Ann Clin Microbiol.  2017 Dec;20(4):90-96. 10.5145/ACM.2017.20.4.90.

Assessment of Efflux Activity Using H33342 Accumulation in Tigecycline-Resistant Acinetobacter baumannii Clinical Isolates

• Kim CM
• Choi IS
• Jang SJ
• Yun NR
• Kim DM
• Lim D
• Ahn YJ
• Kang SH
• Park G
• Moon DS

• KMID: 2398124
• DOI: http://doi.org/10.5145/ACM.2017.20.4.90

Abstract

BACKGROUND
Tigecycline resistance has emerged recently and has shown diverse mechanisms. The aim of this study was to assess the role of efflux activity in tigecycline resistance in 120 clinical isolates of A. baumannii using two methods: the H33342 accumulation assay and adeB real-time reverse transcriptase polymerase chain reaction. In addition, we analyzed the correlation between the expression level of adeB and H33342 accumulation level.
METHODS
A. baumannii clinical isolates was divided into tigecycline-resistant (49 strains), intermediate (40 strains), and susceptible (31 strains) groups. The H33342 accumulation was measured in the absence or presence of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Real-time RT-PCR was performed to determine the relative expression of the adeB gene in A. baumannii clinical isolates.
RESULTS
The level of H33342 accumulation in the resistant group was relatively lower than those in the other groups. The addition of CCCP caused a significantly increased fold change in H33342 accumulation in the tigecycline-resistant group. Significant difference in the fold change level in H33342 accumulation was found between tigecycline-susceptible and resistant isolates. Those findings support the role of efflux pumps of which substrates are H33342 in the resistance of tigecycline. Significant differences in the relative expression levels of adeB were shown between tigecycline-susceptible and resistant groups also.
CONCLUSION
The results showed that several efflux pumps of which substrates were H33342 can contribute to tigecycline resistance. The adeB overexpression can also contribute to tigecycline resistance. It is possible that efflux pumps other than adeB efflux pumps contribute to tigecycline resistance because there was no correlation between fold change level in H33342 accumulation and adeB expression level.

Keyword

Acinetobacter baumannii; Efflux pump inhibitor; Tigecycline

MeSH Terms

Acinetobacter baumannii*
Acinetobacter*
Carbonyl Cyanide m-Chlorophenyl Hydrazone
Reverse Transcriptase Polymerase Chain Reaction
Carbonyl Cyanide m-Chlorophenyl Hydrazone

Figure

• 

  Fig. 1. Pattern of H33342 accumulation (A) and the H33342 accumulation ratio (B) over a 70 min incubation period with 6 Acinetobacter baumannii isolates, including ATCC 19606, 2 tigecycline-susceptible (15136S, 15986S), and 3 resistant (17296R, 18167R, 18997R) strains. The H33342 accumulation ratio reflects the fold changes in H33342 fluorescence accumulation after addition of CCCP, relative to basal level without CCCP addition. The error bars represent the standard error of the mean. Abbreviations: H33342, Hoechst 33342; CCCP, carbonyl cyanide 3-chlorophenylhydrazone.

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  Fig. 2. Comparison of the mean values of H33342 accumulation in the presence and absence of CCCP (A) and the H33342 accumulation ratio (B) of tigecycline-susceptible (S), intermediate (I), and resistant (R) groups in 120 Acinetobacter baumannii clinical isolates at 50 min of exposure (steady state). The error bars represent the standard error of the mean. **P＜0.001; ***P＜0.0001. Abbreviations: H33342, Hoechst 33342; CCCP, carbonyl cyanide 3-chlorophenylhydrazone.

• 

  Fig. 3. Relative expression of the efflux pump gene, adeB, determined by real-time reverse transcriptase PCR in 31 tigecycline-susceptible (S), 40 intermediate (I), and 49 resistant (R) Acinetobacter baumannii strains. The normalized expression of adeB was calibrated with the expression of A. baumannii ATCC 19606. The error bars represent the standard error of the mean. * P＜0.05.

Reference

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