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. 2021 Dec;52(4):1981-1989.
doi: 10.1007/s42770-021-00600-5. Epub 2021 Aug 30.

Genotyping of paired KPC-producing Klebsiella pneumoniae isolates with and without divergent polymyxin B susceptibility profiles

Suely Carlos Ferreira Sampaio  1   2 , Rozane de Lima Bigelli Carvalho  3 , Marcelo Jenné Mimica  3 , Cely Barreto da Silva  4 , Lycia Mara Jenné Mimica  3 , Aline Valerio de Lima  5 , Keila de Oliveira Lima  5 , Darlan Augusto da Costa Rocha  5 , Jorge Luiz Mello Sampaio  5   6
Affiliations

Genotyping of paired KPC-producing Klebsiella pneumoniae isolates with and without divergent polymyxin B susceptibility profiles

Suely Carlos Ferreira Sampaio et al. Braz J Microbiol. 2021 Dec.

Abstract

Polymyxins are still used mainly in treating infections caused by carbapenem-resistant Klebsiella pneumoniae worldwide. The most frequent mechanism of acquired resistance to polymyxins in Gram-negative bacilli is the occurrence of mutations in chromosomal genes regulating operons responsible for lipopolysaccharide modification. As we observed at Santa Casa de São Paulo hospital the occurrence of infections caused by isolates resistant to polymyxins in patients previously treated with this antimicrobial, and new infections caused by the same polymyxin-susceptible species, in this study, we aimed to determine the clonality of consecutive K. pneumoniae isolates from the same patients and characterize the molecular determinants of polymyxin resistance in paired or clonal isolates. A total of 24 pairs and one trio of K. pneumoniae isolates were included in this study. Species identification was achieved by mass spectrometry and multiplex PCR. Polymyxin B minimal inhibitory concentrations were determined by broth microdilution. Clonality was evaluated using pulsed-field gel electrophoresis. The presence of insertions in mgrB gene was tested by PCR, and mutations on pmrA, pmrB, phoP, and phoQ were evaluated by PCR and complete nucleotide sequencing. A fraction of 23.8% of strains resistant to polymyxin B had an insertion in mgrB. Amino acid substitution F204L in PmrB may be implicated in polymyxin resistance. Substitutions T246A and R256G in PmrB were not implicated in polymyxin resistance. In this study, polymyxin resistance after a first susceptible isolate was detected was most frequently due to an infection caused by a distinct clone.

Keywords: Carbapenem-resistant; KPC; Klebsiella pneumoniae; Polymyxin.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Agarose gel electrophoresis of mgrB gene PCR products. Lanes 1 and 12, 1 Kb Plus DNA ladder (Invitrogen); lanes 5 (isolate 1b), 8 (isolate 3a) and 9 (isolate 3b), amplification products of the mgrB genes without insertions. Lanes 4 (isolate 1a), 6 (isolate 2a), 7 (isolate 2b), 10 (isolate 4b), and 11 (isolate 4d), amplification products of the mgrB genes with insertion
Fig. 2
Fig. 2
Dendrogram of PFGE profiles of KPC-producing K. pneumoniae. Strains with a Dice´s similarity index ≥ 80% were considered part of the same clonal group. N, intact mgrB gene; I, presence of insertion in the mgrB gene; BAL, bronchoalveolar lavage
See this image and copyright information in PMC

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