RESEARCH ARTICLE
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The role of RND efflux pump and global regulators in tigecycline resistance in clinical Acinetobacter baumannii isolates Henan Li1, Xiaojuan Wang1, Yawei Zhang1, Chunjiang Zhao1, Hongbin Chen1, Sen Jiang1, Feifei Zhang1 & Hui Wang*,1
ABSTRACT Aim: To analyze the expression and regulation of resistance-nodulationdivision (RND) efflux systems in clinical tigecycline-nonsusceptible (TNS) Acinetobacter baumannii. Materials & methods: Comparisons of molecular and clinical characteristics were performed between 52 TNS and 53 tigecycline-susceptible isolates. Expression of RND efflux pumps and global regulators were analyzed by real-time RT-PCR. A complementation experiment was performed to evaluate the contribution of the adeRS mutations. Results: Mechanical ventilation and prior use of carbapenems were more common among patients with TNS strains. The relative expression of adeB and adeJ was increased significantly in TNS isolates. Complementarity to the adeR or adeS mutations decreased tigecycline susceptibility by ≤2-fold. Decreased expression of marR and soxR was detected in TNS isolates. Conclusion: A correlation between tigecycline MIC and expression level of adeB and adeJ was identified. The influence of adeRS mutation on adeB expression was limited. Global regulators marR and soxR may be involved in tigecycline resistance. Acinetobacter baumannii has emerged as a major cause of nosocomial infections, especially in intensive care units. Multidrug-resistant A. baumannii, resistant to carbapenems, has been increasingly reported worldwide, which raises serious concerns about the limited remaining antimicrobial treatment options [1] . Our previous study indicated that the percentage of imipenem- and meropenem-resistant A. baumannii had increased from 4.5 to 61.7% and 62.8%, respectively, during 2003–2010 in China. In 2011, according to the Chinese Antimicrobial Resistance Surveillance of Nosocomial infections, the susceptibility rates of A. baumanii to imipenem and meropenem were 27.7 and 25.9%, respectively [2] . Tigecycline is a novel 9-t-butyl-substituted minocycline derivative that overcomes several major tetracycline resistance mechanisms [3] . It demonstrates broad-spectrum antimicrobial effects against Gram-positive, Gram-negative, anaerobic and atypical pathogens [4,5] . However, tigecycline resistance has increasingly been reported [6,7] . Tigecycline nonsusceptibility in A. baumannii isolates has been associated with overexpression of resistance-nodulation-division (RND) efflux pumps, such as adeABC [8] , adeIJK [9] and adeFGH [10] . These efflux pumps display broad substrate specificity, including tigecycline. The first RND system adeABC efflux pump confers resistance to aminoglycosides, tetracyclines, fluoroquinolones and chloramphenicol, and reduced susceptibility to tigecycline [11,12] . The expression of the adeABC is tightly regulated by an upstream two-component system, which contains a sensor kinase adeS and a response regulator adeR [13] . Previous studies have indicated that mutations in adeRS, selected in vitro [14] or in vivo [15-17] , correlated with constitutive expression of the adeABC efflux system. AdeIJK was found to act synergistically with adeABC to extrude tigecycline [9] . Overexpression of adeFGH confers high-level resistance to most antibiotics except β-lactams and aminoglycosides [10] .
KEYWORDS
• Acinetobacter baumannii • adeB • adeRS • marR • soxR • tigecycline
Department of Clinical Laboratory, Peking University People’s Hospital, Beijing 100044, People’s Republic of China *Author for correspondence: Tel.: +86 108 832 6300; [email protected] 1
10.2217/FMB.15.7 © 2015 Future Medicine Ltd
Future Microbiol. (2015) 10(3), 337–346
part of
ISSN 1746-0913
337
Research Article Li, Wang, Zhang et al. In this study, we focused on the role of RND efflux system and global regulators in tigecycline resistance in A. baumannii. Comparisons of molecular and clinical characteristics were performed between tigecycline-nonsusceptible (TNS) and tigecycline-susceptible (TS) A. baumannii. Expression of RND efflux pumps and global regulators was analyzed, and one adeB inactivation and complementary mutants were constructed. We also analyzed the contribution of mutations in adeRS to the expression of adeB. Materials & methods ●●Bacterial strains
A total of 105 clinical isolates of A. baumannii were collected from Chinese Antimicrobial Resistance Surveillance of Nosocomial infections in 2011, including 52 TNS isolates (MIC ≥4 mg/l) and 53 TS isolates (MIC ≤2 mg/l) according to the US FDA criteria. The TS isolates were selected with different tigecycline MICs (≤0.125–2 mg/l), and the source of the strains used was consistent with the TNS isolates. A. baumannii were primarily identified using the VITEK2 system (bioMérieux, Marcy L’toile, France) and confirmed by the presence of blaOXA51-like. A case patient was defined as a patient with A. baumannii isolated from clinical specimens during the study period. The study was approved by the Institutional Review Board of Peking University People’s Hospital. ●●Antimicrobial susceptibility testing
Tigecycline MIC was determined by the broth microdilution method [18] . Susceptibility testing of other antimicrobials was performed by the agar dilution method. Fresh bacterial cultures were prepared on the day of testing. Tigecycline powder (Pfizer, Inc., NY, USA) was prepared in a solution with sterile water. The tigecycline breakpoint was interpreted according to the FDA criteria (susceptible: ≤2 mg/l; resistant: ≥8 mg/l). Other antimicrobial breakpoints were interpreted according to the Clinical and Laboratory Standards Institute criteria M100-S23. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality-control strains. ●●PCR & nucleotide sequencing
PCR amplification was performed using a 7300 thermocycler (Applied Biosystems, CA, USA). The PCRs for adeR and adeS were undertaken as described previously [17] . Sequencing of the products was performed by an ABI 3730 DNA analyzer (Applied Biosystems, CA, USA) and
338
Future Microbiol. (2015) 10(3)
analyzed using CLC sequence viewer software (CLC bio, Aarhus, Denmark). ●●Gene expression analysis using real-time
reverse transcription PCR
Primers for adeB, adeJ, adeG and a housekeeping gene rpoB, were used as described previously [10,19] . Primers were designed in this study for marR (F-ACCGTCGAGTATTCA A AACTTCCCC and R-CCAGCTCAACACCTT TGGCCGT 5′-3′) and soxR (F-TCTA ATGACAAACAACCA CATCC and R-CGCCGTTTTACTCTA GTGGTG 5′-3′). Bacterial cells were grown aerobically in Luria-Bertani (LB) broth until mid-log phase. DNase-treated RNA templates were prepared using an RNeasy kit (QIAGEN Sciences, MD, USA). The concentrations of the RNA were quantified with a spectrophotometer and rpoB RNA was used as a housekeeping gene to normalize levels of adeB, adeJ, adeG, marR and soxR transcripts. cDNA was generated from total RNA using a random primer hexamer. The reverse transcription PCR (RT-PCR) was performed with a 7300 thermocycler (Applied Biosystems, CA, USA) with SYBR green PCR master mix (TaKaRa, Tokyo, Japan). The Taq activation step of 5 s at 95°C was followed by 40 cycles of 15 s at 95°C and 31 s at 58°C. Twenty TNS isolates, which belong to the predominant clone ST92, and 23 TS isolates with different tigecycline MICs (≤0.125–2 mg/l) were selected for real-time RT-PCR. Each sample was run in triplicate. ●●Construction of the complemented strains
The full-length adeR, adeS or adeB genes were amplified by PCR from tigecycline-resistant isolate AB566, both of which contained BamHI sites. The PCR products were ligated into BamHI digested pWH1266, which is an Escherichia coli-Acinetobacter shuttle plasmid [20] , using the In-Fusion HD Cloning kit (Clontech, Saint-Germain-en-Laye, France) and transformed in E. coli JM109 (TaKaRa, Tokyo, Japan). Plasmids were isolated from transformants and used to transform electrocompetent A. baumannii selected on Mueller–Hinton agar containing 100μg/ml ticarcillin. ●●Construction of adeB deletion mutant
The kanamycin resistance gene was ligated with the pUC18 vector. The resulting plasmid, pUC18Kan, was modified by cloning an internal fragment of adeB into the SmaI site of pUC18Kan
future science group
Tigecycline resistance in Acinetobacter baumannii as described previously [21] . The recombinant plasmids obtained were introduced into one tigecycline-resistant and kanamycin-susceptible isolate AB566 by electrotransformation.
Research Article
Ticarcillin- and kanamycin-resistant transformants were selected and the appropriate deletions were confirmed by PCR and genomic DNA sequencing.
Table 1. Demographic and clinical characteristics of patients infected with tigecycline-nonsusceptible and tigecycline-susceptible A. baumannii. Characteristic
Gender: – Male – Female Age (mean ± SD); years Location: – ICU – Medicine – Surgery – Emergency – Pediatrics Site of isolates: – Sputum – Blood – Ascites – Bile – Drainage – Bronchoalveolar lavage fluid Underlying diseases: – Pulmonary disease – Cancer – Hepatic disease – Heart disease – Nervous system disease – Kidney disease – Diabetes mellitus Risk factor: – Surgery within 1 month – Use of cortisol – Immunosuppressant –Mechanical ventilation Prior antibiotics use before the isolation: – Carbapenems – Cephalosporins – Quinolones – Aminoglycosides – Tetracyclines Outcome: – Discharge – Died – Lost to follow-up
Patients; n (%) With TNS A. baumannii With TS A. infection (n = 52) baumannii infection (n = 53)
p-value
33 (63.5) 19 (36.5) 56.37 ± 2.511 31 (59.6) 6 (11.5) 13 (25) 1 (1.9) 1 (1.9) 25 (48.1) 17 (32.7) 5 (9.6) 0 3 (5.8) 2 (3.8) 12 (23.1) 12 (23.1) 9 (17.3) 10 (19.2) 9 (17.3) 4 (7.7) 5 (9.6) 21 (40.4) 7 (13.5) 4 (7.7) 29 (55.8)
39 (73.6) 14 (26.4) 59.55 ± 2.548 22 (41.5) 14 (26.4) 14 (26.4) 1 (1.9) 2 (3.8) 26 (49.1) 19 (35.8) 3 (5.7) 4 (7.5) 0 1 (1.9) 17 (32.1) 11 (20.8) 4 (7.5) 17 (32.1) 6 (11.3) 2 (3.8) 3 (5.7) 15 (28.3) 5 (9.4) 4 (7.5) 19 (35.8)
0.2981 0.2981 0.3759 0.0799 0.0806 0.9999 0.9999 0.9999 0.9999 0.8377 0.4882 0.1179 0.1179 0.6178 0.3837 0.8169 0.1497 0.1805 0.4165 0.4370 0.4882 0.2213 0.5553 0.9999 0.0405*
20 (38.5) 10 (19.2) 3 (5.8) 2 (3.8) 0 41 (78.8) 10 (19.2) 1 (1.9)
4 (7.5) 15 (28.3) 3 (5.7) 0 0 36 (67.9) 6 (11.3) 1 (1.9)
0.0002* 0.3603 0.9999 0.2429 – 0.6069 0.2897 0.9999
*p < 0.05. SD: Standard deviation; TS: Tigecycline susceptible; TNS: Tigecycline nonsusceptible.
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Research Article Li, Wang, Zhang et al.
5 Relative expression of adeJ
Relative expression of adeB
A 5000 4500 2000 1500 1000 500 0 TNS 40 30 10 8 6 4 2 0 TNS
3 2 1 0
TS Relative expression of soxR
Relative expression of marR
B
4
TS
TNS
TS
TNS
TS
45 40 15 10 5 0
Figure 1. Relative expression of RND efflux pump and global regulators in Acinetobacter baumanii. Relative expression of (A) RND efflux pump (adeB and adeJ); and (B) global regulators (marR and soxR). TNS: Tigecycline nonsusceptible; TS: Tigecycline susceptible. ●●Statistical analysis
SPSS 17.0 (SPSS, Inc., IL, USA) was used for all statistical analyses. Comparative analyses were executed by χ2 or Fisher exact tests for categorical variables and by the Student t-test for continuous variables. All tests were two tailed, and p-values of 8-fold) and quinolones (5.3-fold), but did not influence susceptibility to ceftazidime, imipenem or trimethoprim. The complementation experiment for adeB restored resistance to tigecycline, aminoglycosides, quinolones, tetracycline and chloramphenicol in complementary mutant AB566△adeB/adeB when compared with the adeB deletion mutant AB566△adeB (Table 2) .
Research Article
Three TS isolates, AB459, AB563 and AB314, with different adeR or adeS sequences were transformed with the plasmids pWH-adeR or pWH-adeS, which carried adeR or adeS main mutations from TNS isolates. The contribution of adeR or adeS mutations to tigecycline resistance was determined in terms of the tigecycline MIC for the transformed isolates. Complementation of adeR or adeS mutations decreased the susceptibility to tigecycline slightly (≤2-fold), and also resulted in decreased susceptibility to ciprofloxacin (≤2-fold; Table 4). Quantitative analysis revealed that expression of adeB increased 1.2- to 3.6-fold in complementary mutants, when compared with the parental strains. ●●Analysis of expression of the RND efflux
●●Nucleotide sequencing of the regulatory
pump global regulators marR & soxR
genes adeR & adeS
MarR and soxR are global repressor proteins involved in bacterial antibiotic resistance. In our study, marR and soxR existed in all isolates by PCR. The relative expression of marR and soxR decreased in TNS isolates by real-time RT-PCR (Figure 1B) . Quantitative analysis revealed that expression of marR and soxR decreased 11.6- and 10.4-fold in TNS isolates, when compared with the TS isolates.
To further assess the mechanism of increased adeB expression, mutations in the two-component regulatory system of the AdeABC efflux pump, comprising adeR and adeS, were investigated. There were several mutations found in all TNS isolates, including Ile14Val, Val120Ile, Ala136Val in AdeR and Gly186Val, Asn213Asp, Asn268His, Ile285Leu in AdeS (p < 0.05; Table 3 ). We also identified another two AdeS mutations in three TNS isolates with tigecycline MIC of 8 mg/l, including Thr156Met in one strain and Ala130Thr in two strains. Meanwhile, AdeR and AdeS were absent in one TNS and eight TS isolates.
Discussion The increasing prevalence of multidrug-resistant A. baumannii has led to limited therapeutic options, and tigecycline is considered one of the few therapies available. In our study, more
Table 2. Antibiotic susceptibility of AdeB inactivated and complement strains. Antibiotics
MIC (mg/l) values of antibiotics
AB566
AB566ΔadeB†
AB566ΔadeB/adeB‡
Gentamicin Amikacin Ciprofloxacin Levofloxacin Tetracycline Chloramphenicol Trimethoprim Imipenem Ceftazidime Tigecycline
>256 12 >32 8 32 256 >32 >32 >256 6
4 1.5 6 1.5 2 8 >32 >32 >256 0.19
96 6 >32 12 64 256 >32 >32 >256 4
AdeB deletion mutant. Complementary to adeB in adeB deletion mutant.
† ‡
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341
Research Article Li, Wang, Zhang et al. Table 3. Amino acid mutations of adeR and adeS. Gene
Amino acid
Isolates (n)
p-value
Location
Coding protein
TNS (n = 52)
TS (n = 23)
adeR adeS
14 120 136 186 213 268 285
Ile Val Val Ile Ala Val Gly Val Asn Asp Asn His Ile Leu
0 51 0 50 0 50 0 49 0 49 0 49 0 49
5 10 6 9 7 8 8 7 4 11 6 9 4 11
0.0003
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The role of RND efflux pump and global regulators in tigecycline resistance in clinical Acinetobacter baumannii isolates Henan Li1, Xiaojuan Wang1, Yawei Zhang1, Chunjiang Zhao1, Hongbin Chen1, Sen Jiang1, Feifei Zhang1 & Hui Wang*,1
ABSTRACT Aim: To analyze the expression and regulation of resistance-nodulationdivision (RND) efflux systems in clinical tigecycline-nonsusceptible (TNS) Acinetobacter baumannii. Materials & methods: Comparisons of molecular and clinical characteristics were performed between 52 TNS and 53 tigecycline-susceptible isolates. Expression of RND efflux pumps and global regulators were analyzed by real-time RT-PCR. A complementation experiment was performed to evaluate the contribution of the adeRS mutations. Results: Mechanical ventilation and prior use of carbapenems were more common among patients with TNS strains. The relative expression of adeB and adeJ was increased significantly in TNS isolates. Complementarity to the adeR or adeS mutations decreased tigecycline susceptibility by ≤2-fold. Decreased expression of marR and soxR was detected in TNS isolates. Conclusion: A correlation between tigecycline MIC and expression level of adeB and adeJ was identified. The influence of adeRS mutation on adeB expression was limited. Global regulators marR and soxR may be involved in tigecycline resistance. Acinetobacter baumannii has emerged as a major cause of nosocomial infections, especially in intensive care units. Multidrug-resistant A. baumannii, resistant to carbapenems, has been increasingly reported worldwide, which raises serious concerns about the limited remaining antimicrobial treatment options [1] . Our previous study indicated that the percentage of imipenem- and meropenem-resistant A. baumannii had increased from 4.5 to 61.7% and 62.8%, respectively, during 2003–2010 in China. In 2011, according to the Chinese Antimicrobial Resistance Surveillance of Nosocomial infections, the susceptibility rates of A. baumanii to imipenem and meropenem were 27.7 and 25.9%, respectively [2] . Tigecycline is a novel 9-t-butyl-substituted minocycline derivative that overcomes several major tetracycline resistance mechanisms [3] . It demonstrates broad-spectrum antimicrobial effects against Gram-positive, Gram-negative, anaerobic and atypical pathogens [4,5] . However, tigecycline resistance has increasingly been reported [6,7] . Tigecycline nonsusceptibility in A. baumannii isolates has been associated with overexpression of resistance-nodulation-division (RND) efflux pumps, such as adeABC [8] , adeIJK [9] and adeFGH [10] . These efflux pumps display broad substrate specificity, including tigecycline. The first RND system adeABC efflux pump confers resistance to aminoglycosides, tetracyclines, fluoroquinolones and chloramphenicol, and reduced susceptibility to tigecycline [11,12] . The expression of the adeABC is tightly regulated by an upstream two-component system, which contains a sensor kinase adeS and a response regulator adeR [13] . Previous studies have indicated that mutations in adeRS, selected in vitro [14] or in vivo [15-17] , correlated with constitutive expression of the adeABC efflux system. AdeIJK was found to act synergistically with adeABC to extrude tigecycline [9] . Overexpression of adeFGH confers high-level resistance to most antibiotics except β-lactams and aminoglycosides [10] .
KEYWORDS
• Acinetobacter baumannii • adeB • adeRS • marR • soxR • tigecycline
Department of Clinical Laboratory, Peking University People’s Hospital, Beijing 100044, People’s Republic of China *Author for correspondence: Tel.: +86 108 832 6300; [email protected] 1
10.2217/FMB.15.7 © 2015 Future Medicine Ltd
Future Microbiol. (2015) 10(3), 337–346
part of
ISSN 1746-0913
337
Research Article Li, Wang, Zhang et al. In this study, we focused on the role of RND efflux system and global regulators in tigecycline resistance in A. baumannii. Comparisons of molecular and clinical characteristics were performed between tigecycline-nonsusceptible (TNS) and tigecycline-susceptible (TS) A. baumannii. Expression of RND efflux pumps and global regulators was analyzed, and one adeB inactivation and complementary mutants were constructed. We also analyzed the contribution of mutations in adeRS to the expression of adeB. Materials & methods ●●Bacterial strains
A total of 105 clinical isolates of A. baumannii were collected from Chinese Antimicrobial Resistance Surveillance of Nosocomial infections in 2011, including 52 TNS isolates (MIC ≥4 mg/l) and 53 TS isolates (MIC ≤2 mg/l) according to the US FDA criteria. The TS isolates were selected with different tigecycline MICs (≤0.125–2 mg/l), and the source of the strains used was consistent with the TNS isolates. A. baumannii were primarily identified using the VITEK2 system (bioMérieux, Marcy L’toile, France) and confirmed by the presence of blaOXA51-like. A case patient was defined as a patient with A. baumannii isolated from clinical specimens during the study period. The study was approved by the Institutional Review Board of Peking University People’s Hospital. ●●Antimicrobial susceptibility testing
Tigecycline MIC was determined by the broth microdilution method [18] . Susceptibility testing of other antimicrobials was performed by the agar dilution method. Fresh bacterial cultures were prepared on the day of testing. Tigecycline powder (Pfizer, Inc., NY, USA) was prepared in a solution with sterile water. The tigecycline breakpoint was interpreted according to the FDA criteria (susceptible: ≤2 mg/l; resistant: ≥8 mg/l). Other antimicrobial breakpoints were interpreted according to the Clinical and Laboratory Standards Institute criteria M100-S23. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality-control strains. ●●PCR & nucleotide sequencing
PCR amplification was performed using a 7300 thermocycler (Applied Biosystems, CA, USA). The PCRs for adeR and adeS were undertaken as described previously [17] . Sequencing of the products was performed by an ABI 3730 DNA analyzer (Applied Biosystems, CA, USA) and
338
Future Microbiol. (2015) 10(3)
analyzed using CLC sequence viewer software (CLC bio, Aarhus, Denmark). ●●Gene expression analysis using real-time
reverse transcription PCR
Primers for adeB, adeJ, adeG and a housekeeping gene rpoB, were used as described previously [10,19] . Primers were designed in this study for marR (F-ACCGTCGAGTATTCA A AACTTCCCC and R-CCAGCTCAACACCTT TGGCCGT 5′-3′) and soxR (F-TCTA ATGACAAACAACCA CATCC and R-CGCCGTTTTACTCTA GTGGTG 5′-3′). Bacterial cells were grown aerobically in Luria-Bertani (LB) broth until mid-log phase. DNase-treated RNA templates were prepared using an RNeasy kit (QIAGEN Sciences, MD, USA). The concentrations of the RNA were quantified with a spectrophotometer and rpoB RNA was used as a housekeeping gene to normalize levels of adeB, adeJ, adeG, marR and soxR transcripts. cDNA was generated from total RNA using a random primer hexamer. The reverse transcription PCR (RT-PCR) was performed with a 7300 thermocycler (Applied Biosystems, CA, USA) with SYBR green PCR master mix (TaKaRa, Tokyo, Japan). The Taq activation step of 5 s at 95°C was followed by 40 cycles of 15 s at 95°C and 31 s at 58°C. Twenty TNS isolates, which belong to the predominant clone ST92, and 23 TS isolates with different tigecycline MICs (≤0.125–2 mg/l) were selected for real-time RT-PCR. Each sample was run in triplicate. ●●Construction of the complemented strains
The full-length adeR, adeS or adeB genes were amplified by PCR from tigecycline-resistant isolate AB566, both of which contained BamHI sites. The PCR products were ligated into BamHI digested pWH1266, which is an Escherichia coli-Acinetobacter shuttle plasmid [20] , using the In-Fusion HD Cloning kit (Clontech, Saint-Germain-en-Laye, France) and transformed in E. coli JM109 (TaKaRa, Tokyo, Japan). Plasmids were isolated from transformants and used to transform electrocompetent A. baumannii selected on Mueller–Hinton agar containing 100μg/ml ticarcillin. ●●Construction of adeB deletion mutant
The kanamycin resistance gene was ligated with the pUC18 vector. The resulting plasmid, pUC18Kan, was modified by cloning an internal fragment of adeB into the SmaI site of pUC18Kan
future science group
Tigecycline resistance in Acinetobacter baumannii as described previously [21] . The recombinant plasmids obtained were introduced into one tigecycline-resistant and kanamycin-susceptible isolate AB566 by electrotransformation.
Research Article
Ticarcillin- and kanamycin-resistant transformants were selected and the appropriate deletions were confirmed by PCR and genomic DNA sequencing.
Table 1. Demographic and clinical characteristics of patients infected with tigecycline-nonsusceptible and tigecycline-susceptible A. baumannii. Characteristic
Gender: – Male – Female Age (mean ± SD); years Location: – ICU – Medicine – Surgery – Emergency – Pediatrics Site of isolates: – Sputum – Blood – Ascites – Bile – Drainage – Bronchoalveolar lavage fluid Underlying diseases: – Pulmonary disease – Cancer – Hepatic disease – Heart disease – Nervous system disease – Kidney disease – Diabetes mellitus Risk factor: – Surgery within 1 month – Use of cortisol – Immunosuppressant –Mechanical ventilation Prior antibiotics use before the isolation: – Carbapenems – Cephalosporins – Quinolones – Aminoglycosides – Tetracyclines Outcome: – Discharge – Died – Lost to follow-up
Patients; n (%) With TNS A. baumannii With TS A. infection (n = 52) baumannii infection (n = 53)
p-value
33 (63.5) 19 (36.5) 56.37 ± 2.511 31 (59.6) 6 (11.5) 13 (25) 1 (1.9) 1 (1.9) 25 (48.1) 17 (32.7) 5 (9.6) 0 3 (5.8) 2 (3.8) 12 (23.1) 12 (23.1) 9 (17.3) 10 (19.2) 9 (17.3) 4 (7.7) 5 (9.6) 21 (40.4) 7 (13.5) 4 (7.7) 29 (55.8)
39 (73.6) 14 (26.4) 59.55 ± 2.548 22 (41.5) 14 (26.4) 14 (26.4) 1 (1.9) 2 (3.8) 26 (49.1) 19 (35.8) 3 (5.7) 4 (7.5) 0 1 (1.9) 17 (32.1) 11 (20.8) 4 (7.5) 17 (32.1) 6 (11.3) 2 (3.8) 3 (5.7) 15 (28.3) 5 (9.4) 4 (7.5) 19 (35.8)
0.2981 0.2981 0.3759 0.0799 0.0806 0.9999 0.9999 0.9999 0.9999 0.8377 0.4882 0.1179 0.1179 0.6178 0.3837 0.8169 0.1497 0.1805 0.4165 0.4370 0.4882 0.2213 0.5553 0.9999 0.0405*
20 (38.5) 10 (19.2) 3 (5.8) 2 (3.8) 0 41 (78.8) 10 (19.2) 1 (1.9)
4 (7.5) 15 (28.3) 3 (5.7) 0 0 36 (67.9) 6 (11.3) 1 (1.9)
0.0002* 0.3603 0.9999 0.2429 – 0.6069 0.2897 0.9999
*p < 0.05. SD: Standard deviation; TS: Tigecycline susceptible; TNS: Tigecycline nonsusceptible.
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Research Article Li, Wang, Zhang et al.
5 Relative expression of adeJ
Relative expression of adeB
A 5000 4500 2000 1500 1000 500 0 TNS 40 30 10 8 6 4 2 0 TNS
3 2 1 0
TS Relative expression of soxR
Relative expression of marR
B
4
TS
TNS
TS
TNS
TS
45 40 15 10 5 0
Figure 1. Relative expression of RND efflux pump and global regulators in Acinetobacter baumanii. Relative expression of (A) RND efflux pump (adeB and adeJ); and (B) global regulators (marR and soxR). TNS: Tigecycline nonsusceptible; TS: Tigecycline susceptible. ●●Statistical analysis
SPSS 17.0 (SPSS, Inc., IL, USA) was used for all statistical analyses. Comparative analyses were executed by χ2 or Fisher exact tests for categorical variables and by the Student t-test for continuous variables. All tests were two tailed, and p-values of 8-fold) and quinolones (5.3-fold), but did not influence susceptibility to ceftazidime, imipenem or trimethoprim. The complementation experiment for adeB restored resistance to tigecycline, aminoglycosides, quinolones, tetracycline and chloramphenicol in complementary mutant AB566△adeB/adeB when compared with the adeB deletion mutant AB566△adeB (Table 2) .
Research Article
Three TS isolates, AB459, AB563 and AB314, with different adeR or adeS sequences were transformed with the plasmids pWH-adeR or pWH-adeS, which carried adeR or adeS main mutations from TNS isolates. The contribution of adeR or adeS mutations to tigecycline resistance was determined in terms of the tigecycline MIC for the transformed isolates. Complementation of adeR or adeS mutations decreased the susceptibility to tigecycline slightly (≤2-fold), and also resulted in decreased susceptibility to ciprofloxacin (≤2-fold; Table 4). Quantitative analysis revealed that expression of adeB increased 1.2- to 3.6-fold in complementary mutants, when compared with the parental strains. ●●Analysis of expression of the RND efflux
●●Nucleotide sequencing of the regulatory
pump global regulators marR & soxR
genes adeR & adeS
MarR and soxR are global repressor proteins involved in bacterial antibiotic resistance. In our study, marR and soxR existed in all isolates by PCR. The relative expression of marR and soxR decreased in TNS isolates by real-time RT-PCR (Figure 1B) . Quantitative analysis revealed that expression of marR and soxR decreased 11.6- and 10.4-fold in TNS isolates, when compared with the TS isolates.
To further assess the mechanism of increased adeB expression, mutations in the two-component regulatory system of the AdeABC efflux pump, comprising adeR and adeS, were investigated. There were several mutations found in all TNS isolates, including Ile14Val, Val120Ile, Ala136Val in AdeR and Gly186Val, Asn213Asp, Asn268His, Ile285Leu in AdeS (p < 0.05; Table 3 ). We also identified another two AdeS mutations in three TNS isolates with tigecycline MIC of 8 mg/l, including Thr156Met in one strain and Ala130Thr in two strains. Meanwhile, AdeR and AdeS were absent in one TNS and eight TS isolates.
Discussion The increasing prevalence of multidrug-resistant A. baumannii has led to limited therapeutic options, and tigecycline is considered one of the few therapies available. In our study, more
Table 2. Antibiotic susceptibility of AdeB inactivated and complement strains. Antibiotics
MIC (mg/l) values of antibiotics
AB566
AB566ΔadeB†
AB566ΔadeB/adeB‡
Gentamicin Amikacin Ciprofloxacin Levofloxacin Tetracycline Chloramphenicol Trimethoprim Imipenem Ceftazidime Tigecycline
>256 12 >32 8 32 256 >32 >32 >256 6
4 1.5 6 1.5 2 8 >32 >32 >256 0.19
96 6 >32 12 64 256 >32 >32 >256 4
AdeB deletion mutant. Complementary to adeB in adeB deletion mutant.
† ‡
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341
Research Article Li, Wang, Zhang et al. Table 3. Amino acid mutations of adeR and adeS. Gene
Amino acid
Isolates (n)
p-value
Location
Coding protein
TNS (n = 52)
TS (n = 23)
adeR adeS
14 120 136 186 213 268 285
Ile Val Val Ile Ala Val Gly Val Asn Asp Asn His Ile Leu
0 51 0 50 0 50 0 49 0 49 0 49 0 49
5 10 6 9 7 8 8 7 4 11 6 9 4 11
0.0003
