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Antimicrobial Agents and Chemotherapy, October 2008, p. 3826-3827, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00419-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Antimicrobial Studies with the Pseudomonas aeruginosa Two-Allele Library Require Caution{triangledown}


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LETTER
 
The increasing prevalence of antimicrobial resistance requires a continuous effort to find new antimicrobials. The availability of genome sequences for a variety of microbes has facilitated the development of whole-genome, single-gene knockout libraries for systematic gene function analysis. These libraries, such as the Keio library of Escherichia coli (1) and the University of Washington two-allele library of Pseudomonas aeruginosa (3), can be used in high-throughput phenotypic array studies (7); thus, they seem well suited for identifying new antimicrobial targets. However, we recently noticed with the P. aeruginosa two-allele mutant library that intrinsic, unidentified factors may affect susceptibility for some antimicrobials with many randomly picked mutants, thereby obscuring effects attributed to specific candidate mutants.

In E. coli, a phoU knockout mutation confers hypersensitivity to a variety of antimicrobials and other stresses (5). To determine whether inactivation of a phoU homologue in P. aeruginosa confers a similar phenotype, we obtained a phoU mutant and the parental wild-type strain (PAO1, lacking a transposon insertion) from the University of Washington two-allele library. When quinolone susceptibility was tested, a 4- to 16-fold decrease in MIC was observed for both ciprofloxacin and oxolinic acid (Table 1), as expected (5). However, when 10 unrelated mutants were tested, each of those mutants also exhibited hypersusceptibility to quinolones (Table 1). Moreover, PCR analysis failed to confirm transposition being inside the phoU allele. Thus, increased susceptibility of the phoU mutant could not be specifically attributed to the phoU mutation.


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  		TABLE 1. Antimicrobial susceptibility of mutants obtained from the P. aeruginosa two-allele library

Colony color with most of these mutants was darker (bluish green) than with the wild-type strain, indicating production of more phenazine pigments in the mutants. Since phenazines, such as pyocyanin, may be involved in oxidative stress and thereby affect antimicrobial susceptibility (2), we tested two additional mutants (the phzS and phzM mutants) that do not produce pyocyanin (6). Reduced MICs, similar to those seen with the other mutants, were observed. Thus, variation in pyocyanin production was not responsible for reduced quinolone susceptibility. Since the mutants tested had transposon insertions at a variety of chromosomal locations, some of which were far outside open reading frames, transposon-mediated polarity is also an unlikely explanation for reduced MIC. When other antimicrobial classes were tested, hypersusceptibility was observed with chloramphenicol (4- to 64-fold) but not with tobramycin, meropenem, or ceftazidime (Table 1). Thus, the mutants tested were universally hypersusceptible to some, but not all, antimicrobials for reasons that are unknown. This lack of specificity may not be unique to the University of Washington library, because several lux-insertional mutants obtained from a different library (4) also conferred quinolone hypersusceptibility (data not shown).

The results described above suggest that the transposition/insertion of a DNA fragment into the P. aeruginosa chromosome may itself have a general effect on susceptibility for some, but not all, antimicrobials. Had we simply compared the phoU mutant with the parental wild-type strain, which is standard practice with many antimicrobial studies, we would have been misled. Thus, interpretation of antimicrobial experiments involving mutants from the P. aeruginosa insertional libraries requires caution.


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ACKNOWLEDGMENTS
 
We thank Karl Drlica and Richard Pine for valuable comments.

This work is supported by NIH grants AI068014 and AI073491.


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FOOTNOTES
 
{triangledown} Published ahead of print on 11 August 2008. Back


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REFERENCES
 
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  1. Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko, M. Tomita, B. L. Wanner, and H. Mori. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2:2006.0008.
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  2. Dietrich, L. E., A. Price-Whelan, A. Petersen, M. Whiteley, and D. K. Newman. 2006. The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Mol. Microbiol. 61:1308-1321.[CrossRef][Medline]
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  4. Lewenza, S., R. K. Falsafi, G. Winsor, W. J. Gooderham, J. B. McPhee, F. S. Brinkman, and R. E. Hancock. 2005. Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes. Genome Res. 15:583-589.[Abstract/Free Full Text]
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  5. Li, Y., and Y. Zhang. 2007. PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli. Antimicrob. Agents Chemother. 51:2092-2099.[Abstract/Free Full Text]
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  6. Mavrodi, D. V., R. F. Bonsall, S. M. Delaney, M. J. Soule, G. Phillips, and L. S. Thomashow. 2001. Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J. Bacteriol. 183:6454-6465.[Abstract/Free Full Text]
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  7. Zhou, L., X. Lei, B. Bochner, and B. Wanner. 2003. Phenotype microarray analysis of Escherichia coli K-12 mutants with deletions of all two-component systems. J. Bacteriol. 185:4956-4972.[Abstract/Free Full Text]
Xiangli Wu
Hexiang Wang
State Key Laboratory for Agrobiotechnology and Department of Microbiology
China Agricultural University
Beijing 100094, China

Xilin Zhao*
Public Health Research Institute
New Jersey Medical School-UMDNJ
Newark, New Jersey 07103

* Phone: (973) 854-3364, Fax: (973) 854-3101, E-mail: zhaox5{at}umdnj.edu

Antimicrobial Agents and Chemotherapy, October 2008, p. 3826-3827, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00419-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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