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Lipid A modication-induced colistin-resistant Klebsiella variicola from healthy adults

We identied two Klebsiella variicola isolates from fecal samples: one is colistin-resistant, and another is colistin-susceptible. The colistin-resistant K. variicola isolate showed no mutations in phoPQ, pmrAB, and mgrB, and crrAB and mcr were not identied. However, its phoQ and pbgP expression was signicantly higher and amino-arabinosylated lipid A with hexa-acylated species in lipopolysaccharide was identied.


Introduction
Colistin is a long-known drug, but is now considered one of the last resorts for treating bacterial infections caused by multidrug-resistant gram-negative pathogens. Colistin resistance in Klebsiella pneumoniae has emerged and is now a great concern in clinical settings as well as in public health (Gipson et al., 2020). However, colistin resistance in other species of the K. pneumoniae complex has not been actively explored. Klebsiella variicola, formerly known as a benign endosymbiont, has become an emerging pathogen that can cause life-threatening infections in immunocompromised individuals (Rodríguez-Medina et al., 2019). In this study, we investigated the resistance mechanism of a colistinresistant K. variicola isolate obtained from a healthy adult in South Korea.

Materials And Methods
We identi ed two K. variicola isolates in the fecal samples of healthy Korean adults, collected between June and September 2014 (Joo et al., 2018). Species identi cation was performed based on 16S rRNA and rpoB sequences, and the isolates were designated 464-1 and 475-3, respectively. Antimicrobial susceptibility testing was performed using the broth microdilution method according to the CLSI guidelines (2018). For colistin, we further performed a disk diffusion assay using Sensi-Disc™ (Becton, Dickinson and Company, Sparks, MD, USA) and the Epsilometer test (E-test) using ETEST® strip (bioMérieux, Marcy l'Etoile, France).
We investigated the structural differences in the lipid A of the lipopolysaccharide (LPS) of the colistinresistant and colistin-susceptible K. variicola isolates. Lipid A sample for a MALDI-TOF MS analysis was prepared as previously described (Yi and Hackett, 2000). MALDI-TOF analyses were performed on the MALDI TOF-TOF 5800 system (AB SCIEX, USA). The raw m/z values were processed and analyzed using Mass-Up (López-Fernández et al., 2015). The baseline value was corrected using TopHat transform, and the curve was standardized by total ion current and smoothed by Savitzky-Golay ltering.

Results And Discussion
Both K. variicola isolates were susceptible to imipenem (MICs, 0.5 mg/L), cipro oxacin (MICs, ≤0.06 mg/L), cefepime (MICs, ≤0.06 and 0.12 mg/L), gentamicin (MICs, 0.5 mg/L), and tetracycline (MICs, 1 mg/L), and resistant to rifampin (MICs, 16 and 32 mg/L). While 464-1 was resistant to colistin (MIC, 32 mg/L), 475-3 was susceptible to it (MIC, 1 mg/L). The results of the disk diffusion assay and E-test were consistent with those of the broth microdilution method. The colistin-resistant isolate 464-1 showed no zone of inhibition in the disk diffusion assay, and an MIC of 16 mg/L in the E-test. The isolate 475-3 showed a colistin MIC of 0.19.
No nucleotide variation was identi ed between colistin-resistant and colistin-susceptible isolates in phoPQ, pmrAB, and mgrB. crrAB was not detected in either K. variicola isolates. Additionally, mcr-1 was not detected in the isolates. The expression of pmrB did not differ signi cantly between the colistinresistant and colistin-susceptible isolates, but phoQ expression was signi cantly higher in the colistinresistant isolate 464-1 than in the colistin-susceptible isolate 475-3, despite there being no differences in the sequences of the two isolates ( Figure 1). pbgP expression also increased signi cantly in the colistinresistant isolate.
The colistin-susceptible isolate 475-3 showed two distinctive peaks at m/z 1824 and 1840, each representing two glucosamines, phosphates, myristates (C 14 ) with four R-3-hydroxy-myristoyl acyl chains, and a hexa-acylated lipid A consisting of two glucosamines, phosphates, four R-3-hydroxy-myristoyl acyl chains, and one myristate (C 14  Here, we did not nd any mutations in pmrAB, phoPQ, and mgrB, which are known to be associated with colistin resistance in K. pneumoniae, in the colistin-resistant K. variicola isolate. However, an upregulation of phoQ and pbgP expression was identi ed in the colistin-resistant K. variicola isolate, along with lipid A modi cations. Thus, lipid A modi cation due to the two-component regulatory system and an overexpression of the pbgP operon conferred colistin resistance in the K. variicola isolate. Moreover, it has been reported that reduced colistin susceptibility occurs due to lipid A modi cation without amino acid changes in the proteins encoded by pmrAB, phoPQ, mgrB, and crrAB in K. pneumoniae. 6 This may be attributed to adaptive resistance, which involves an increase in antibiotic resistance because of alterations in protein expression levels triggered by environmental conditions, such as nutrient conditions, stress, and sub-inhibitory antibiotic concentrations (Skida et al., 2011). Alternatively, genetic changes in other genes may lead to the upregulation of genes associated with colistin resistance.
In this study, we identi ed a colistin-resistant K. variicola isolate and compared it with a colistinsusceptible K. variicola isolate. We found that colistin resistance in K. variicola was mediated by the modi cation of lipid A, which was associated with an overexpression of the two-component regulatory system and the pbgP operon, as observed in the closest related pathogen, K. pneumoniae. Although the isolate was obtained from fecal samples of healthy adults, colistin-resistant K. variicola challenges public health as an opportunistic pathogen. Figure 1 Quanti cation of pmrB, phoQ, and pbgP expression in colistin-susceptible and colistin-resistant K. variicola isolates. The experiments were repeated with three independent cultures, and rpoB was used as the reference gene. The fold changes were calculated using the comparative threshold cycle (-ΔΔCT) method. Statistical signi cance was determined using Student's unpaired t-test, and statistical analysis was performed using Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, USA). S, colistin-susceptible; R, colistin-resistant; *P < 0.05.