Pseudomonas aeruginosa (P. aeruginosa) is one of the leading pathogens involved in nosocomial pneumonia. In addition, P. aeruginosa infection is associated with significant morbidity and mortality.1 A major problem in P. aeruginosa infection is that this organism exhibits natural and acquired resistance to many structurally and functionally diverse antibiotics.
Ciprofloxacin (CIP) is the most in vitro-active anti-P. aeruginosa fluoroquinolone, but increasing resistance frequently precludes its use. The mechanism of the resistance includes target-based mutation in gyrase and/or topoisomerase and overexpression of efflux pumps.2
As the target of quinolones, mutation in the quinolone-resistance-determining regions (QRDRs) of the corresponding genes, gyrA and parC, appear to play a central role in the acquisition of resistance to fluoroquinolone in clinical isolates.
Efflux pumps present in P. aeruginosa serve physiologic functions such as the removal of intracellular toxic substances including antibiotics. The low susceptibility to antibiotics is mainly the consequence of the presence of several genes encoding multidrug resistance (MDR) efflux pumps in the genome of this bacterial species. Active MDR pumps have been found in P. aeruginosa strains associated with clinical or environmental habitats. Six efflux pumps have been identified: MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM, MexJK-OprM, and MexVW-OprM.3 While each pump has a preferential set of antimicrobial agent substrates, the fluoroquinolones are universal substrates for the first four Mex pumps.
In 2005 American Thoracic Society (ATS) guideline for nosocomial pneumonia, antipseudomonal β-lactam plus either an antipseudomonal quinolone or an aminoglycoside are recommended as initial empiric therapy when MDR pseudomonal infection is suspected.4 The recommended dosage of ciprofloxacin in this guideline is 400 mg every 8 hours which is higher than the doses used in routine clinical practice. Increasing the dosage of ciprofloxacin is demonstrated efficacy when efflux pumps do contributions to the high-level resistance of P. aeruginosa. In this study, we report high-level ciprofloxacin resistance in P. aeruginosa due to interplay of the efflux pump systems and the mutations of DNA gyrase and topoisomerase IV.
Thirty-four P. aeruginosa strains were collected from patients with lower respiratory infections in 2002 in Beijing hospital. Isolates resistant to ciprofloxacin were tested by Etest (AB Biodisk, Solna, Sweden). Control strain was P. aeruginosa (ATCC 27853). According to National Committee for Clinical Laboratory Standards (NCCLS), MIC≤1.0 μg/ml were considered as sensitive strain; 1.0 μg/ml<MIC<4.0 μg/ml were considered as interpose strain; MIC≥4.0 μg/ml were considered as resistant strain. In this study, we divided the resistant strains into common-level resistant strain (4.0 μg/ml ≤ MIC<32.0 μg/ml) and high-level resistant strain (MIC≥ 32.0 μg/ml).
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)
Chromosomal DNA was isolated with a DNeasy tissue kit (Tianwei, Beijing, China). Each PCR was performed with a Perkin-Elmer 9600 thermal cycler by 2×Taq PCR MasterMix (Tianwei, Beijing, China). The reaction mixtures contained 1.5 mmol/L MgCl2, 250 μmol/L of each dNTP, 10 mmol/L Tris-HCl (pH 8.3), 1.0 U/10 μl Taq DNA polymerase, 50 mmol/L KCl. The primers of gyrA5 were 5′GGC CTG AAG CCG GTG CAC3′ and 5′CAC GGC GAT ACC GCT GGA3′. The primers of parC6 were 5′TCC AAG CAA GAA ACT G3′ and 5′AGC AGC ACC TCG GAA TAG3′. The cycling parameters for the PCR were as follows: 94°C for 4 minutes, 32 cycles at 94°C for 1 minute, 62°C (gyrA)/59°C (parC) for 30 seconds and 72°C for 1 minute, and followed by the final step at 72°C for 10 minutes. The amplified PCR products of gyrA(410 bp) and parC (227 bp) were digested with Sac II, Hinf I (TaKaRa, Japan) respectively for 2 hours at 37°C. The digestion products were separated by electrophoresis on 8% polyacrylamide gels.
P. aeruginosa strains of sensitive group and high-level resistance group were cultured in Luria-Bertani (LB) medium at 250 r/min for 16 hours at 37°C. Then total RNA was isolated respectively using Trizol (Invitrogen, USA). RT-PCR was performed as described in the RT-PCR kit (TaKaRa, Japan). The primers of endogenous control7 were 5′ GTG CCT GCA GCC GCG GTA AT3′ and 5′TGC GCC ACT AAG ATG TCA AG3′. The primers of mexB were 5′ CCA TGG TGC TCT CGG CGG TAT TCC T3′ and 5′CTG TTC TCG CCA CCG GGA CGC TCT T3′. The primers of mexD were 5′ GCC AAC GCG ATC CAG ACC CTA CCC T3′ and 5′CGC GCC ACC AGC TTC GAG TTG AG3′. The primers of mexF were 5′ CCA GGT CAA CGT GCC GCA GA-T3′ and 5′CCT CCT GCT TGT CCT TGG CGA ACT C3′. The primers of mexY were 5′ CCC GGC CAA GCT GAC CTC GAT GAA C3′ and 5′CCC AGG CCG AGC ATC ACC GTG AAG3′. The cycling parameters for the PCR were as follows: 94°C for 2 minutes, 30 cycles at 94°C for 30 seconds, 62°C for 30 seconds and 72°C for 1 minute, and followed by the final step at 72°C for 10 minutes. The RT-PCR products were run on 15 g/L agarose gel and visualized by ethidium bromide staining.
Data were analyzed using SPSS 11.0 and expressed as mean±standard deviation (SD). The continuous variables were tested with one-way analysis of variance (ANOVA) and q test. Others were tested with Kruskal-Wallis test. A P value less than 0.05 was considered statistically significant.
Mutation in gyrase and topoisomerase
The mutation rate at gyrA83 in sensitive group, interpose group, common-level resistance group, high-level resistance group were 0% (0/0), 0% (0/0), 50% (2/4) and 100% (23/23) respectively (Fig. 1); at parC80 were 0% (0/0), 0%(0/0), 0% (0/0) and 60.87% (14/23) respectively (Fig. 2). ParC80 mutation strains all had mutated at gyrA83.
Expression of efflux pumps
High-level resistance was divided into two groups: resistances group with gyrA83 single mutation; resistanced group with gyrA83 and parC80 double mutations. Data about the expression level of RND transporters were shown in the table and Fig. 3.
P. aeruginosa is a leading cause of nosocomial infections that are difficult to eradicate because of the organism's resistance to a multitude of antibiotics. The mechanism of resistance is very complex. Only its resistance to CIP are involved in biofilm, integron, target-based mutation and overexpression of efflux pumps. It is useful to study the relationships between different mechanisms of resistance to a particular antibiotic that can coexist in the same bacterial cell. In this study, we researched the interplay between efflux-based and target-mediated resistance to CIP in P. aeruginosa.
Our study and others5,6 got the similar outcome that mutation with Thr83→Ile in gyrA gene was associated with the principal mutation for CIP. Additional mutation in the parC gene (Ser80→Leu) contributed to a higher level of CIP resistance than a single mutation.
Multidrug transporters belonging to the resistancenodulation-cell division (RND) family interact with a membrane fusion protein (MFP) and an outer membrane protein to allow drug transport across both the inner and outer membrane of gram-negative bacteria. P. aeruginosa is a clinically important opportunistic pathogen characterized by relatively high intrinsic resistance to a variety of antimicrobial agents. This property is now recognized to result from at least four RND efflux systems.
In this study, we inspected the transporters (MexB, MexD, MexE, MexF) not outer membrane protein to express the pump's level due in part to the phenomenon that OprM contributes to antibiotic resistance in P. aeruginosa independent of MexAB.8 The result was the RNA levels of MexB, MexF, sum of the four Mex in high-level resistance strains had higher expression than in sensitive strains. Nakajima et al9 had reported high-level fluoroquinolone resistance in P. aeruginosa due to interplay of the MexAB-OprM efflux pump and the DNA gyrase mutation. Jalal et al10 had found alterations in two efflux systems, MexCD-OprJ and MexEF-OprN, were the predominant mechanisms of fluoroquinolones resistance in P. aeruginosa strains from the lungs of CF patients. We did not find the difference of levels between resistance and sensitive isolates in our clinic isolates. Since the four efflux systems can pump quinolones, we analyzed the difference in expressions of sum of the pumps. The result was sum RNA levels of MexB, MexD, MexF and MexY in high-level resistance strains with single mutation at gyrA83 were higher than in sensitive strains. That means high-level fluoroquinolone resistance in P. aeruginosa due to interplay of the efflux pump systems and the mutations of DNA gyrase and topoisomerase IV.
In conclusion, efflux pumps contribute to high-level resistance of P. aeruginosa to ciprofloxacin.
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Keywords:© 2007 Chinese Medical Association
Pseudomonas aeruginosa; drug resistance; efflux pumps; ciprofloxacin