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Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli
J Infect Chemother xxx (2014) 1e5
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Original article
Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli Eiki Yamasaki a, *, Chihiro Yamada b, Xinghua Jin b, G. Balakrish Nair c, Hisao Kurazono a, Shingo Yamamoto b a b c
Division of Food Hygiene, Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan The Department of Urology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan Translational Health Science and Technology Institute, Gurgaon, Haryana, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 June 2014 Received in revised form 8 October 2014 Accepted 10 October 2014 Available online xxx
Background: Analyses of efflux pumps overexpression and mutations in quinolone resistance determining region (QRDR) in early stage of development of resistance to fluoroquinolones (FQs) are valuable to discuss countermeasures against them. We induced levofloxacin (LVFX)-resistant strains from susceptible uropathogenic Escherichia coli in vitro to analyze the mechanisms of development of FQsresistance. Methods: 89 strains were exposed to discontinuous elevation of LVFX dose, and mRNA level of efflux pumps and their regulators as well as mutations developed in QRDR of LVFX-resistant strains were analyzed. Results: In 5 strains, a stepwise increase in MIC to LVFX (up to >128 mg/ml)was observed. Compared to the parent strains, additional mutations in QRDR were observed in the strains developing high MIC. Remarkable increase of marA expression was observed even in the early stage of LVFX-resistance development, and it lasted until high-level resistance was developed. On the other hand, moderate increase in acrB expression but only low increase in yhiU, yhiV, mdfA, tolC and sdiA were observed. Conclusions: These results suggested that marA expression is a sensitive marker for early detection of development of LVFX-resistance. © 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Fluoroquinolones-resistant Escherichia coli Quinolone resistant determining region Efflux pump Urinary tract infection
1. Introduction Since Escherichia coli (E. coli) is the bacteria most commonly isolated from patient with urinary tract infection (UTI), the emergence of E. coli isolates that are resistant to some of the most commonly used antimicrobial drugs has become a major problem in the management of UTI [1e3]. Fluoroquinolones (FQs) are the most widely used antibiotics worldwide, and are the drugs of choice for empirical therapy for UTI. Increasing emergence of FQsresistant E. coli has been reported worldwide [3e5]. It has been reported that >10% of E. coli isolated from uncomplicated UTI and 30e40 % of E. coli isolated from complicated UTI were resistant to
* Corresponding author. Nishi 2-11, Inada-cho, Obihiro City, Hokkaido, 080-8555 Japan. Tel./fax: +81 155 49 5390. E-mail address: [email protected] (E. Yamasaki).
FQs [1,2]. It is not hard to understand that these high frequencies of FQs-resistant strains affect therapeutic strategy for UTI. FQs can directly bind to DNA gyrase and topoisomerase IV, and inhibit the activity of these enzymes that are essential for DNA replication and transcription. The mechanism of resistance to FQs mainly consists of two separate strategies: a decrease in drug accumulation in the bacterial cells through an induction of efflux pumps overexpression and mutations in the genes encoding DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Previous reports demonstrated that multi-step process is responsible for the development of high-level resistance to FQs [6]. In the early step, low-level resistance is developed by overexpression of efflux-pumps which facilitate active extraction of FQs [7]. Five superfamilies of efflux pumps (RND (resistance-nodulation-division), MF (major facilitator), ABC (ATP binding cassette), MATE (multidrug and toxic efflux) and SMR (small multidrug resistance)) that
http://dx.doi.org/10.1016/j.jiac.2014.10.007 1341-321X/© 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yamasaki E, et al., Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.007
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E. Yamasaki et al. / J Infect Chemother xxx (2014) 1e5
promote resistance to various antimicrobial drugs had been described in E. coli [8,9]. And, AcrA-AcrB-TolC and YhiU-YhiV-TolC pumps belonging to RND superfamily and MdfA-Cmr pump belonging to MS superfamily were reported to be involved in excretion of FQs [9,10]. In addition, it has been reported that overexpression of sdiA and marA, both of them are the positive regulator for AcrA-AcrB-TolC pump expression, were associated with the resistance to FQs in E. coli [11,12]. SdiA is a quorum-sensing regulated transcription factor, and is involved in the regulation of cell division. There is increasing evidence that molecules involved in quorum-sensing participate in development of multidrug resistance [13,14]. Deletion of sdiA led to decreased level of the AcrB expression, which resulted in the decreased level of drug resistance [13]. MarA is the global activator, which modulates the expression of many genes including acrA, acrB and tolC [12]. It is known that expression of marA is affected by the presence of salicylate through the inactivation of transcription repressor MarR, and by the redoxinducible SoxS which is the positive regulator of marA expression. Although these efflux pumps expressions are rapid response observed in early stage of drug-resistant development, it conferred only 2- to 8-fold elevation in MIC [7] while, in the late step, highlevel resistance is developed by the mutation in quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE. Recently, we and others has reported that mutation at Ser83 and Asp87 in gyrA and Ser80 in parC are frequently observed in FQsresistant strains isolated from UTI patient although single point mutation at Ser83 or Asp87 in gyrA was not sufficient to develop resistance to FQs [6,15]. Further, we have also reported that there is a phylogenetic preference among FQs-resistant strains [15]. Phylogenic group B2 was dominant among FQs-resistant strains (49.4%) although the dominancy was lower than that in FQssusceptible strains (77.5%), and phylogenic group D which was second most prevalent phylogenic group among FQs-resistant strains (34.8%) is more observed among FQs-resistant strains than among FQ-susceptible strains (12.4%), suggesting that the acquisition of FQs-resistance might be frequent in the limited strains such as phylogenic group B2 or D rather than other phylogenic groups. The mechanisms of FQs-resistance development have been mostly discussed based on statistical comparison between FQssensitive and FQs-resistant clinical isolates. Whereas the status during resistance development process has been much less investigated [7,11]. In the present study, we induced drug-resistant E. coli strains in vitro by exposure to levofloxacin (LVFX), which is the drug commonly used for treating UTIs, cystitis and pyelonephritis. The expressions of efflux pumps and their transcriptional regulators, and mutation in QRDR in early and late stage of resistance development were analyzed. 2. Materials and methods 2.1. Bacterial strains Eighty nine FQ-susceptible E. coli strains, including 5, 4, 69, and 11 strains in phylogenic groups A, B1, B2, and D, respectively, were isolated from the patient with cystitis as described in previous study [15]. MICs for LVFX and mutation profiles in QRDR of these FQ-susceptible E. coli strains are summarized in Table 1. 2.2. LVFX-resistant strain development FQ-susceptible E. coli strains were inoculated onto LB agar plate containing 0.5, 1, 2, 4 and 8 mg/ml LVFX (DAIICHI SANKYO PROPHARMA), and incubated at 37 C for 7 days (first selection). Lowresistant isolates obtained in first selection were inoculated onto LB agar plate containing 4, 8, 16, 32 and 64 mg/ml LVFX and incubated
Table 1 MICs for LVFX and mutation profiles in QRDR of FQs-susceptible E. coli strains used in this study. MIC for Mutation profiles LVFX in QRDR (mg/ml) 0.016 0.03 0.06 0.13
0.25 0.5 1 Total
No. of Phylogenic parent A B1 B2 D strain
None 9 gyrA: S83L 1 None 25 parE: D475E 1 None 34 parE: D475E 1 None 2 gyrA: S83L 2 gyrA: D87G 2 gyrA: D87G, parE: D475E 1 None 1 gyrA: S83L, parE: D475E 1 None 1 gyrA: S83L 6 None 1 gyrA: S83L 1 89
1 0 1 0 2 0 0 0 0 0 1 0 0 0 0 0 5
0 0 2 0 0 0 1 1 0 0 0 0 0 0 0 0 4
7 1 1 0 20 2 0 1 30 2 0 1 1 0 1 0 2 0 0 1 0 0 0 1 1 0 5 1 0 1 1 0 69 11
No. of resistant strains developed 0 0 0 0 0 0 0 0 0 0 0 1 0 3 1 0 5
at 37 C for 7 days (second selection). Further, intermediateresistant isolates obtained in second selection were inoculated onto LB agar plate containing LVFX of up to 128 mg/ml. All the developed strains were maintained on the agar containing LVFX at the same concentration with MICs of each strain until further analysis. 2.3. Sequence analysis of QRDR Sequences of QRDRs of gyrA, gyrB, parC and parE in strains that developed LVFX-resistance were analyzed as described previously [15]. 2.4. Quantitative analysis of mRNA expression of efflux pumps and their regulators From the overnight cultures of E. coli strains in liquid LB medium containing LVFX at the same concentration with MICs of each strain, the total RNA were isolated using RNeasy Mini Kit (QIAGEN) according to the manufacture's instructions. Quality of isolated total RNA were analyzed by electrophoresis in 1.3% NuSieve3:1 Agarose (Lonza)/1 TAE gel following visualization by LAS-1000 (Fujifilm). The samples for electrophoresis were prepared by using RNA loading buffer AGþ (BioDynamics Laboratory Inc.). The cDNA were prepared from the obtained total RNA by using PrimeScript RT reagent Kit (Perfect Real Time) (Takara Bio, Inc.) according to the manufacture's instructions. The expression level of mRNA for components of efflux pumps (yhiU, yhiV, mdfA, acrB and tolC), transcription regulators of efflux pumps (marA and sidA) and a housekeeping gene, gapA (as an internal control) were analyzed by quantitative real-time PCR using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio, Inc.) in 7500 Real-Time PCR System (Applied Biosystems). The gene-specific primers for yhiU, yhiV, mdfA, marA and gapA were designed as described by Yasufuku et al. [16] and the gene-specific primers for acrB, tolC and sidA were designed as described by Tavio et al. [11]. The PCR reaction mixture for gapA, yhiU and yhiV were subjected to the following PCR program: 30 s at 95 C, and then 40 cycles of denaturation at 95 C for 5 s and annealing/extension at 62 C for 35 s. The PCR reaction mixture for marA, mdfA, sidA, acrB and tolC were subjected to the following PCR program: 30 s at 95 C, and then 40 cycles of denaturation at 95 C for 5 s, annealing at 55 C for 30 s and extension at 72 C for 35 s. The specificities of PCR
Please cite this article in press as: Yamasaki E, et al., Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.007
E. Yamasaki et al. / J Infect Chemother xxx (2014) 1e5
reactions were confirmed by subjecting the PCR products to electrophoresis in 3.0% NuSieve3:1 Agarose/1 TAE gel. The relative expression levels of each gene were normalized by dividing them by expression of gapA in the same cell, and the changes of expression levels of each genes were defined as the fold increase compared with the control strain E. coli ATCC 25922. 3. Results 3.1. Induction of LVFX-resistant isolates It was previously reported that sequential escalation of exposure dose to antimicrobial drugs encouraged the development of high-level drug-resistant strain [7]. In this study, we employed the discontinuous cultivation by using two different series of LB agar containing serially diluted LVFX. Total 89 E. coli strains that had MICs for LVFX between 0.016 and 1.00 mg/ml were used as the parent strains (Table 1). In the first selection, all parent strains were inoculated onto the LB agar containing 0.5, 1, 2, 4 and 8 mg/ml of LVFX. After an incubation period of 7 days at 37 C, we observed 5 isolates growing on agars containing 2 mg/ml of LVFX. These lowresistant isolates were derived from 5 different parent strains (2 in phylogenic group B2 and 3 in D) that originally indicated relatively high MICs (0.25e1.00 mg/ml) (Table 1). Next, to develop higher resistant strains, the developed 5 low-resistant isolates were inoculated onto LB agar plate containing 4e64 mg/ml LVFX, and incubated at 37 C for 7 days. All 5 E. coli isolates grew on the LB agar containing 4e32 mg/ml LVFX (2 isolates on 4 mg/ml, 2 isolates on 8 mg/ml, and 1 isolate on 32 mg/ml). And then, obtained intermediate-resistant strains were further subjected to incubation onto LB agar plate containing LVFX of up to 128 mg/ml, resulting in development of two high-resistant isolates (one isolate each in phylogenic groups B2 and D) growing on agars containing 128 mg/ ml LVFX (Table 2).
3
isolates with MICs for ciprofloxacin of 4 mg/ml possess mutation at more than 3 sites within QRDRs [15]. In the parent strains used in this study, no isolates have mutations at more than 2 sites within QRDRs (Table 1). When the sequences of QRDRs in developed resistant isolates were examined, additional mutation was not found in all low-resistant isolates (>2 mg/ml) developed in the first selection as well as in 4 out of 5 intermediate-resistant isolates (>4 or 8 mg/ml) developed in the second selection whereas strain GFCS1, which grew on agar containing 32 mg/ml of LVFX, acquired an additional mutation in gyrA in the second selection (Table 2). Further, another additional mutation was found in gyrB, parC or parE in 2 high-resistant isolates (>128 mg/ml) (Table 2). These results were reasonably consistent with previous reports suggested the importance of multiple mutations in QRDRs to acquire highlevel resistance. 3.3. mRNA expression of efflux pumps and their regulators in lowto high-resistant isolates It has previously been reported that, even in the absence of mutation in QRDR, low-level elevation in MIC could be achieved by overexpression of efflux pumps [7]. When mRNA expressions of genes encoding efflux pumps and their transcriptional regulator in developed resistant isolates were examined, expression of yhiU, yhiV, mdfA, tolC and sdiA showed slight increase in the low- to highresistant isolates compared with their parent strains, however, the expression levels were still similar or lower than those of the control strain 25922 (Fig. 1). On the other hand, moderately high expression of acrB in almost intermediate- and high-resistant isolates were observed compared with their parents as well as the control strain 25922. It is noteworthy that remarkable increase in expression of marA was observed in low-resistant isolates and it was increased along with the bacteria obtained higher LVFXresistance.
3.2. Additional mutations in QRDR of LVFX-resistant isolates
4. Discussion
We previously reported that single point mutation in QRDR was not sufficient to develop resistance to FQs and that clinical E. coli
There are several surveillance or retrospective studies with clinical isolates, and these studies revealed the characteristic features observed in drug-resistant strains in addition to epidemics caused by them. Whereas, there are not many in vitro studies that can provide the information about the process or early stage of resistance development. In this study, we did in vitro study to analyze the early stage of FQs resistance-acquiring process. To induce LVFX-resistance in the initial setting, we elevated the LVFX concentrations from 0.5 mg/ml corresponding to the range in the urine of LVFX-administrated patient [17]. This condition is also consistent with the previous report that moderate improvement of drug-resistance (2e8-fold) was frequent incident whereas significant improvement (>64-fold) was occasional [7], as observed that LVFX-resistant strains were developed from the parent strains with relatively high MIC (0.25e1.0 mg/ml) in this study. These results strongly support the alert that administration of insufficient dose of FQs facilitates the emergence of FQs-resistant strains. There is a consideration that use of FQs at the lower dose may contribute to the more frequent isolation of resistant strains [18]. Efflux pump(s) overexpression and mutation in QRDR are the main molecular mechanisms involved in FQs-resistant strain development. It has been previously reported that these two events occur independently [6]. Changes in the expression of efflux pumps are more rapid response than mutations in QRDR, although efflux pumps overexpression confer only low-level (usually 2- to 8-fold) increase of resistance [7,19]. In the present study, consistent with the previous reports, we observed increase of marA expression in all low- to intermediate-resistant isolates,
Table 2 Profiles of developed resistant isolates. Parents LVFX Growth conc. Mutation profiles in QRDR resistant of LVFX Strain Phylogenic MIC for levela (mg/ml) LVFX (mg/ml) UUS5
B2
0.5
UCS14 D
0.25
GCS8
D
1
GUC9
B2
0.5
GFCS1 D
0.5
S L I S L I S L I S L I H
0 2 4 0 2 4 0 2 8 0 2 8 128
S L I H
0 2 32 128
gyrA: S83L gyrA: S83L gyrA: S83L gyrA: S83L, parE: D475E gyrA: S83L, parE: D475E gyrA: S83L, parE: D475E None None None gyrA: S83L gyrA: S83L gyrA: S83L gyrA: S83L, gyrB: E466D, parE: V466E gyrA: S83L gyrA: S83L gyrA: S83L/D87G gyrA: S83L/D87G, parC: S80I
a S: susceptible (<1 mg/ml), L: low-resistant (>2 mg/ml), I: intermediate-resistant (>4e32 mg/ml), H: high-resistant (>128 mg/ml).
Please cite this article in press as: Yamasaki E, et al., Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.007
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E. Yamasaki et al. / J Infect Chemother xxx (2014) 1e5
and high expression of marA was noted even after bacteria obtained high LVFX-resistance as >128 mg/ml with acquisition of QRDR mutations. In this regard, Yasufuku et al. have reported that the increased expression of marA in clinical isolates showed a
Fig. 1. Comparison of the mRNA expression level of efflux pumps and their regulators. The mRNA levels of indicated genes in susceptible parent strains (white bar), low-level LVFX-resistant (light gray), intermediate-resistant (dark gray), and high-resistant (black) isolates developed by first, second, and final selections were indicated as fold increase compared with the control strain E. coli ATCC 25922. The data were representatives of two or three independent experiments.
significant correlation with MICs for FQs, which strongly supports the results investigated in vitro in this study [16]. On the other hand, other efflux pump component and regulator such as mdfA and sdiA which were indicated to be correlated with FQs resistance was not elevated in this study [11,16]. In previous reports, it has been revealed that, while MarA regulates expressions of multiple genes including acrA/B and tolC, expression of marA is intricately regulated by multiple elements including MarR, SoxS, Rob and MarA [12,20]. Future studies would be required to understand the genetic event and mechanisms underlying in the early stage of development of LVFX-resistance associated with remarkable increase of marA expression. In the previous study, it was reported that the strains with single or double point mutation in QRDRs indicated no-resistance [15]. In the present study, marA expression conferred only low- to intermediate-resistance even if the strains had single or double point mutations. This data indicated that, consistent with the previous report [7,15], marA expression was not sufficient to develop high-level resistance and it required additional or more than 3 point mutations in QRDR. With the accumulation of evidences that indicate the importance of efflux pumps in drug-resistant strains, efflux pumps become a promising target in the regulation of drug-resistant bacteria [21,22]. It has been demonstrated that efflux pump inhibitors synergistically increase the activity of antimicrobial drugs. In addition to the significance of efflux pumps as a molecular target in the fight against drug-resistant stains, our data suggest the possible beneficial effect of them as an early marker of drugresistant strain development. As shown in Fig. 1, especially in marA, significant increase of gene expression could be observed. The increase of marA expression seemed to be an early-stage detection marker for FQs-resistance development because even in the low-resistant isolates with MIC >2.0 mg/ml LVFX, which regard as “intermediate resistant” strain according to Clinical Laboratory and Standards Institute (CLSI) susceptibility breakpoints categorization, expression of marA is more than 10 times higher than in control strain and other efflux pumps. Utility of marA as a marker for drug-resistant strain is supported by previous data in various studies with clinical isolates. For example, although the status before high-resistant strain establishment was unclear, it had been indicated that the expression of marA in drug-resistant strain showed a significant correlation with MICs for FQs whereas no correlation between other efflux pumps and MICs were observed in clinical isolates [6,16], and Keeney et al. had indicated that elevation of marA expression in drug-resistant strains were much higher than other regulators and efflux pumps expressions [23]. From these results, we believe that monitoring of marA expression helps early recognition of drug-resistant strain development. In addition, it may helps to know approximate time to administration of efflux pump inhibitors that are expected to potentiate the antimicrobial activity of FQs and contribute to prevention of the emergence of high-resistant strains. Future monitoring study of clinical isolates throughout the duration of antibiotic therapy is required to verify the effectiveness of marA as an early marker of drug-resistant strain emergence. In this study, we indicated that, throughout FQs-resistant strain development, the high expression of marA drove the induction of FQ-resistance, and could be a sufficient marker for detection of it. We presume that it would be important for effective control of FQsresistant strain emergence to detect the early stage of FQsresistance development before development of irreversible highlevel resistance conferred by mutation in QRDR. In addition, it is presumed that suppression or inhibition of the efflux pumps exert a positive effect on antibiotic activity of FQs in early stage of FQsresistance development as well as late stage of it.
Please cite this article in press as: Yamasaki E, et al., Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.007
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Competing interests The authors declare that there is no conflict of interests regarding the publication of this article.
Acknowledgment This study was supported in part by JSPS KAKENHI Grant Number 22591803 and 26462455.
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Original article
Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli Eiki Yamasaki a, *, Chihiro Yamada b, Xinghua Jin b, G. Balakrish Nair c, Hisao Kurazono a, Shingo Yamamoto b a b c
Division of Food Hygiene, Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan The Department of Urology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan Translational Health Science and Technology Institute, Gurgaon, Haryana, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 June 2014 Received in revised form 8 October 2014 Accepted 10 October 2014 Available online xxx
Background: Analyses of efflux pumps overexpression and mutations in quinolone resistance determining region (QRDR) in early stage of development of resistance to fluoroquinolones (FQs) are valuable to discuss countermeasures against them. We induced levofloxacin (LVFX)-resistant strains from susceptible uropathogenic Escherichia coli in vitro to analyze the mechanisms of development of FQsresistance. Methods: 89 strains were exposed to discontinuous elevation of LVFX dose, and mRNA level of efflux pumps and their regulators as well as mutations developed in QRDR of LVFX-resistant strains were analyzed. Results: In 5 strains, a stepwise increase in MIC to LVFX (up to >128 mg/ml)was observed. Compared to the parent strains, additional mutations in QRDR were observed in the strains developing high MIC. Remarkable increase of marA expression was observed even in the early stage of LVFX-resistance development, and it lasted until high-level resistance was developed. On the other hand, moderate increase in acrB expression but only low increase in yhiU, yhiV, mdfA, tolC and sdiA were observed. Conclusions: These results suggested that marA expression is a sensitive marker for early detection of development of LVFX-resistance. © 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Fluoroquinolones-resistant Escherichia coli Quinolone resistant determining region Efflux pump Urinary tract infection
1. Introduction Since Escherichia coli (E. coli) is the bacteria most commonly isolated from patient with urinary tract infection (UTI), the emergence of E. coli isolates that are resistant to some of the most commonly used antimicrobial drugs has become a major problem in the management of UTI [1e3]. Fluoroquinolones (FQs) are the most widely used antibiotics worldwide, and are the drugs of choice for empirical therapy for UTI. Increasing emergence of FQsresistant E. coli has been reported worldwide [3e5]. It has been reported that >10% of E. coli isolated from uncomplicated UTI and 30e40 % of E. coli isolated from complicated UTI were resistant to
* Corresponding author. Nishi 2-11, Inada-cho, Obihiro City, Hokkaido, 080-8555 Japan. Tel./fax: +81 155 49 5390. E-mail address: [email protected] (E. Yamasaki).
FQs [1,2]. It is not hard to understand that these high frequencies of FQs-resistant strains affect therapeutic strategy for UTI. FQs can directly bind to DNA gyrase and topoisomerase IV, and inhibit the activity of these enzymes that are essential for DNA replication and transcription. The mechanism of resistance to FQs mainly consists of two separate strategies: a decrease in drug accumulation in the bacterial cells through an induction of efflux pumps overexpression and mutations in the genes encoding DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Previous reports demonstrated that multi-step process is responsible for the development of high-level resistance to FQs [6]. In the early step, low-level resistance is developed by overexpression of efflux-pumps which facilitate active extraction of FQs [7]. Five superfamilies of efflux pumps (RND (resistance-nodulation-division), MF (major facilitator), ABC (ATP binding cassette), MATE (multidrug and toxic efflux) and SMR (small multidrug resistance)) that
http://dx.doi.org/10.1016/j.jiac.2014.10.007 1341-321X/© 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yamasaki E, et al., Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.007
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promote resistance to various antimicrobial drugs had been described in E. coli [8,9]. And, AcrA-AcrB-TolC and YhiU-YhiV-TolC pumps belonging to RND superfamily and MdfA-Cmr pump belonging to MS superfamily were reported to be involved in excretion of FQs [9,10]. In addition, it has been reported that overexpression of sdiA and marA, both of them are the positive regulator for AcrA-AcrB-TolC pump expression, were associated with the resistance to FQs in E. coli [11,12]. SdiA is a quorum-sensing regulated transcription factor, and is involved in the regulation of cell division. There is increasing evidence that molecules involved in quorum-sensing participate in development of multidrug resistance [13,14]. Deletion of sdiA led to decreased level of the AcrB expression, which resulted in the decreased level of drug resistance [13]. MarA is the global activator, which modulates the expression of many genes including acrA, acrB and tolC [12]. It is known that expression of marA is affected by the presence of salicylate through the inactivation of transcription repressor MarR, and by the redoxinducible SoxS which is the positive regulator of marA expression. Although these efflux pumps expressions are rapid response observed in early stage of drug-resistant development, it conferred only 2- to 8-fold elevation in MIC [7] while, in the late step, highlevel resistance is developed by the mutation in quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE. Recently, we and others has reported that mutation at Ser83 and Asp87 in gyrA and Ser80 in parC are frequently observed in FQsresistant strains isolated from UTI patient although single point mutation at Ser83 or Asp87 in gyrA was not sufficient to develop resistance to FQs [6,15]. Further, we have also reported that there is a phylogenetic preference among FQs-resistant strains [15]. Phylogenic group B2 was dominant among FQs-resistant strains (49.4%) although the dominancy was lower than that in FQssusceptible strains (77.5%), and phylogenic group D which was second most prevalent phylogenic group among FQs-resistant strains (34.8%) is more observed among FQs-resistant strains than among FQ-susceptible strains (12.4%), suggesting that the acquisition of FQs-resistance might be frequent in the limited strains such as phylogenic group B2 or D rather than other phylogenic groups. The mechanisms of FQs-resistance development have been mostly discussed based on statistical comparison between FQssensitive and FQs-resistant clinical isolates. Whereas the status during resistance development process has been much less investigated [7,11]. In the present study, we induced drug-resistant E. coli strains in vitro by exposure to levofloxacin (LVFX), which is the drug commonly used for treating UTIs, cystitis and pyelonephritis. The expressions of efflux pumps and their transcriptional regulators, and mutation in QRDR in early and late stage of resistance development were analyzed. 2. Materials and methods 2.1. Bacterial strains Eighty nine FQ-susceptible E. coli strains, including 5, 4, 69, and 11 strains in phylogenic groups A, B1, B2, and D, respectively, were isolated from the patient with cystitis as described in previous study [15]. MICs for LVFX and mutation profiles in QRDR of these FQ-susceptible E. coli strains are summarized in Table 1. 2.2. LVFX-resistant strain development FQ-susceptible E. coli strains were inoculated onto LB agar plate containing 0.5, 1, 2, 4 and 8 mg/ml LVFX (DAIICHI SANKYO PROPHARMA), and incubated at 37 C for 7 days (first selection). Lowresistant isolates obtained in first selection were inoculated onto LB agar plate containing 4, 8, 16, 32 and 64 mg/ml LVFX and incubated
Table 1 MICs for LVFX and mutation profiles in QRDR of FQs-susceptible E. coli strains used in this study. MIC for Mutation profiles LVFX in QRDR (mg/ml) 0.016 0.03 0.06 0.13
0.25 0.5 1 Total
No. of Phylogenic parent A B1 B2 D strain
None 9 gyrA: S83L 1 None 25 parE: D475E 1 None 34 parE: D475E 1 None 2 gyrA: S83L 2 gyrA: D87G 2 gyrA: D87G, parE: D475E 1 None 1 gyrA: S83L, parE: D475E 1 None 1 gyrA: S83L 6 None 1 gyrA: S83L 1 89
1 0 1 0 2 0 0 0 0 0 1 0 0 0 0 0 5
0 0 2 0 0 0 1 1 0 0 0 0 0 0 0 0 4
7 1 1 0 20 2 0 1 30 2 0 1 1 0 1 0 2 0 0 1 0 0 0 1 1 0 5 1 0 1 1 0 69 11
No. of resistant strains developed 0 0 0 0 0 0 0 0 0 0 0 1 0 3 1 0 5
at 37 C for 7 days (second selection). Further, intermediateresistant isolates obtained in second selection were inoculated onto LB agar plate containing LVFX of up to 128 mg/ml. All the developed strains were maintained on the agar containing LVFX at the same concentration with MICs of each strain until further analysis. 2.3. Sequence analysis of QRDR Sequences of QRDRs of gyrA, gyrB, parC and parE in strains that developed LVFX-resistance were analyzed as described previously [15]. 2.4. Quantitative analysis of mRNA expression of efflux pumps and their regulators From the overnight cultures of E. coli strains in liquid LB medium containing LVFX at the same concentration with MICs of each strain, the total RNA were isolated using RNeasy Mini Kit (QIAGEN) according to the manufacture's instructions. Quality of isolated total RNA were analyzed by electrophoresis in 1.3% NuSieve3:1 Agarose (Lonza)/1 TAE gel following visualization by LAS-1000 (Fujifilm). The samples for electrophoresis were prepared by using RNA loading buffer AGþ (BioDynamics Laboratory Inc.). The cDNA were prepared from the obtained total RNA by using PrimeScript RT reagent Kit (Perfect Real Time) (Takara Bio, Inc.) according to the manufacture's instructions. The expression level of mRNA for components of efflux pumps (yhiU, yhiV, mdfA, acrB and tolC), transcription regulators of efflux pumps (marA and sidA) and a housekeeping gene, gapA (as an internal control) were analyzed by quantitative real-time PCR using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio, Inc.) in 7500 Real-Time PCR System (Applied Biosystems). The gene-specific primers for yhiU, yhiV, mdfA, marA and gapA were designed as described by Yasufuku et al. [16] and the gene-specific primers for acrB, tolC and sidA were designed as described by Tavio et al. [11]. The PCR reaction mixture for gapA, yhiU and yhiV were subjected to the following PCR program: 30 s at 95 C, and then 40 cycles of denaturation at 95 C for 5 s and annealing/extension at 62 C for 35 s. The PCR reaction mixture for marA, mdfA, sidA, acrB and tolC were subjected to the following PCR program: 30 s at 95 C, and then 40 cycles of denaturation at 95 C for 5 s, annealing at 55 C for 30 s and extension at 72 C for 35 s. The specificities of PCR
Please cite this article in press as: Yamasaki E, et al., Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.007
E. Yamasaki et al. / J Infect Chemother xxx (2014) 1e5
reactions were confirmed by subjecting the PCR products to electrophoresis in 3.0% NuSieve3:1 Agarose/1 TAE gel. The relative expression levels of each gene were normalized by dividing them by expression of gapA in the same cell, and the changes of expression levels of each genes were defined as the fold increase compared with the control strain E. coli ATCC 25922. 3. Results 3.1. Induction of LVFX-resistant isolates It was previously reported that sequential escalation of exposure dose to antimicrobial drugs encouraged the development of high-level drug-resistant strain [7]. In this study, we employed the discontinuous cultivation by using two different series of LB agar containing serially diluted LVFX. Total 89 E. coli strains that had MICs for LVFX between 0.016 and 1.00 mg/ml were used as the parent strains (Table 1). In the first selection, all parent strains were inoculated onto the LB agar containing 0.5, 1, 2, 4 and 8 mg/ml of LVFX. After an incubation period of 7 days at 37 C, we observed 5 isolates growing on agars containing 2 mg/ml of LVFX. These lowresistant isolates were derived from 5 different parent strains (2 in phylogenic group B2 and 3 in D) that originally indicated relatively high MICs (0.25e1.00 mg/ml) (Table 1). Next, to develop higher resistant strains, the developed 5 low-resistant isolates were inoculated onto LB agar plate containing 4e64 mg/ml LVFX, and incubated at 37 C for 7 days. All 5 E. coli isolates grew on the LB agar containing 4e32 mg/ml LVFX (2 isolates on 4 mg/ml, 2 isolates on 8 mg/ml, and 1 isolate on 32 mg/ml). And then, obtained intermediate-resistant strains were further subjected to incubation onto LB agar plate containing LVFX of up to 128 mg/ml, resulting in development of two high-resistant isolates (one isolate each in phylogenic groups B2 and D) growing on agars containing 128 mg/ ml LVFX (Table 2).
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isolates with MICs for ciprofloxacin of 4 mg/ml possess mutation at more than 3 sites within QRDRs [15]. In the parent strains used in this study, no isolates have mutations at more than 2 sites within QRDRs (Table 1). When the sequences of QRDRs in developed resistant isolates were examined, additional mutation was not found in all low-resistant isolates (>2 mg/ml) developed in the first selection as well as in 4 out of 5 intermediate-resistant isolates (>4 or 8 mg/ml) developed in the second selection whereas strain GFCS1, which grew on agar containing 32 mg/ml of LVFX, acquired an additional mutation in gyrA in the second selection (Table 2). Further, another additional mutation was found in gyrB, parC or parE in 2 high-resistant isolates (>128 mg/ml) (Table 2). These results were reasonably consistent with previous reports suggested the importance of multiple mutations in QRDRs to acquire highlevel resistance. 3.3. mRNA expression of efflux pumps and their regulators in lowto high-resistant isolates It has previously been reported that, even in the absence of mutation in QRDR, low-level elevation in MIC could be achieved by overexpression of efflux pumps [7]. When mRNA expressions of genes encoding efflux pumps and their transcriptional regulator in developed resistant isolates were examined, expression of yhiU, yhiV, mdfA, tolC and sdiA showed slight increase in the low- to highresistant isolates compared with their parent strains, however, the expression levels were still similar or lower than those of the control strain 25922 (Fig. 1). On the other hand, moderately high expression of acrB in almost intermediate- and high-resistant isolates were observed compared with their parents as well as the control strain 25922. It is noteworthy that remarkable increase in expression of marA was observed in low-resistant isolates and it was increased along with the bacteria obtained higher LVFXresistance.
3.2. Additional mutations in QRDR of LVFX-resistant isolates
4. Discussion
We previously reported that single point mutation in QRDR was not sufficient to develop resistance to FQs and that clinical E. coli
There are several surveillance or retrospective studies with clinical isolates, and these studies revealed the characteristic features observed in drug-resistant strains in addition to epidemics caused by them. Whereas, there are not many in vitro studies that can provide the information about the process or early stage of resistance development. In this study, we did in vitro study to analyze the early stage of FQs resistance-acquiring process. To induce LVFX-resistance in the initial setting, we elevated the LVFX concentrations from 0.5 mg/ml corresponding to the range in the urine of LVFX-administrated patient [17]. This condition is also consistent with the previous report that moderate improvement of drug-resistance (2e8-fold) was frequent incident whereas significant improvement (>64-fold) was occasional [7], as observed that LVFX-resistant strains were developed from the parent strains with relatively high MIC (0.25e1.0 mg/ml) in this study. These results strongly support the alert that administration of insufficient dose of FQs facilitates the emergence of FQs-resistant strains. There is a consideration that use of FQs at the lower dose may contribute to the more frequent isolation of resistant strains [18]. Efflux pump(s) overexpression and mutation in QRDR are the main molecular mechanisms involved in FQs-resistant strain development. It has been previously reported that these two events occur independently [6]. Changes in the expression of efflux pumps are more rapid response than mutations in QRDR, although efflux pumps overexpression confer only low-level (usually 2- to 8-fold) increase of resistance [7,19]. In the present study, consistent with the previous reports, we observed increase of marA expression in all low- to intermediate-resistant isolates,
Table 2 Profiles of developed resistant isolates. Parents LVFX Growth conc. Mutation profiles in QRDR resistant of LVFX Strain Phylogenic MIC for levela (mg/ml) LVFX (mg/ml) UUS5
B2
0.5
UCS14 D
0.25
GCS8
D
1
GUC9
B2
0.5
GFCS1 D
0.5
S L I S L I S L I S L I H
0 2 4 0 2 4 0 2 8 0 2 8 128
S L I H
0 2 32 128
gyrA: S83L gyrA: S83L gyrA: S83L gyrA: S83L, parE: D475E gyrA: S83L, parE: D475E gyrA: S83L, parE: D475E None None None gyrA: S83L gyrA: S83L gyrA: S83L gyrA: S83L, gyrB: E466D, parE: V466E gyrA: S83L gyrA: S83L gyrA: S83L/D87G gyrA: S83L/D87G, parC: S80I
a S: susceptible (<1 mg/ml), L: low-resistant (>2 mg/ml), I: intermediate-resistant (>4e32 mg/ml), H: high-resistant (>128 mg/ml).
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and high expression of marA was noted even after bacteria obtained high LVFX-resistance as >128 mg/ml with acquisition of QRDR mutations. In this regard, Yasufuku et al. have reported that the increased expression of marA in clinical isolates showed a
Fig. 1. Comparison of the mRNA expression level of efflux pumps and their regulators. The mRNA levels of indicated genes in susceptible parent strains (white bar), low-level LVFX-resistant (light gray), intermediate-resistant (dark gray), and high-resistant (black) isolates developed by first, second, and final selections were indicated as fold increase compared with the control strain E. coli ATCC 25922. The data were representatives of two or three independent experiments.
significant correlation with MICs for FQs, which strongly supports the results investigated in vitro in this study [16]. On the other hand, other efflux pump component and regulator such as mdfA and sdiA which were indicated to be correlated with FQs resistance was not elevated in this study [11,16]. In previous reports, it has been revealed that, while MarA regulates expressions of multiple genes including acrA/B and tolC, expression of marA is intricately regulated by multiple elements including MarR, SoxS, Rob and MarA [12,20]. Future studies would be required to understand the genetic event and mechanisms underlying in the early stage of development of LVFX-resistance associated with remarkable increase of marA expression. In the previous study, it was reported that the strains with single or double point mutation in QRDRs indicated no-resistance [15]. In the present study, marA expression conferred only low- to intermediate-resistance even if the strains had single or double point mutations. This data indicated that, consistent with the previous report [7,15], marA expression was not sufficient to develop high-level resistance and it required additional or more than 3 point mutations in QRDR. With the accumulation of evidences that indicate the importance of efflux pumps in drug-resistant strains, efflux pumps become a promising target in the regulation of drug-resistant bacteria [21,22]. It has been demonstrated that efflux pump inhibitors synergistically increase the activity of antimicrobial drugs. In addition to the significance of efflux pumps as a molecular target in the fight against drug-resistant stains, our data suggest the possible beneficial effect of them as an early marker of drugresistant strain development. As shown in Fig. 1, especially in marA, significant increase of gene expression could be observed. The increase of marA expression seemed to be an early-stage detection marker for FQs-resistance development because even in the low-resistant isolates with MIC >2.0 mg/ml LVFX, which regard as “intermediate resistant” strain according to Clinical Laboratory and Standards Institute (CLSI) susceptibility breakpoints categorization, expression of marA is more than 10 times higher than in control strain and other efflux pumps. Utility of marA as a marker for drug-resistant strain is supported by previous data in various studies with clinical isolates. For example, although the status before high-resistant strain establishment was unclear, it had been indicated that the expression of marA in drug-resistant strain showed a significant correlation with MICs for FQs whereas no correlation between other efflux pumps and MICs were observed in clinical isolates [6,16], and Keeney et al. had indicated that elevation of marA expression in drug-resistant strains were much higher than other regulators and efflux pumps expressions [23]. From these results, we believe that monitoring of marA expression helps early recognition of drug-resistant strain development. In addition, it may helps to know approximate time to administration of efflux pump inhibitors that are expected to potentiate the antimicrobial activity of FQs and contribute to prevention of the emergence of high-resistant strains. Future monitoring study of clinical isolates throughout the duration of antibiotic therapy is required to verify the effectiveness of marA as an early marker of drug-resistant strain emergence. In this study, we indicated that, throughout FQs-resistant strain development, the high expression of marA drove the induction of FQ-resistance, and could be a sufficient marker for detection of it. We presume that it would be important for effective control of FQsresistant strain emergence to detect the early stage of FQsresistance development before development of irreversible highlevel resistance conferred by mutation in QRDR. In addition, it is presumed that suppression or inhibition of the efflux pumps exert a positive effect on antibiotic activity of FQs in early stage of FQsresistance development as well as late stage of it.
Please cite this article in press as: Yamasaki E, et al., Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.007
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Competing interests The authors declare that there is no conflict of interests regarding the publication of this article.
Acknowledgment This study was supported in part by JSPS KAKENHI Grant Number 22591803 and 26462455.
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Please cite this article in press as: Yamasaki E, et al., Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.007