Both K. variicola isolates were susceptible to imipenem (MICs, 0.5 mg/L), ciprofloxacin (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 identified 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 significantly between the colistin-resistant and colistin-susceptible isolates, but phoQ expression was significantly higher in the colistin-resistant 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 significantly in the colistin-resistant isolate.
The colistin-susceptible isolate 475-3 showed two distinctive peaks at m/z 1824 and 1840, each representing two glucosamines, phosphates, myristates (C14) 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 (C14) with 2-hydroxymyristates (C14:OH) (Figure 2). These moieties have also been reported in K. pneumoniae (Leung et al., 2017). Contrastingly, the colistin-resistant isolate 464-1 showed strong intensities between m/z 1400 and 1900 with the two highest peaks at m/z 1643 and 1744, representing amino-arabinosylated lipid A with hexa-acylated species (Trent et al., 2001; Vorachek-Warren et al., 2002).
Colistin resistance in K. variicola has rarely been studied. Recently, colistin-resistant hypervirulent K. variicola isolates (MICs, 8 mg/L and 16 mg/L) have been reported in China and Chile, respectively (Lu et al., 2018; Morales-León et al., 2021). In these isolates, amino acid substitutions in PhoP or PmrB have been suggested to mediate colistin resistance. In a colistin-resistant mutant, mutations in phoP have been confirmed to be associated with colistin resistance in K. variicola in vitro (Janssen et al., 2020). This previous study identified lipid A modifications, such as the hydroxylation of an acyl chain, addition of 4-amino-4-deoxy-L-arabinose, and acylation with palmitate, in the colistin-resistant K. variicola mutant.
Here, we did not find 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 identified in the colistin-resistant K. variicola isolate, along with lipid A modifications. Thus, lipid A modification 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 modification 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 identified a colistin-resistant K. variicola isolate and compared it with a colistin-susceptible K. variicola isolate. We found that colistin resistance in K. variicola was mediated by the modification 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.