Efects of the order of exposureto antimicrobials on the incidenceof multidrug‑resistant Pseudomonas aeruginosa
Transfered by: Dr. Khaled Kamal Hussain

Efects of the order of exposureto antimicrobials on the incidence
of multidrug‑resistant Pseudomonas aeruginosa

Transfered by: Dr. Khaled Kamal Hussain

NamiYasuda1,5, Tomoko Fujita1,5, Takahiro Fujioka1, MeiTagawa1
, Naoki Kohira1 KenshoTorimaru2 , Sumiko Shiota3 , Takanori Kumagai Daichi Morita1,2, Wakano Ogawa1,4,TomofusaTsuchiya1 & Teruo Kuroda1,2*

Multidrug-resistant Pseudomonas aeruginosa (MDRP) is one of the most important pathogens in
clinical practice. To clarify the mechanisms contributing to its emergence, we isolated MDRPs using
the P. aeruginosa PAO1, the whole genome sequence of which has already been elucidated. Mutant
strains resistant to carbapenems, aminoglycosides, and new quinolones, which are used to treat P.
aeruginosa infections, were isolated; however, none met the criteria for MDRPs. Then, PAO1 strains
were exposed to these antimicrobial agents in various orders and the appearance rate of MDRP
varied depending on the order of exposure; MDRPs more frequently appeared when gentamicin was
applied before ciprofoxacin, but were rarely isolated when ciprofoxacin was applied frst. Exposure
to ciprofoxacin followed by gentamicin increased the expression of MexCD-OprJ, an RND-type
multidrug efux pump, due to the NfxB mutation. In contrast, exposure to gentamicin followed
by ciprofoxacin resulted in more mutations in DNA gyrase. These results suggest that the type of
quinolone resistance mechanism is related to the frequency of MDRP and that the risk of MDRP
incidence is highly dependent on the order of exposure to gentamicin and ciprofoxacin.
Pseudomonas aeruginosa is an opportunistic pathogen that exhibits high intrinsic resistance to antimicrobial
agents. P. aeruginosa may acquire resistance through the inappropriate use and/or long-term administration
of antimicrobial agents. Many multidrug-resistant P. aeruginosa strains (MDRPs) have been identifed and
are highly resistant to three antimicrobial agents, namely, broad-spectrum β-lactams, aminoglycosides, and
fuoroquinolones1–3
. Since the number of antibiotics that are efective against MDRP is limited, countermeasures
are important.
To promote the appropriate use of antimicrobial agents, it is important to clarify the relationship between
the antimicrobial agents used and the mechanisms underlying the acquisition of resistance. Analyses of clinical isolates have progressed in recent years, resulting in a more detailed understanding of MDRPs1–3
. However,
it is impossible to fnd the parental strain of an isolated MDRP in clinical settings because once the MDRP is
isolated, the susceptible parent strain has already disappeared. Terefore, analyses of clinically isolated MDRPs
alone cannot reveal a direct relationship between the type of antimicrobial used in treatment and the mechanism
underlying the acquisition of resistance to it.
In the present study, we isolated and analyzed MDRPs using the P. aeruginosa PAO1 strain, the whole genomic
DNA sequence of which is available. We were unable to isolate MDRPs with an exposure to one or two antimicrobial agents, but were successful with a sequential exposure to three antimicrobial agents. Terefore, MDRPs
emerged by stacking multiple resistance mechanisms. We also found that the order in which antimicrobials are
used may afect the emergence of MDRPs.