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

Toğrul Nağıyev , Tülay Kandemir , Fatih Köksal

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.

Keywords

DNA dizi analizi, Gram-negatif bakteri, karbapenem direnci, mcr-1 geni, PCR

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

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.

Keywords

DNA sequence analysis, Gram-negative bacteria, carbapenem resistance, mcr-1 gene, PCR

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