Abstract
Of 26 tigecycline-nonsusceptible Klebsiella pneumoniae (TNSKP) clinical isolates, 25 had nonsynonymous mutations in ramR and/or acrR (23 in ramR and 10 in acrR). Eight TNSKP isolates possessed overexpression of ramA, acrB, rarA, and oqxB simultaneously, while 8 and 1 TNSKP strains had upregulation of ramA and acrB and of rarA and oqxB, respectively. Thus, resistance mechanisms of 9 TNSKP isolates cannot be explained by the present pathways. This study underscores the role of RamA in TNSKP and suggests the presence of novel tigecycline resistance mechanisms.
TEXT
Tigecycline, the first member of glycylcyclines, can overcome the two main resistance mechanisms of tetracycline (ribosomal protection and activity of efflux pumps) due to its long side chain and high affinity to ribosome (1). However, it is intrinsically resistant to Pseudomonas aeruginosa due to efflux. Although tigecycline resistance is not yet common in Enterobacteriaceae (except in species with intrinsic resistance), it has been described in several species, including Escherichia coli (2), Klebsiella pneumoniae (3), Enterobacter spp. (4), and Salmonella enterica (5) because of AcrAB efflux pump overexpression.
The AcrAB efflux pump is regulated by its local transcriptional repressor, AcrR, and a global transcriptional activator, RamA, in tigecycline-nonsusceptible K. pneumoniae (TNSKP) isolates (6). High-level expression of acrAB can result from mutation in acrR and upregulation of ramA. The latter can be caused by a mutation in ramR, which encodes a local transcriptional repressor of ramA. Moreover, overexpression of RarA, functioning as a transcriptional activator of the efflux pump OqxAB, can confer low-level resistance to tigecycline in K. pneumoniae as well (7). In summary, the RamA and AcrAB pathway and RarA together with the AcrAB and OqxAB pathways have been implicated mainly in tigecycline resistance in K. pneumoniae.
To date, studies on tigecycline resistance mechanisms in K. pneumoniae are limited and involve only a small number of isolates. In this study, we investigated the tigecycline resistance mechanisms in 26 unique TNSKP clinical isolates, including 3 isolates that were highly resistant to tigecycline, with an MIC of 16 μg/ml.
Screening of TNSKP clinical isolates.
Tigecycline-nonsusceptible isolates were screened from 2,605 consecutive nonduplicate Enterobacteriaceae isolates collected at our hospital between January 2012 and January 2013. The MIC of tigecycline (Pfizer Inc.) was determined by the broth microdilution methodology as described previously (8). Tigecycline MICs were tested in triplicate for isolates with reduced susceptibility to tigecycline. The results were interpreted according to the U.S. Food and Drug Administration breakpoints for tigecycline (≤2.0 μg/ml, susceptible; 4.0 μg/ml, intermediate; ≥8.0 μg/ml, resistant) (9).
Of the 2,605 Enterobacteriaceae isolates, 141 (5.4%) had a tigecycline MIC of ≥4 μg/ml (Table 1). Twenty-six TNSKP isolates were obtained, a tigecycline nonsusceptibility rate of 2.3% (26/1,116) for K. pneumoniae (Table 1), which was similar to the rates observed in the Asia-Western Pacific region and Latin America (10, 11).
TABLE 1.
Tigecycline-nonsusceptible clinical isolates in Enterobacteriaceae
| Bacterium | Total no. of isolates | No. of nonsusceptible isolates with indicate tigecycline MIC (μg/ml) |
No. (%) of nonsusceptible isolates | ||
|---|---|---|---|---|---|
| 4 | 8 | 16 | |||
| Klebsiella spp. | 1,152 | 15 | 8 | 3 | 26 (2.3) |
| K. pneumoniae | 1,116 | 15 | 8 | 3 | 26 (2.3) |
| Others | 36 | 0 | 0 | 0 | 0 (0) |
| Escherichia coli | 832 | 1 | 1 | 0 | 2 (0.2) |
| Serratia spp. | 164 | 4 | 1 | 0 | 5 (3.0) |
| S. marcescens | 161 | 4 | 1 | 0 | 5 (3.1) |
| S. liquefaciens | 3 | 0 | 0 | 0 | 0 (0) |
| Enterobacter spp. | 171 | 2 | 3 | 0 | 5 (2.9) |
| E. cloacae | 93 | 2 | 2 | 0 | 4 (4.3) |
| E. aerogenes | 73 | 0 | 1 | 0 | 1 (1.4) |
| Others | 5 | 0 | 0 | 0 | 0 (0) |
| Citrobacter spp. | 72 | 3 | 1 | 0 | 4 (5.6) |
| C. koseri | 40 | 0 | 1 | 0 | 1 (2.5) |
| C. freundii | 23 | 2 | 0 | 0 | 2 (8.7) |
| C. amalonaticus | 2 | 1 | 0 | 0 | 1 (50.0) |
| Others | 5 | 0 | 0 | 0 | 0 (0) |
| Proteus spp. | 137 | 40 | 39 | 2 | 81 (59.1) |
| P. mirabilis | 121 | 39 | 37 | 2 | 78 (64.5) |
| P. vulgaris | 15 | 1 | 2 | 0 | 3 (20.0) |
| P. penneri | 1 | 0 | 0 | 0 | 0 (0) |
| Providencia spp. | 35 | 14 | 2 | 0 | 16 (45.7) |
| P. stuartii | 30 | 12 | 2 | 0 | 14 (46.7) |
| P. rettgeri | 4 | 2 | 0 | 0 | 2 (50.0) |
| P. alcalifaciens | 1 | 0 | 0 | 0 | 0 (0) |
| Morganella morganii | 42 | 1 | 1 | 0 | 2 (4.8) |
| Total | 2,605 | 80 | 56 | 5 | 141 (5.4) |
Identification of mutations in ramR and acrR.
The presence of mutations in the ramR and acrR genes was assessed by PCR. Primers for the full length of ramR (F-AGTCGTCAAGACGATTTTCAATTTT and R-AGTGTTTCCGGCGTCATTAG) were designed in this study, and published primers were used for acrR (6). PCR products were sequenced and analyzed.
Of the 26 TNSKP isolates, 23 (88.5%) had mutations in ramR. The remaining 3 isolates that had no ramR mutation had a tigecycline MIC of 4 μg/ml, and 2 of these contained a mutation in acrR. Only one isolate lacked any mutation in either ramR or acrR. The various types of mutation are summarized in Table 2; of these, only A19V in ramR was reported previously (6). Hentschke et al. identified several mutations in ramR that were associated with increased tigecycline MICs in K. pneumoniae (12), and a similar observation was also made in Salmonella enterica (13).
TABLE 2.
Mutations of negative regulatory genes ramR and acrR in the 26 TNSKP isolates
| Mutation typea | ramR mutations (n = 23) | acrR mutation(s) (n = 10) |
|---|---|---|
| Insertion | A transposase and an integrase insertion (n = 2) | A transposase insertion (n = 7) |
| Frameshift mutation | Deletion of a sequence of ACAAAGCGAT (n = 4), deletion of a sequence of CTCGACGTCGGCCAT, deletion of a sequence of CACAAAGCGAT, insertion of a sequence of GC, deletion of G | |
| Missense mutation | K5E, A16D, T43M, G96D, I88N, T162I, A19V, A19V+R3P, A19V+A183D | M109I, Y114F+V165I (n = 2) |
| Nonsense mutation | E53Stop, W89Stop, R108Stop | |
| Missense mutation + nonsense mutation | A19V+Q122Stop |
Of 26 TNSKP isolates, 8 harbored ramR and acrR mutations simultaneously, and only one had no mutation in these two genes.
Mutations in acrR were identified in 10 (38.5%) of the 26 TNSKP isolates (Table 2). The most common change resulting from the acrR mutation was a transposase insertion after V94 (n = 7), followed by substitutions Y114F and V165I (n = 2) and substitution M109I (n = 1). Among these mutations in acrR leading to amino acid substitutions, none has been reported previously.
Of 15 TNSKP isolates without mutations in acrR but with mutations in ramR, 7 (46.7%) had tigecycline MICs of ≥8 μg/ml. Of 8 TNSKP isolates with mutations in both ramR and acrR, 5 (62.5%) had tigecycline MICs of ≥8 μg/ml. Taken together, these results further support the main role of ramR mutation in tigecycline resistance in K. pneumoniae and also the potential role of acrR in augmenting the level of resistance conferred by ramR mutation.
qRT-PCR analysis.
Quantitative real-time PCR (qRT-PCR) was used to assess the transcriptional expression level of efflux pump genes (acrB and oqxB) and their regulatory genes (ramA and rarA) in TNSKP isolates. Previously described primers were used for acrB and an endogenous reference gene, rrsE (3, 12), and new primers were designed for ramA (F-ATTTCCGCTCAGGTGATT and R-GTTGCAGATGCCATTTCG), rarA (F-ATTGCCCTCGGCTTTGAC and R-AACAGAGCGGCTGATACTCC), and oqxB (F-TCATTGGCGGCGTGAAGA and R-CGGCGTGTTGGTGAACTGC) in this study. Total RNA was prepared as previously described (3), and qRT-PCR was performed using SYBR Premix Ex Taq (TaKaRa) on the model 7500 real-time PCR system (Applied Biosystems). Reactions were repeated in triplicate, and the fold changes in expression of these genes were calculated as previously described (3). A tigecycline-susceptible K. pneumoniae clinical isolate (TSKP1; MIC, 0.5 μg/ml) was used as a reference isolate for the gene expression analysis.
Of the 26 TNSKP isolates, 8 (TNSKP1 to -8) had uniformly high expression levels of the 4 genes, namely ramA, acrB, rarA, and oqxB (Fig. 1A). Eight (TNSKP9 to -16) TNSKP isolates had elevated expression levels of ramA and acrB but not of rarA and oqxB (Fig. 1B). One isolate (TNSKP17) had increased expression levels of rarA, oqxB, and acrB but a baseline expression level of ramA (0.5-fold) (Fig. 1B). These data indicate that tigecycline nonsusceptibility in these 17 isolates may have been caused by the upregulation of RamA and/or RarA through the AcrAB and/or OqxAB efflux pumps, respectively.
FIG 1.
Expression of ramA, acrB, rarA, and oqxB and relationship between gene upregulation and tigecycline MICs in 26 TNSKP isolates. (A) Overexpression of ramA (10.8- to 222.4-fold) and acrB (11.6- to 128.2-fold) and of rarA (5.2- to 968.8-fold) and oqxB (31.6- to 672.7-fold) in 8 TNSKP isolates. (B) Upregulation of ramA (8.6- to 368.4-fold) and acrB (5.4- to 111.4-fold) but not of rarA or oqxB in 8 isolates (TNSKP9 to -16). TNSKP17 had increased expression levels of rarA and oqxB, as well as acrB, but a baseline expression level of ramA (0.5-fold). (C) Of 9 TNSKP isolates, 5 had overexpression of ramA (3 of them also with upregulation of oqxB) but baseline expression levels of acrB and rarA, and 4 had baseline expression levels of the 4 genes. (D) Among TNSKP isolates with ramA and/or acrB overexpression, isolates with higher tigecycline MICs (8 or 16 μg/ml) possessed higher expression levels of ramA (10.8- to 229.7-fold) and acrB (10.9- to 272.7-fold) than did isolates with tigecycline MICs of 4 μg/ml (ramA, 8.6- to 222.4-fold; acrB, 5.4- to 128.2-fold). (E) Of TNSKP isolates with rarA and/or oqxB upregulation, expression levels of rarA (421.5- to 968.8-fold) and oqxB (10- to 581.9-fold) in isolates with higher MICs (8 or 16 μg/ml) were higher than those (rarA, 5.2- to 532.7-fold; oqxB, 13.2- to 382-fold) in isolates with lower tigecycline MICs (4 μg/ml).
Five (TNSKP18 to -22) of the 26 isolates had upregulation of ramA (3 of them also with upregulation of oqxB) but exhibited baseline expression of acrB and rarA (Fig. 1C). In addition, the remaining 4 TNSKP isolates (TNSKP23 to -26) exhibited baseline expression of these 4 efflux-related genes (Fig. 1C). Taken together, the reported regulatory pathways of tigecycline resistance were partially and completely absent in 5 and 4 TNSKP isolates, respectively, which indicated that tigecycline resistance mechanisms were not limited to the upregulation of RamA or RarA and that alternative regulatory pathways may exist.
Of TNSKP isolates with ramA and/or acrB overexpression, isolates with higher tigecycline MICs (8 or 16 μg/ml) had higher expression levels of ramA and acrB than did isolates with MICs of 4 μg/ml (Fig. 1D). Similarly, among TNSKP isolates with rarA and/or oqxB upregulation, expression of rarA and oqxB in isolates with tigecycline MICs of 8 or 16 μg/ml were higher than those in isolates with lower tigecycline MICs (4 μg/ml) (Fig. 1E). These results together suggested that expression levels of efflux genes (acrB and oqxB) as well as their regulator genes (ramA and rarA) were generally in agreement with the tigecycline MICs in the TNSKP isolates in this study.
Three isolates (TNSKP18, -19, and -21) had remarkable expression levels of ramA and oqxB but baseline expression levels of acrB and rarA (Fig. 1C), suggesting that RamA likely upregulated the OqxAB efflux pump directly. In addition, increased ramA expression has been associated with upregulation of rarA and oqxA in Enterobacter cloacae (4). Nonetheless, whether RamA has an activator effect on the OqxAB efflux pump is still uncertain. Therefore, further research is needed to confirm the relationship between RamA and OqxAB, which will help clarify the regulatory networks involved in tigecycline resistance in K. pneumoniae and other Enterobacteriaceae.
Exclusion of other resistance mechanisms.
Although tetX and its orthologous genes have been reported to confer tigecycline resistance in Enterobacteriaceae and Acinetobacter baumannii (14, 15), they were not found in TNSKP isolates in this study. Recently, a mutation in rpsJ, coding for ribosomal protein S10, was reported to mediate tigecycline resistance in K. pneumoniae (16); however, no mutation in rpsJ was detected in the 26 TNSKP isolates. In addition, tigecycline MICs were not significantly inhibited by the efflux pump inhibitor Phe-Arg-β-naphthylamide (PAβN) in any of the TNSKP isolates in this study.
In conclusion, this study underscores the key role RamA plays in TNSKP. However, the reported modulation of regulatory pathways was absent in 9 of the 26 TNSKP isolates, which suggests that novel mechanisms mediating tigecycline resistance exist. Therefore, further studies are needed to elucidate the tigecycline resistance mechanisms of TNSKP isolates.
ACKNOWLEDGMENTS
We thank Yohei Doi for his critical review of the manuscript.
This work was supported by grant no. 81102509, 81120108024, and 81273559 from the National Natural Science Foundation of China and grant no. 10ZR1405600 from the Shanghai Municipal Science and Technology Commission. This study was also supported by the Innovation Personnel Training Plan of Key Discipline, Shanghai Medical College, Fudan University.
Footnotes
Published ahead of print 2 September 2014
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