detection of extended-spectrum β-lactamases, carbapenemases, plasmid-mediated AmpC β-lactamases and OXA β-lactamases. Int J Antimicrob Agents. 2011;37:356-9.
  • Salman M, Aamir Ali A, Haque A. A novel multiplex PCR for detection of Pseudomonas aeruginosa: A major cause of wound infections. Pak J Med Sci. 2013;29:957-61.
  • Liu Y, Liu C, Zheng W, Zhang X, Yu J, Gao Q et al. PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer. Int J Food Microbiol. 2008;125:230-5.
  • van der Zwaluw K, de Haan A, Pluister GN, Bootsma HJ, de Neeling AJ, Schouls LM. The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods. PLoS One. 2015;10:e0123690.
  • The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 11.0, 2021. http://www.eucast.org. (cited 2023 January 10).
  • Guven Gokmen T, Nagiyev T, Meral M, Onlen C, Heydari F, Koksal F. NDM-1 and rmtC-Producing Klebsiella pneumoniae Isolates in Turkey. Jundishapur J Microbiol. 2016;9:e33990.
  • Sanger F, Nicklen S, Coulson AR. DNA Sequencing by chain termination with dideoxynucleotides. Proc Natl Acad Sci USA. 1977;74:5463-7.
  • Fawaz AS. Methods for Sanger Sequencing in Diagnostic and Research. (serial online) 2018 (cited 2023 January 27). https://www.researchgate.net/publication/326672981.
  • Kurekci C, Aydin M, Nalbantoglu OU, Gundogdu A. First report of Escherichia coli carrying the mobile colistin resistance gene mcr-1 in Turkey. J Glob Antimicrob Resist. 2018;15:169-70.
  • Adiguzel MC, Baran A, Wu Z, Cengiz S, Dai L, Oz C, Oz et all. Prevalence of Colistin resistance in Escherichia coli in Eastern Turkey and Genomic Characterization of an mcr-1 Positive Strain from Retail Chicken Meat. Microb Drug Resist. 2021;27:424-32.
  • Arabacı Ç, Dal T, Başyiğit T, Genişel N, Durmaz R. Investigation of carbapenemase and mcr-1 genes in carbapenem-resistant Klebsiella pneumoniae isolates. J Infect Dev Ctries. 2019;13:504-09.
  • Hazırolan G, Karagöz A. Emergence of carbapenemase-producing and colistin resistant Klebsiella pneumoniae ST101 high-risk clone in Turkey Acta Microbiol Immunol Hung. 2020;67:216-21.
  • Özkaya E, Buruk CK, Tosun İ, Toraman B, Kaklıkkaya N, Aydın F. Investigation of Plasmid Mediated mcr Colistin Resistance Gene in Clinical Enterobacterales Isolates. Mikrobiyol Bul. 2020;54:191-02.
  • Tarhan G, Şahin F, Cesur S. Investigation of the presence of OXA 48 and mcr-1 genes in carbapenem-resistant Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa strains by in house-PCR method. J Med Palliat Care. 2021;2:118-23.
  • Mmatli M, Leshaba TMS, Skosana LB, Mbelle NM, Osei Sekyere J. Molecular screening of clinical multidrug-resistant gram-negative bacteria shows endemicity of carbapenemases, coexistence of multiple carbapenemases, and rarity of mcr in south africa. Microb Drug Resist. 2022;28:1028-36.
  • Al-Kadmy IMS, Ibrahim SA, Al-Saryi N, Aziz SN, Besinis A, Hetta HF. Prevalence of genes involved in colistin resistance in acinetobacter baumannii: first report from Iraq. Microb Drug Resist. 2020;26:616-22.
  • Rahman M, Ahmad S. First report for emergence of chromosomal borne colistin resistance gene mcr-1 in a clinical Acinetobacter baumannii Isolates from India. Open Forum Infect Dis. 2019;6:261–2.
  • Falgenhauer L, Waezsada SE, Gwozdzinski K, Ghosh H, Doijad S, Bunk B et al. Chromosomal locations of mcr-1 and bla CTX-M-15 in Fluoroquinolone-resistant Escherichia coli ST410. Emerg Infect Dis. 2016;22:1689-91.
  • Strepis N, Voor in ’t holt AF, Vos MC, Zandijk WHA, Heikema AP, Hays JP et al. Genetic analysis of mcr-1-carrying plasmids from gram-negative bacteria in a dutch tertiary care hospital: evidence for intrapatient and interspecies transmission events. Front Microbiol. 2021;12:727435.
  • Zhong YM, Liu WE, Zheng ZF. Epidemiology and molecular characterization of mcr-1 in Escherichia coli recovered from patients with bloodstream infections in Changsha, central China. Infect Drug Resist. 2019;12:2069–76.
  • McGann P, Snesrud E, Maybank R, Corey B, Ong AC, Clifford R et al. Escherichia coli Harboring mcr-1 and blaCTX-M on a Novel IncF Plasmid: First Report of mcr-1 in the United States. Antimicrob Agents Chemother. 2016;60:4420-21.
  • Xiang Q, Hu S, Ke Y, Hu S. Multiplex PCR for detection of MCR genes in clinical fecal samples. E3S Web Conf. 2021;269:01019

Misdiagnosis in molecular detection of colistin resistance: false mcr-1-PCR positivity among the colistin-susceptible Acinetobacter baumannii isolates

Year 2023, Volume: 48 Issue: 3, 1139 - 1147, 30.09.2023
https://doi.org/10.17826/cumj.1348548

Abstract

Purpose: The aim of this study was to investigate the presence of the mcr-1 gene, which is responsible for colistin resistance, in carbapenem-resistant Gram-negative bacteria that cause difficult-to-treat infections in a research hospital in Turkey.
Materials and Methods: The mcr-1 gene was examined using PCR in 103 carbapenem-resistant isolates, including 75 Acinetobacter baumannii, 19 Pseudomonas aeruginosa, and 9 Klebsiella pneumoniae. DNA sequencing was performed to confirm the mcr-1 positivity. Other antimicrobial resistance genes were investigated in isolates that were found to be mcr-1-positive by PCR and colistin-resistant isolates.
Results: Four (3.9% of the 103 carbapenem-resistant isolates and 5.3% of the 75 A. baumannii isolates) A. baumannii isolates, all susceptible to colistin, were found to be mcr-1-positive by PCR, whereas mcr-1 was not detected in four colistin-resistant isolates, one in A. baumannii and three in K. pneumoniae. DNA sequencing analysis determined that none of the amplification products was the targeted fragment, but they matched more than 70% with the chromosomal DNA fragments of A. baumannii strains. Therefore, these results were considered false-positive. Although these false-positive isolates were susceptible to colistin, they were extensively drug-resistant (XDR). Two of them were found to carry blaOXA23-like and blaTEM genes, another blaOXA23-like, blaTEM and blaOXA48-like genes, and the fourth one to have blaOXA23-like and blaCTXM genes.
Conclusion: Although the specificity of the primers used to detect the mcr-1 gene by PCR was reported as 100% in most studies, we concluded that PCR tests are insufficient yet to use alone or with antibiotic susceptibility tests in rapid routine diagnosis. Confirming at least PCR-positive samples using DNA sequence analysis would be appropriate for a certain period.

References

  • Prim N, Turbau M, Rivera A, Navarro JR, Coll P, Mirelis B. Prevalence of colistin resistance in clinical isolates of Enterobacteriaceae: A four-year cross-sectional study. J Infect. 2017;75:493–8.
  • Baron S, Hadjadj L, Rolain JM, Olaitan AO. Molecular mechanisms of polymyxin resistance: knowns and unknowns. Int J Antimicrob Agents. 2016;48:583–91.
  • Bakthavatchalam YD, Pragasam AK, Biswas I, Veeraraghavan B. Polymyxin susceptibility testing, interpretative breakpoints and resistance mechanisms: An update. J Glob Antimicrob Resist. 2018;12:124–36.
  • Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16:161–8.
  • MacNair CR, Stokes JM, Carfrae LA, Fiebig-Comyn AA, Coombes BK, Mulvey MR et al. Overcoming mcr-1 mediated colistin resistance with colistin in combination with other antibiotics. Nat. Commun. 2018;9:458.
  • Caldes C, de Waard JH, Salgado MS, Villacís MJ, Coral-Almeida M, Yamamoto Y et al. Worldwide prevalence of mcr-mediated colistin-resistance escherichia coli in isolates of clinical samples, healthy humans, and livestock—a systematic review and meta-analysis. Pathogens. 2022;11:659.
  • Hussein NH, AL-Kadmy IMS, Taha BM, Hussein JD. Mobilized colistin resistance (mcr) genes from 1 to 10: a comprehensive review. Mol Biol Rep. 2021;48:2897–07.
  • Xiaomin S, Yiming L, Yuying Y, Zhangqi S, Yongning W, Shaolin W. Global impact of mcr-1-positive Enterobacteriaceae bacteria on “one health”. Crit Rev Microbiol. 2020;46:565-77.
  • Pathak A, Singh S, Kumar A, Prasad KN. Emergence of chromosome borne colistin resistance gene, mcr-1 in clinical isolates of Pseudomonas aeruginosa. Int J Infect Dis. 2020;101:22.
  • Chen H, Mai H, Lopes B, Wen F, Patil S. Novel Pseudomonas aeruginosa Strains Co-Harbouring blaNDM-1 Metallo β-Lactamase and mcr-1 Isolated from Immunocompromised Paediatric Patients. Infect Drug Resist. 2022;15:2929-36.
  • Hameed F, Khan MA, Muhammad H, Sarwar T, Bilal H, Rehman TU. Plasmid-mediated mcr-1 gene in Acinetobacter baumannii and Pseudomonas aeruginosa: first report from Pakistan. Rev Soc Bras Med Trop. 2019;5:52.
  • Özkaya E, Buruk CK, Tosun İ, Toraman B, Kaklıkkaya N, Aydın F. Investigation of plasmid mediated mcr colistin resistance gene in clinical Enterobacterales isolates Mikrobiyol Bul. 2020;54:191-202.
  • Li A, Yang Y, Miao M, Chavda KD, Mediavilla JR, Xie X et al. Complete sequences of mcr-1-harboring plasmids from extended-spectrum-lactamase- and carbapenemase-producing Enterobacteriaceae. Antimicrob Agents Chemother. 2016;60:4351–4.
  • Xie J, Liang B, Xu X, Yang L, Li H, Li P et al. Identification of mcr-1-positive multidrug-resistant Escherichia coli isolates from clinical samples in Shanghai, China. J Glob Antimicrob Resist. 2022;29:88-96.
  • Karakonstantis S, Kritsotakis EI, Gikas A. Pandrug-resistant Gram-negative bacteria: a systematic review of current epidemiology, prognosis and treatment options. J Antimicrob Chemother. 2020;75:271-82.
  • Voets GM, Fluit AC, Scharringa J, Cohen Stuart J, Leverstein-van Hall MA. A set of multiplex PCRs for genotypic detection of extended-spectrum β-lactamases, carbapenemases, plasmid-mediated AmpC β-lactamases and OXA β-lactamases. Int J Antimicrob Agents. 2011;37:356-9.
  • Salman M, Aamir Ali A, Haque A. A novel multiplex PCR for detection of Pseudomonas aeruginosa: A major cause of wound infections. Pak J Med Sci. 2013;29:957-61.
  • Liu Y, Liu C, Zheng W, Zhang X, Yu J, Gao Q et al. PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer. Int J Food Microbiol. 2008;125:230-5.
  • van der Zwaluw K, de Haan A, Pluister GN, Bootsma HJ, de Neeling AJ, Schouls LM. The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods. PLoS One. 2015;10:e0123690.
  • The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 11.0, 2021. http://www.eucast.org. (cited 2023 January 10).
  • Guven Gokmen T, Nagiyev T, Meral M, Onlen C, Heydari F, Koksal F. NDM-1 and rmtC-Producing Klebsiella pneumoniae Isolates in Turkey. Jundishapur J Microbiol. 2016;9:e33990.
  • Sanger F, Nicklen S, Coulson AR. DNA Sequencing by chain termination with dideoxynucleotides. Proc Natl Acad Sci USA. 1977;74:5463-7.
  • Fawaz AS. Methods for Sanger Sequencing in Diagnostic and Research. (serial online) 2018 (cited 2023 January 27). https://www.researchgate.net/publication/326672981.
  • Kurekci C, Aydin M, Nalbantoglu OU, Gundogdu A. First report of Escherichia coli carrying the mobile colistin resistance gene mcr-1 in Turkey. J Glob Antimicrob Resist. 2018;15:169-70.
  • Adiguzel MC, Baran A, Wu Z, Cengiz S, Dai L, Oz C, Oz et all. Prevalence of Colistin resistance in Escherichia coli in Eastern Turkey and Genomic Characterization of an mcr-1 Positive Strain from Retail Chicken Meat. Microb Drug Resist. 2021;27:424-32.
  • Arabacı Ç, Dal T, Başyiğit T, Genişel N, Durmaz R. Investigation of carbapenemase and mcr-1 genes in carbapenem-resistant Klebsiella pneumoniae isolates. J Infect Dev Ctries. 2019;13:504-09.
  • Hazırolan G, Karagöz A. Emergence of carbapenemase-producing and colistin resistant Klebsiella pneumoniae ST101 high-risk clone in Turkey Acta Microbiol Immunol Hung. 2020;67:216-21.
  • Özkaya E, Buruk CK, Tosun İ, Toraman B, Kaklıkkaya N, Aydın F. Investigation of Plasmid Mediated mcr Colistin Resistance Gene in Clinical Enterobacterales Isolates. Mikrobiyol Bul. 2020;54:191-02.
  • Tarhan G, Şahin F, Cesur S. Investigation of the presence of OXA 48 and mcr-1 genes in carbapenem-resistant Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa strains by in house-PCR method. J Med Palliat Care. 2021;2:118-23.
  • Mmatli M, Leshaba TMS, Skosana LB, Mbelle NM, Osei Sekyere J. Molecular screening of clinical multidrug-resistant gram-negative bacteria shows endemicity of carbapenemases, coexistence of multiple carbapenemases, and rarity of mcr in south africa. Microb Drug Resist. 2022;28:1028-36.
  • Al-Kadmy IMS, Ibrahim SA, Al-Saryi N, Aziz SN, Besinis A, Hetta HF. Prevalence of genes involved in colistin resistance in acinetobacter baumannii: first report from Iraq. Microb Drug Resist. 2020;26:616-22.
  • Rahman M, Ahmad S. First report for emergence of chromosomal borne colistin resistance gene mcr-1 in a clinical Acinetobacter baumannii Isolates from India. Open Forum Infect Dis. 2019;6:261–2.
  • Falgenhauer L, Waezsada SE, Gwozdzinski K, Ghosh H, Doijad S, Bunk B et al. Chromosomal locations of mcr-1 and bla CTX-M-15 in Fluoroquinolone-resistant Escherichia coli ST410. Emerg Infect Dis. 2016;22:1689-91.
  • Strepis N, Voor in ’t holt AF, Vos MC, Zandijk WHA, Heikema AP, Hays JP et al. Genetic analysis of mcr-1-carrying plasmids from gram-negative bacteria in a dutch tertiary care hospital: evidence for intrapatient and interspecies transmission events. Front Microbiol. 2021;12:727435.
  • Zhong YM, Liu WE, Zheng ZF. Epidemiology and molecular characterization of mcr-1 in Escherichia coli recovered from patients with bloodstream infections in Changsha, central China. Infect Drug Resist. 2019;12:2069–76.
  • McGann P, Snesrud E, Maybank R, Corey B, Ong AC, Clifford R et al. Escherichia coli Harboring mcr-1 and blaCTX-M on a Novel IncF Plasmid: First Report of mcr-1 in the United States. Antimicrob Agents Chemother. 2016;60:4420-21.
  • Xiang Q, Hu S, Ke Y, Hu S. Multiplex PCR for detection of MCR genes in clinical fecal samples. E3S Web Conf. 2021;269:01019
There are 37 citations in total.

Details

Primary Language English
Subjects Medical Bacteriology
Journal Section Research
Authors

Toğrul Nağıyev 0000-0002-5719-370X

Tülay Kandemir 0000-0001-9002-699X

Fatih Köksal 0000-0003-0790-1525

Early Pub Date September 26, 2023
Publication Date September 30, 2023
Acceptance Date September 21, 2023
Published in Issue Year 2023 Volume: 48 Issue: 3

Cite

MLA Nağıyev, Toğrul et al. “Misdiagnosis in Molecular Detection of Colistin Resistance: False Mcr-1-PCR Positivity Among the Colistin-Susceptible Acinetobacter Baumannii Isolates”. Cukurova Medical Journal, vol. 48, no. 3, 2023, pp. 1139-47, doi:10.17826/cumj.1348548.