Mutant prevention concentrations of ciprofloxacin for urinary tract infection isolates of Escherichia coli

Linda L. Marcusson1, Sara K. Olofsson2, Patricia Komp Lindgren1, Otto Cars2 and Diarmaid Hughes1,*

1 Department of Cell and Molecular Biology, Box 596, Biomedical Center, Uppsala University, S-751 24, Uppsala, Sweden; 2 Department of Medical Sciences, Clinical Bacteriology, Box 552, Uppsala University, S-751 22, Uppsala, Sweden


* Corresponding author. Email: diarmaid.hughes{at}icm.uu.se

Received 21 December 2004; accepted 23 March 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To measure the mutant prevention concentration (MPC) of ciprofloxacin for a set of urinary tract infection (UTI) Escherichia coli isolates with different levels of susceptibility and determine whether MPC can be predicted from MIC.

Methods: MPC was defined as the lowest ciprofloxacin concentration that prevented the growth of resistant colonies when 1010 bacteria were spread on solid medium and incubated for 96 h at 37°C. MIC was measured by Etest. Bacteria surviving (persisting) at MPC were isolated and quantified from agar plugs taken after 96 h. The genes hipA and hipB were amplified by PCR from persisters and sequenced.

Results: Isolates with MICs above the NCCLS breakpoint for ciprofloxacin resistance (4 mg/L) typically have MPCs greater than 32 mg/L. Isolates with MICs below the breakpoint for ciprofloxacin susceptibility (1 mg/L) have MPCs up to 5 mg/L. MPC/MIC is ~16 for most susceptible isolates but there are several notable exceptions (MPC/MIC > 100). Resistant colonies arising one dilution step below MPC often had MIC > MPC. In every case tested, a proportion of cells survived (persisted), but did not grow into colonies, at MPC, without any increase in MIC.

Conclusions: MPCs were determined for all ciprofloxacin-susceptible isolates. MPC is not accurately predicted from MIC. Colonies selected below MPC frequently have MIC > MPC, suggesting multiple mutations. A small fraction of cells from all strains tested survived for 96 h at MPC, without any associated increase in MIC. These survivors/persisters are not hipAB mutants.

Keywords: MPC , UTI , E. coli , mutant selection window


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A major goal of antimicrobial therapy is to achieve a sufficient drug exposure in relation to MIC, at the site of infection, for optimal efficacy. However, a bacterial infection may contain subpopulations of mutant variants with reduced susceptibility to the antimicrobial agent. Thus, a therapy effective against the major part of the population might select for growth of less susceptible mutants. The mutant prevention concentration (MPC)1 is the concentration of drug that prevents the growth of the least susceptible single-step mutant present in a large bacterial population.14 The antibiotic concentration range between MIC and MPC, the mutant selection window (MSW),3,5 is where single-step mutants will be enriched.2,6 The rationale of the MPC concept is to use antimicrobial concentrations above the MSW to restrict selective enrichment.

The MPC concept has been tested in in vitro static studies,1,715 in in vitro pharmacodynamic studies1621 and in vivo.22,23 These previous studies have examined MPC and the MSW in relation to Streptococcus pneumoniae, Staphylococcus aureus, Mycobacteria spp. and Haemophilus influenzae. Here we examine MPC in relation to the selection of ciprofloxacin resistance among urinary tract infection (UTI) isolates of Escherichia coli. The fluoroquinolone ciprofloxacin is one of the preferred treatment options for UTI.2426 Resistance to fluoroquinolones in E. coli is usually caused by an accumulation of mutations in gyrA, parC, parE and marR.2732 Quinolone resistance can also be caused by qnrA, a gene coding for a protein that protects DNA gyrase from inhibition by ciprofloxacin,33 and found associated with class I integron-like sequences on a transmissible plasmid.3336 We have used as a test panel a set of E. coli UTI isolates with levels of susceptibility to fluoroquinolones that cover the range of MICs from fully susceptible to highly resistant.29 We asked whether MIC was a good predictor of MPC, whether mutants arising within the MSW had MICs below the MPC, and whether any bacteria remained alive at the MPC.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

Thirty-six E. coli UTI isolates with different levels of susceptibility to fluoroquinolones29 were originally obtained from the AB Biodisk Culture Collection (Solna, Sweden). A fluoroquinolone-susceptible clinical UTI isolate, E. coli Nu14,3739 was used as the standard wild-type strain. E. coli ATCC 25922 (American Type Culture Collection), S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains for susceptibility testing.

Media and growth conditions

The liquid and solid media used for bacterial growth were Mueller–Hinton broth and Mueller–Hinton agar, respectively (Difco Becton Dickinson, MD, USA), except for assays of cell survival where Luria agar (LA; LB supplemented with 1.5% agar; Oxoid Ltd, Basingstoke, UK) was used. Strains were grown at 37°C, and liquid cultures were aerated by shaking.

Antibiotics

Ciprofloxacin, a gift from Bayer AG (Wuppertal, Germany), was dissolved in 0.1 M NaOH at concentrations between 0.1 and 5 mg/mL before incorporation into Mueller–Hinton agar to the final concentrations used in the MPC assays.

MIC determinations

The MIC of ciprofloxacin was determined by Etest according to the instructions of the manufacturer (AB Biodisk, Solna, Sweden). Etest was used with Mueller–Hinton agar plates incubated for 16–18 h at 37°C. The performance of the materials and methods was controlled by testing in parallel the recommended National Committee for Clinical Laboratory Standards (NCCLS) quality control reference strains.

MPC determination

For each E. coli UTI isolate, 100 µL of an overnight culture was inoculated into 100 mL of Mueller–Hinton broth and then incubated at 37°C with aeration for ~6 h until an OD540 of ~1.0 was reached (Novaspec II, Pharmacia LKB, Uppsala, Sweden), corresponding to ~109 cells/mL. Aliquots (10 mL) of culture were then centrifuged at 3000 g for 15 min in a Sorvall Instruments RC-3B. The supernatant was discarded and the pellet containing ≥1010 cells was resuspended in the remaining liquid and spread onto an agar plate containing a defined concentration of ciprofloxacin. Each strain was tested at 11 different ciprofloxacin concentrations (concentration step range 1 = 0.1; 0.15; 0.3; 0.5; 1.0; 2.5; 5; 10; 16; 25; 32 mg/L). Plates were incubated at 37°C in closed plastic bags for a total of 96 h and examined every 24 h for the appearance of colonies. Strains which failed to give colonies at any of these concentrations were subsequently tested on nine additional ciprofloxacin concentrations (concentration step range 2 = 0.02; 0.04; 0.06; 0.08; 0.125; 0.175; 0.225; 0.25; 0.275 mg/L), also with incubation for 96 h. MPC was recorded as the lowest antibiotic concentration at which no colonies grew on an agar plate. For each strain, MPC was determined in at least three independent experiments. The variation between experiments was not more than one concentration step.

Assays of cell survival

(i) At 96 h, bacteria were scraped from a large area of the MPC agar plate (the lowest antibiotic concentration that gave no visible colonies), streaked directly onto LA and incubated overnight at 37°C. Plates were examined for growth of colonies, indicating that some cells had survived exposure to the ciprofloxacin MPC. (ii) To quantify survival at MPC, agar plugs were taken (in total 10% of the plate area), resuspended in 0.9% NaCl and spread on LA. Colonies were counted after overnight incubation at 37°C and tested for MIC using Etest.

PCR amplification and DNA sequencing of hipA and hipB

The E. coli K-12 and E. coli CFT073 genome sequences (National Center for Biotechnology Information GenBank, accession numbers NC_000913 and NC_004431, respectively) were used to design the PCR and sequencing primers for hipA and hipB (Table 1). PCR was carried out as described previously.29 PCR product concentrations were quantified using a Nanodrop NO-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA). Products were purified prior to sequencing using the QIAquick PCR purification kit (Qiagen, VWR International AB, Stockholm, Sweden) according to the manufacturer's instructions. DNA sequencing was performed by cycle sequencing at the DNA Sequencing Facility (Rudbeck Laboratory, Uppsala University, Sweden) on an ABI Prism 3700 DNA Analyzer. Each sequencing reaction contained 10 ± 5 ng of PCR product, 1.6 pmol of sequencing primer, 4 µL of Terminator Ready Reaction mix, with water added to bring the total volume to 10 µL.


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Table 1. Primers

 

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 Abstract
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 Materials and methods
 Results
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Ratio of MPC to MIC

Thirty-six E. coli UTI isolates were assayed for ciprofloxacin MIC using Etest. The results (Table 2) were in good agreement with published MICs for these strains.29 Fourteen strains have MICs placing them at or above the NCCLS breakpoint for ciprofloxacin resistance (4 mg/L) whereas the remaining 22 have MICs below the NCCLS breakpoint for susceptibility (1 mg/L). MPC assays were performed on all 36 UTI isolates (Materials and methods) essentially as described by others,10 with the exception that the incubation time allowed for colonies to grow was extended to 96 h. This extension was made because a standard incubation time of 48 h was insufficient to allow the growth of resistant colonies from all clinical isolates in the test panel. Three of the 22 ciprofloxacin-susceptible isolates required 72 or 96 h of incubation before resistant colonies were observed. Three strains (C2, C149 and C88) did not give rise to colonies using concentration step range 1, but did when assayed using concentration step range 2, which has lower and smaller concentration intervals. MPC and MIC values are listed in Table 2.


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Table 2. Ciprofloxacin MPC and MIC values for clinical UTI strains and derived resistant mutants

 
Thirteen of the 14 resistant strains (C1172, C1212, C1199, C1208, C1192, C1200, C1195, C1204, C1197, C1191, C1211, C139, C1180;29 have MPCs greater than 32 mg/L, the highest concentration tested. The remaining resistant strain, C1177, has an MPC of 25 mg/L. Because the MPCs for these 14 resistant strains were several-fold higher than the maximum plasma concentrations of ciprofloxacin that can be achieved with currently approved dosing practices,25,40 they were not analysed further.

The MPCs for the 22 ciprofloxacin-susceptible strains ranged from 0.1 to 5 mg/L (Table 2). There is only a weak correlation between MIC and MPC with a linear regression R2 value of 0.58. There are clear exceptions to the correlation between MIC and MPC. In particular, three strains (C120, C150 and C124) with low MICs (0.016–0.023) have amongst the highest MPCs at 2.5 mg/L, more than 100-fold MIC (Table 2). Excluding these three strains from consideration, the mean value for MPC/MIC for ciprofloxacin-susceptible E. coli is 16.1 (median, 13) with a standard error of 2.3.

MIC for mutants selected in the MSW

Colonies growing on the antibiotic concentration one step below MPC were picked from each of the 22 susceptible strains and tested for MIC using Etest. Two resistant colonies were tested from each strain. The MICs were increased (Table 2) as expected for resistant mutants. However, for more than half the strains, the mutant MIC values were equal to, or greater than, the MPC (Table 2). This result is not expected of single-step mutants.

Survival (persistence) of bacteria at MPC

We assayed strains for the presence of living bacteria after 96 h of incubation at MPC (Table 3). For each strain, we recovered dozens to hundreds of cfu. We quantified the fraction of bacteria surviving at MPC for four strains (C47, C50, C97 and Nu14) in 4–5 independent MPC experiments (see the Materials and methods section). For each strain, cfu were recovered from many different agar plugs, showing that surviving cells were distributed over the plate and did not represent the survival of a single clone. The surviving cfu in all cases retained their original MICs. The frequency of cells surviving 96 h at MPC as cfu was ~10–8 for C47, C50 and Nu14, and ~10–7 for C97 (Table 3).


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Table 3. Bacteria surviving at MPC

 
From each of the four strains, MPC survivors were processed through a second MPC assay. The MPC, the fraction of survivors and the MIC for survivors were again measured. In each case, MPC was unchanged, some cells survived, and the surviving cells retained their original MIC values. In one case, we identified an MPC-survivor, C97-1, with an increased survival relative to its parent strain (Table 3). The frequency of C97 survival is 1.2 x 10–7, and increased 6-fold to 7.3 x 10–7 in C97-1. This is statistically significant (P=0.005, t-test). Improved survival was stable in repeated MPC assays, consistent with a stable genetic change that improves survival at MPC.

Previously, it was reported that a specific double mutation of the hipA gene improves survival (persistence) of E. coli in the presence of ß-lactam and quinolone antibiotics.4143 hipA and the adjacent hipB code for a toxin–antitoxin pair.44 We PCR-amplified and sequenced hipA and hipB from each of the four strains C47, C50, C97 and Nu14, and from derivatives that had survived one or two successive selections at MPC, including C97-1. The only differences found in the hipA and hipB sequences reflect the phylogenetic groups these strains belong to (B1-1: C50 and C97; B2-1: C47 and Nu14; A1: MG1655 K-12), as supported by PCR analysis.45 Among survivors at MPC, including C97-1, there were no new mutations in hipA or hipB (data not shown). Interestingly, the sequence of C50 and C97 in the immediate neighbourhood of the ‘persistence mutation’ in hipA744 (Asp-291->Ala) is CTT CGC GAT for codons 289–291 while in each of the other strains it is CTG AAA GAT (underlining indicates the region that differs between sequences).


    Discussion
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The concept of MPC could be used to make decisions about dosing regimen with respect to the potential for the selection and enrichment of mutants. In particular, it has been suggested that MPC could be useful in telling, for given isolates, whether it is still reasonable to use monotherapy with little chance of resistance emerging or whether one needs to shift to a combination therapy.3 We have shown that all E. coli UTI isolates in our test panel with MICs above the NCCLS breakpoint for ciprofloxacin susceptibility had MPCs of 25 mg/L or greater. For these strains, dosing above MPC during monotherapy would not be possible with approved dosing procedures25,40 and evaluations of drug toxicity.46 In contrast, for 22 isolates with MICs below the breakpoint for ciprofloxacin susceptibility, MPC values were between 0.1 and 5 mg/L. For most of these susceptible strains, it should be possible to dose above MPC using monotherapy with currently approved dosing procedures,25,40 with little chance of selecting resistance. Reducing the MIC breakpoint for susceptibility, as recently decided by EUCAST47 will increase the number of strains for which MPC may be achieved during therapy, but may not solve the problem because, as discussed below, some isolates with low MICs have unusually high MPCs.

An interesting finding is that MIC is not a good predictor of MPC, i.e. the size of the MSW varies greatly between strains. For most isolates, the ratio MPC/MIC is ~16. However, among the most susceptible isolates, four with low MICs of 0.016–0.023 mg/L had unusually high MPCs of 1–2.5 mg/L, giving an MPC/MIC > 100. This shows that the MPC cannot be accurately predicted from MIC and emphasizes the need to make measurements of MPC on clinical isolates if MPC is to be useful as a clinical tool.

A potential consequence of suboptimal therapy that selects for resistance is illustrated by the mutants selected one concentration step below MPC. These mutants frequently had MICs equal to or greater than the MPC for that strain. This implies that they are not the product of single-step mutations. A logical explanation for this is that within the MSW, some mutants that survive grow poorly, and are subject to selection for second-step mutations that enhance growth by increasing the level of resistance. Multi-step selection for colony growth has been well documented in the phenomenon of adaptive mutation.4850 At MPC, in contrast, the growth of first-step mutants is completely inhibited and a multi-step evolution process will be prevented.

We isolated surviving cfu after 96 h of incubation at MPC at a frequency of ~10–8 for C47, C50 and Nu14, and ~10–7 for C97, and also isolated C97-1, a strain with a heritable enhanced survival frequency relative to its parent C97. Survival at MPC was not associated with an increase in MIC, nor was it associated with mutation in the hipA or hipB genes where a mutation that enhances survival in the presence of ß-lactams and fluoroquinolones has previously been identified.4143 These results rule out mutations in hipA or hipB as the cause of enhanced survival at MPC in these isolates. Thus, although some cells survive prolonged exposure to MPC, there were no resistant mutants among the survivors, showing that MPC is working as expected.

In the in vitro assay of MPC described here, constant antibiotic concentrations were used throughout the experiments. As has been pointed out many times,1,3,10,16,1821 to be clinically useful, the MPC cannot be used without consideration of the drug's pharmacokinetic properties, i.e. the critical magnitude of the drug exposure in relation to the MPC needs to be determined.


    Acknowledgements
 
We thank Eva Ferngren for her kind help and expertise in making media for this project. D. H. thanks Bayer AG for the gift of ciprofloxacin. This work was supported by grants from Vetenskapsrådet (The Swedish Science Council) to D. H., from the European Union Fifth Framework Programme project DEAR (QLK2-CT-2001-00873) to O. C. and D. H., and from Bayer AG to O. C.


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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