2020 / 03

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DOI: 10.24075/brsmu.2020.032

ORIGINAL RESEARCH

Colistin resistance of carbapenem-resistant Klebsiella pneumoniae strains: molecular mechanisms and bacterial fitness

Shamina OV1, Kryzhanovskaya OA1, Lazareva AV1, Alyabieva NM1, Mayanskiy NA2
About authors

1 National Medical Research Center for Children's Health, Moscow, Russia

2 Pirogov Russian National Research Medical University, Moscow, Russia

Correspondence should be addressed: Olga V. Shamina
Lomonosovskiy prospect, 2, str. 1, Moscow, 119991; ur.xobni@animahs.aglo

About paper

Funding: the study was supported by the Russian Science Foundation (Project ID 20-15-00235).

Acknowledgements: the authors thank Polikarpova SV of Filatov City Clinical Hospital № 15 and Karaseva OV of the Research Institute of Emergency Pediatric Surgery and Traumatology for K. pneumoniae isolates.

Author contributionThe increasing use of colistin in the clinic has led to the emergence and spread of colistin resistance. According to the literature, antibiotic resistance can have a metabolic cost, resulting in poor adaptation and survival, i.e. reduced bacterial fitness. The aim of this study was to investigate molecular mechanisms underlying resistance to colistin and their effect on the bacterial fitness of carbapenem-resistant (carba-R) strains of K. pneumoniae isolated from the patients of Moscow hospitals in 2012–2017. Of 159 analyzed carba-R isolates, 71 (45%) were resistant to colistin (minimum inhibitory concentration over 2 mg/L). By conducting Sanger sequencing, we were able to identify the mechanisms underlying colistin resistance in 26 (37%) isolates. Growth curves were constructed by measuring optical density at 600 nm wavelength for 15 hours. The competitive growth of colistin-resistant (col-R) K. pneumoniae isolates was assessed relative to the colistinsusceptible (col-S) isolate. Col-R and col-S cultures harvested in the exponential phase were combined at the ratio of 1:1, incubated in the Luria-Bertani medium and plated onto Luria-Bertani agar plates with 10 mg/L colistin and without it. The competition index was calculated as the ratio of grown col-R and col-S colonies. Resistance to colistin did not affect the growth kinetics of K. pneumoniae, but did reduce the competitive ability of the bacteria as compared to the col-S isolates. However, some col-R isolates were more competitive than the col-S strains of the same sequence type. Further research is needed to elucidate the effects of colistin resistance on bacterial fitness.: Shamina OV planned and conducted the study, analyzed the literature, analyzed and interpreted the obtained data, and wrote the manuscript; Kryzhanovskaya OA, Lazareva AV, Alyabieva NM planned and conducted the study; Mayanskiy NA planned, conducted and supervised the study, analyzed the literature, collected, analyzed and interpreted the obtained data, and wrote the manuscript.

Received: 2020-05-18 Accepted: 2020-06-02 Published online: 2020-06-12
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Fig. 1. Typical growth curves for K. pneumoniae isolates in the colistin-free culture medium. The shaded region on the graph represents an area under the growth curve (AUGC). The growth curve for col-S isolates is shown in red; the growth curve for col-R isolates is shown in green
Fig. 2. Evaluation of the competitive ability of K. pneumoniae (a representative experiment). Col-S — colistin-susceptible isolates; col-R — colistin-resistant isolates; col-S /col-R — a mixture of susceptible and resistant strains; CFU — colony forming units. The photos of Petri dishes demonstrate the growth pattern for the col-S isolates (A, D), col-R isolates (B, E) and the mixture of col-R/col-S isolates (C, F) of K. pneumoniae on LB agar plates without colistin (A–C) and supplemented with 10 mg/L colistin (D–F). Numbers indicate the CFU count on each plate. The competition index (CI) is calculated as (the number of CFU on LB + colistin) divided by (the number of CFU on LB minus the number of CFU on LB + colistin), i.e. CFU F : (CFU C-CFU F)
Table 1. Genotypes, phenotypes and mechanisms underlying colistin resistance in carba-R K. pneumoniae isolates (n = 26)
Note: ST — sequence type; MIC — minimum inhibitory concentration; a — the position of the insertion sequence is specified in brackets; b — PmrB alteration (T157P); c — PmrA (A141T) and PmrB (L213M, G256R) alterations; d — PmrB alteration (deletion at 27–30 (QLIS)).
Table 2. Effects of colistin resistance on the bacterial fitness (growth kinetics and competition index) of carba-R K. pneumoniae isolates
Note: MIC — minimum inhibitory concentration; AUGC — area under growth curve; Ме — median; Р25 and Р75 — the 25th and 75th percentiles; CI — competition index; SD — standard deviation; col-S — colistin-susceptible isolates; col-R — colistin-resistant isolates; n/a — not applicable; a — p = 0.842 for comparison with col-S AUGC; b — p = 0.19 for comparison with col-R AUGC at 0 mg/L colistin; c — p = 0.016 for comparison with col-R AUGC at 0 mg/L colistin; d — p < 0.001 for comparison with col-R AUGC at 0 mg/L colistin; e — р > 0.05 for comparison with col-R AUGC for isolates with disrupted mgrB; f — n = 26; g — n = 11; h — n = 15; i — p = 0.283 for comparison with CI of the isolates with disrupted mgrB.
Table 3. The competition index of col-R and col-S isolates of carba-R K. pneumoniae representing the same sequence types
Note: CI — competition index; SD — standard deviation.