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Antimicrobial Agents and Chemotherapy, February 2005, p. 791-793, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.791-793.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

AcrAB Efflux Pump Plays a Role in Decreased Susceptibility to Tigecycline in Morganella morganii

Alexey Ruzin,^* David Keeney, and Patricia A. Bradford

Wyeth Research, Pearl River, New York

Received 30 June 2004/ Returned for modification 1 September 2004/ Accepted 7 October 2004

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Transposon mutagenesis of a clinical isolate of Morganella morganii, G1492 (tigecycline MIC of 4 µg/ml), yielded two insertion knockout mutants for which tigecycline MICs were 0.03 µg/ml. Transposon insertions mapped to acrA, which is constitutively overexpressed in G1492, suggesting a role of the AcrAB efflux pump in decreased susceptibility to tigecycline in M. morganii.

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Tigecycline is a novel expanded broad-spectrum glycylcycline antibiotic (1, 8, 9). Tigecycline circumvents classical tetracycline resistance mechanisms such as ribosomal protection and efflux by tetracycline-specific pumps (3, 9). Nevertheless, several species of gram-negative bacteria are intrinsically less susceptible to tigecycline; among them are Pseudomonas aeruginosa, Proteus spp., Providencia spp., and Morganella morganii. In addition, a few strains of Klebsiella pneumoniae have been isolated with acquired decreased susceptibility to tigecycline.

Previous studies showed that decreased tigecycline susceptibility is associated with multidrug efflux systems such as MexXY in P. aeruginosa and AcrAB in Proteus mirabilis and K. pneumoniae (4, 10, 14). These pumps belong to the resistance/nodulation/division family and are often associated with multidrug resistance (MDR). As determined previously, the constitutive overexpression of the pump components correlated with reduced susceptibility to tigecycline in P. aeruginosa and K. pneumoniae (4, 10).

This study was performed to identify the mechanism of decreased susceptibility to tigecycline in many strains of M. morganii. For the majority of M. morganii strains, the tigecycline MIC was 1 to 4 µg/ml. Two clinical isolates of M. morganii, G858 and G1492, which represent the lower and higher ends of the range of tigecycline MICs, were selected for this study.

Bacterial strains and plasmids used in this study are shown in Table 1. The strains were propagated at 37°C in Luria-Bertani broth or agar. Standard DNA manipulations such as restriction digestion and molecular cloning were performed as described previously (11). Chemically competent Escherichia coli strains TOP10 and INV110 (Invitrogen, Carlsbad, Calif.) were used for cloning experiments. DNA transformations were performed by electroporation with the Gene Pulser II system (Bio-Rad, Hercules, Calif.), using the optimal electroporation settings of 2.5 kV, 25 µF, 200 , and 5 ms. Transposon mutagenesis was done with the EZ::TN <R6ori/KAN-2> transposome kit (Epicentre, Madison, Wis.) according to the manufacturer's protocol. The selection of tigecycline-susceptible transposon mutants, rescue cloning, and mapping of transposon insertions were performed as described previously (14). The nucleotide sequence was determined with an ABI 3700 automated sequencer (Applied Biosystems, Foster City, Calif.). The MICs of various antibacterial agents were determined by standard broth microdilution tests (6). MICs of tigecycline were determined with fresh Mueller-Hinton broth (<12 h old).

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TABLE 1. Bacterial strains and plasmids used in this study

Two tigecycline-susceptible transposon mutants, GC7676 and GC7677, were obtained upon transposon mutagenesis of a clinical isolate of M. morganii G1492. The sites of transposon insertion were mapped by rescue cloning and sequencing to a homolog of the acrA gene. GC7676 had an insertion at nucleotide 453, and GC7677 had an insertion at nucleotide 946. The nucleotide sequence of a 6,104-bp genomic DNA fragment containing the entire acrRAB locus of G1492 was determined from the rescue clones. The acrRAB genes of M. morganii were 648, 1,194, and 3,177 bp long, respectively, and had 63, 57, and 81% amino acid sequence identity to acrRAB genes of P. mirabilis (14). The orientation of genes was identical to that of P. mirabilis, with the acrR and acrAB genes being divergently transcribed from a common intergenic region.

Transposon insertion into the acrA gene of G1492 resulted in a substantial decrease in the MIC of tigecycline (128-fold reduction from 4 to 0.03 µg/ml; Table 2). In addition, the MDR phenotype was suppressed, as demonstrated by the decrease in the MICs of minocycline, acriflavine, ethidium bromide, sodium dodecyl sulfate, erythromycin, chloramphenicol, nalidixic acid, novobiocin, and trimethoprim for GC7676 and GC7677 (Table 2).

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TABLE 2. Antibiotic susceptibility for M. morganii strains

For transcomplementation studies, a DNA fragment containing the full-length acrAB of G1492 was amplified by PCR with the primers listed in Table 3. Genomic DNA of G1492 was isolated by using the Puregene tissue kit (Gentra Systems, Inc., Minneapolis, Minn.) and served as a template for PCR. The gel-purified PCR fragment was cloned into the pCR2.1-TOPO vector (Invitrogen) as specified by the manufacturer. The resulting plasmid, pCLL3443, was modified by cloning an 878-bp XbaI fragment containing the gentamicin resistance cassette from pUCGm into the XbaI site of pCLL3443. The resulting plasmid, pCLL3444, was used in transcomplementation studies. Introduction of pCLL3444 into transposon mutants resulted in tigecycline MICs of 4 to 8 µg/ml and in the restoration of the MDR phenotype. It should be noted that both acrA and acrB were required for transcomplementation because these genes are cotranscribed; therefore, both were inactivated by transposon insertions in acrA. These results indicated that the acrAB locus was linked to the decreased tigecycline susceptibility and MDR phenotype of G1492.

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TABLE 3. Primers and fluorescent probes used for PCR and RT-PCR

To determine whether the decreased tigecycline susceptibility of G1492 was associated with acrAB overexpression, the transcriptional levels of acrA were analyzed by Taqman quantitative real-time PCR (RT-PCR) on the iCycler iQ real-time PCR detection system (Bio-Rad). DNase-treated RNA templates were prepared from mid-log-phase bacterial cultures by using the RNAeasy kit (Qiagen, Valencia, Calif.). Oligonucleotide primers and probes used for RT-PCR are shown in Table 3. RT-PCR and the quantification of the target gene expression (acrA) relative to an endogenous reference (rrsE) were performed as described previously (10). As shown in Table 4, the level of acrA expression was approximately 48.5-fold higher in G1492 (tigecycline MIC = 4 µg/ml) than in G858 (tigecycline MIC = 1 µg/ml). In addition, the elevated level of acrA expression in G1492 in comparison to that in G858 was confirmed by Northern blot hybridization (data not shown), further suggesting that reduced susceptibility to tigecycline was linked to acrA overexpression in M. morganii. It should be noted that strains G858 and G1492 are nonisogenic, and, therefore, an additional analysis of acrAB expression levels in the same-strain background would be beneficial to confirm the correlation between increased levels of acrAB expression and decreased susceptibility to tigecycline in M. morganii.

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TABLE 4. RT-PCR analysis of acrA expression

In conclusion, the mechanism of efflux-mediated decreased susceptibility to tigecycline in M. morganii appears to be similar to that identified earlier in P. aeruginosa, P. mirabilis, and K. pneumoniae (4, 10, 14). Future studies will be directed toward understanding the molecular mechanisms of constitutive pump overexpression and the regulation of pump production in M. morganii. Based on previously published studies, the overexpression of the pump components is commonly caused by inactivation of the pump repressor or overproduction of transcriptional activator(s) (2, 4, 5, 7, 12). The possibility that any of these mechanisms caused the increased production of AcrAB in M. morganii will be an area for future study.

Only limited data on genetic characterization of M. morganii are available. The present study provides the first sequence and description of acrRAB in this species.

Nucleotide sequence accession number. The nucleotide and protein sequences of the acrRAB genes of M. morganii have been registered in GenBank under accession no. AY669147.

 
* Corresponding author. Mailing address: Wyeth Research, Department of Infectious Disease, 401 North Middletown Rd., Bld. 200, Rm. 3219, Pearl River, NY 10965. Phone: (845) 602-4592. Fax: (845) 602-5671. E-mail: ruzina{at}wyeth.com.

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Antimicrobial Agents and Chemotherapy, February 2005, p. 791-793, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.791-793.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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