Molecular epidemiology and mechanisms of tigecycline resistance in carbapenem‐resistant Klebsiella pneumoniae isolates

Abstract Background The emergence and transmission of tigecycline‐ and carbapenem‐resistant Klebsiella pneumoniae (TCRKP) have become a major concern to public health globally. Here, we investigated the molecular epidemiology and mechanisms of tigecycline resistance in carbapenem‐resistant K pneumoniae (CRKP) isolates. Methods Forty‐five non‐duplicate CRKP isolates were collected from January 2017 to June 2019. We performed antimicrobial susceptibility tests, multilocus sequence typing (MLST), and pulsed‐field gel electrophoresis (PFGE). PCR and DNA sequencing were performed for the detection and mutation analysis of acrR, oqxR, ramR, rpsJ, tet(A), and tet(X) genes, which are related to tigecycline resistance. The expression levels of efflux pump genes acrB and oqxB and their regulator genes rarA, ramA, soxS, and marA were assessed by quantitative real‐time PCR. Results The resistance rate to tigecycline in CRKP isolates was 37.8% (17/45). K pneumoniae ST307 was a predominant clone type (70.6%, 12/17) among the TCRKP isolates. The expression levels of acrB (P < .001) and marA (P = .009) were significantly higher in the tigecycline‐resistant group than in the tigecycline‐intermediate and tigecycline‐susceptible groups. Increased expression of acrB was associated with marA expression (r = 0.59, P = .013). Conclusions We found that the activated MarA‐induced overexpression of AcrAB efflux pump plays an important role in the emergence of tigecycline resistance in CRKP isolates.

such as CRKP. [5][6][7][8][9] According to an international ESCMID cross-sectional survey, tigecycline monotherapy is the most commonly used treatment for patients with intra-abdominal infections and skin and soft-tissue infections caused by CRE. 10 However, CRKP isolates with tigecycline resistance have been reported. [11][12][13] According to Taiwan's national surveillance study, the resistance rate of tigecycline (minimum inhibitory concentration, MIC > 2 mg/L) in carbapenem non-susceptible K pneumoniae was reported to be 9%. 14 In South Korea, the resistance rate of tigecycline (MIC > 2 mg/L) among carbapenemase-producing K pneumoniae was found to be approximately 14.5%. 15 Tigecycline resistance in CRKP has thus become a serious problem that can eventually lead to treatment failure. [11][12][13] To date, there are several known mechanisms associated with resistance to tigecycline in K pneumoniae 11,[16][17][18][19][20][21][22][23][24][25] The most common mechanism is the overproduction of non-specific active resistance-nodulation-cell division (RND) efflux pumps such as AcrAB-TolC 11,19 and OqxAB. 17 Expression of the acrAB efflux pump genes is regulated by the global AraC-family transcriptional activators such as RamA, MarA, and SoxS and the local TetR-family transcriptional repressor AcrR. 16,19 The transcription of ramA, marA, and soxS is repressed by RamR, MarR, and SoxR, respectively. 13,26 RamA is also regulated by Lon protease. 27,28 As RamR directly represses the expression of ramA, loss-of-function mutations in ramR can cause the overexpression of ramA. 18,21 The expression of the oqxAB efflux pump genes is also regulated by the global activators RarA, MarA, and SoxS and the local repressor OqxR. 17 Mutations in the rpsJ gene, which encodes the ribosomal protein S10, are associated with reduced tigecycline susceptibility. 24 Moreover, mutations in tet(A), which encodes one of the major facilitator superfamily (MFS) efflux pumps, and tet(X), which encodes a tigecycline-modifying enzyme, are associated with decreased tigecycline susceptibility. 20,22,23,25 The aim of this study was to investigate the phenotypic characteristics, molecular epidemiology, and mechanisms of tigecycline resistance in CRKP isolates from a tertiary care hospital in South Korea.

| Bacterial strains
A total of 3461 non-duplicate K pneumoniae isolates were collected from Chungnam National University Hospital in South Korea from January 2017 to June 2019. The VITEK 2 ID-GNB cards (bioMérieux SA, Marcy l'Étoile, France) were used for the identification of isolates. We retrospectively reviewed the clinical data for each isolate.

| Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE)
MLST and PFGE were used to determine the genetic relatedness among the 45 CRKP isolates. PCR and sequencing for MLST were carried out for seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) for K pneumoniae and the sequences were compared in the MLST database, so that the allelic numbers and sequence types (STs) could be determined. 33 The allelic profiles and STs were assigned using an online database (https://pubml st.org/). For PFGE, bacterial DNA was cleaved with XbaI endonuclease (Roche, Penzberg, Germany), and the XbaI-digested genomic DNA was subjected to PFGE using a CHEF-DR ® III Variable Angle System (Bio-Rad, USA). 34 The PFGE patterns were compared using BioNumerics software (Applied Maths, Kortrijk, Belgium) with dice correlation for band matching at a 1.5% position tolerance and the unweighted pair group method with an arithmetic average (UPGMA). Clusters were defined as DNA patterns sharing >80% similarity.

| Quantitative real-time PCR (qRT-PCR)
The expression levels of the efflux pump genes acrB and oqxB and their regulator genes rarA, ramA, soxS, and marA were assessed

| Detection and mutation analysis of the acrR, oqxR, ramR, rpsJ, tet(A), and tet(X) genes and pI and pII promoter regions
We performed PCR to detect acrR, oqxR, ramR, rpsJ, tet(A), and tet(X) genes and pI (upstream of the romA controlling romA-ramA operon transcription) and pII (located in the open reading frame of romA) promoter regions, which are transcriptional start sites of the ramA. 11,35 The amplicons were sequenced. For mutation analysis, we compared each gene sequence with that of the wild-type reference strains, K pneumoniae MGH78578 (GenBank accession number CP000647) in case of acrR, oqxR, ramR, and rpsJ genes and pI and pII promoter regions and E coli plasmid RP1 (GenBank accession number X00006) for the tet(A) gene. Primers used for PCR are shown in the Table S2.

| Statistical analysis
All statistical analyses were performed using MedCalc statistical software 14.12.0 (MedCalc Software, Mariakerke, Belgium). Data are presented as the mean ± standard deviation (SD) unless otherwise stated. In groups with a non-normal distribution, we evaluated the intergroup comparisons using either a Mann-Whitney rank sum test or Kruskal-Wallis test followed by Dunn's multiple comparison test. To assess the correlations between the expression levels of each gene, linear regressions were calculated. A P value < .05 was considered statistically significant.

| Clinical characteristics of CRKP isolates
Of the 3416 non-duplicate K pneumoniae strains, CRKP accounted were isolated from patients who had previously been treated with tigecycline.

| Antibiotic resistance profile and distribution of resistance genes in CRKP isolates
All CRKP isolates were resistant to ertapenem, cefotaxime, and ciprofloxacin. These isolates were also co-resistant to ceftazi-   Figure 3). There was no significant difference in the expression levels of ramA, soxS, oqxB, and rarA genes among the three groups.

| Mutation analysis of acrR, oqxR, ramR, rpsJ, tet(A), and tet(X) genes and pI and pII promoter regions, and their relationship with gene expression levels
The relative expression levels of acrB, ramA, marA, soxS, rarA, and oqxB genes and mutation analysis of TCRKP isolates are presented in Table 1. The acrR, oqxR, ramR, and rpsJ genes and pI and pII promoter regions were detected in all 17 TCRKP isolates. The tet(A) gene was detected in 52.9% (9/17) of the TCRKP isolates. In contrast, the tet(X) gene was not detected in any of the TCRKP isolates.
Mutations in the efflux pump-encoding acrR and oqxR genes were  (Table S3). There were no mutations in the rpsJ gene.

| D ISCUSS I ON
In the present study, we analyzed phenotypic characteristics including tigecycline susceptibility, molecular epidemiology, and mechanisms of tigecycline resistance in CRKP isolates from a tertiary care hospital in South Korea. According to a multicenter study in the United States, the resistance rate of tigecycline in CRKP isolates was reported to be 18.0%. 36   Some studies have reported that treatment with tigecycline could lead to the development of tigecycline resistance. 11,37 However, in this study, all TCRKP isolates were collected from patients who had not been exposed to tigecycline treatment previously. These findings revealed that tigecycline resistance might occur even without exposure to this antibiotic. According to another study, exposure to other antibiotics that are effluxed by non-specific pumps, such as AcrAB, could indirectly contribute to reduced tigecycline susceptibility. 38 The MLST and PFGE analyses revealed that K pneumoniae ST307, which was divided into five clonal groups based on PFGE, was the predominant clone among the TCRKP isolates in this study.
Moreover, some of the K pneumonia ST307 isolates in this study harbored bla KPC-2 . K pneumoniae ST307 has been internationally reported as a high-risk pathogen associated with high resistance to fluoroquinolones, third generation cephalosporins, and carbapenem. 39,40 Moreover, K pneumoniae ST307 is one of the dominant clonal types, along with ST11, ST768, ST15, ST23, and ST48, among TA B L E 1 Mutation analysis of acrR, ramR, oqxR, and tet(A)genes and pI and pII promoter regions and the relative expression levels of acrB, ramA, marA, soxS, rarA, and oqxB in TCRKP isolates In this study, we confirmed that high-risk TCRKP isolates such as K pneumoniae ST307 had already emerged and are disseminating in this area. Therefore, we should thoroughly monitor these high-risk pathogens to prevent their transmission.
Regarding the mechanisms of tigecycline resistance, we found that tigecycline resistance in most of the CRKP isolates was associated with increased expression of the efflux pump-encoding acrB gene. The upregulation of acrB could be mediated by a local repressor AcrR and/or transcriptional activators such as MarA, RamA, and SoxS. 38,39 Among them, we found that increased acrB expression correlated with overexpression of the transcriptional activator marA in the TCRKP isolates. However, the overexpression of acrB was not detected in the three TCRKP isolates. These findings suggest that tigecycline resistance in these isolates might be due to an alternative pathway or efflux pumps other than AcrAB or OqxAB.
In previous studies, it has been reported that mutations in the ramR gene could contribute to ramA overexpression and subsequent acrAB upregulation. 13,18,21,22 However, the expression levels of ramA in TCRKP isolates harboring mutations in ramR were not significantly increased compared to those in the control strain with a wild-type ramR gene in the present study. Moreover, based on mutation analysis of the transcriptional start sites (pI and pII promoter regions) of the ramA gene, there were no specific mutations that could affect the expression level of ramA. This implied that ramA overexpression might not be required to upregulate acrB and to confer tigecycline resistance. Among mutations, a Q122stop mutant in RamR 13 and V130A mutant of OqxR 22 were reported to confer resistance to tigecycline in previous studies; however, in this study, these mutants were also observed in the tigecycline-susceptible CRKP isolate indicating that it has little impact on tigecycline resistance.
There is one limitation to this study. Our study suggested that the upregulation of acrB mediated by the transcriptional activator MarA plays an important role in the mechanisms of tigecycline resistance. However, the expression of marA could also be regulated by a transcriptional regulator such as MarR. Therefore, further studies on additional regulators such as MarR, SoxR, and Lon protease, which affect the expression of marA, soxS, and ramA genes, respectively, and subsequent acrAB expression will be needed to assess the possible role in tigecycline resistance. In conclusion, although the mechanisms of tigecycline resistance are complex and have not been fully understood, our study indicates that the main mechanisms of tigecycline resistance in the CRKP isolates can be attributed to transcriptional activator MarA-mediated overexpression of AcrAB efflux pump.