JAC Advance Access originally published online on December 20, 2005
Journal of Antimicrobial Chemotherapy 2006 57(2):364-365; doi:10.1093/jac/dki465
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Correspondence |
Evaluation of six fluoroquinolones for their capabilities in restricting the selection of resistant salmonellae
Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China
* Corresponding author. Tel: +852-2632-3333; Fax: +852-2647-3227; E-mail: meilunling{at}cuhk.edu.hk
Keywords: mutant prevention concentration , Salmonella Typhimurium , Salmonella Hadar , gemifloxacin , moxifloxacin
Sir,
Mutant prevent concentration (MPC), a recently developed concept,1 is defined as the minimum concentration of an antibiotic that can inhibit growth of mutants from amongst 1010 colonies. MPC/MIC ratio is an index to predict the size of the mutant selection window (MSW) within which susceptible bacterial cells are inhibited but resistant mutants multiply and grow rapidly.1 The narrower the MSW of a drug, the greater is its potential to restrict selection of resistant mutants.
Fluoroquinolones are now the drugs of choice for treatment of typhoid fever and invasive infections caused by the gastroenteric group of salmonellae.2 However, strains with decreased susceptibility which do not respond to fluoroquinolone treatment as well as having high-level resistance to the fluoroquinolones have increasingly been reported.2
In this study, we aimed to investigate the capabilities of six fluoroquinolones to select resistant mutants from Salmonella enterica serotypes Typhimurium and Hadar by determining the MPCs.
The susceptibility to nalidixic acid and six fluoroquinolones (Table 1) of six clinical isolates and one standard strain (ATCC 13311) of Salmonella Typhimurium and eight clinical isolates of Salmonella Hadar was tested by determining the MIC using the agar dilution method according to recommendations of the National Committee for Clinical Laboratory Standards [NCCLS, now known as the Clinical and Laboratory Standards Institute (CLSI)].3 Clonal relationship of the strains was checked by pulsed-field gel electrophoresis (PFGE) of XbaI-restricted DNA.
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To determine the MPCs, a single overnight colony on Mueller–Hinton agar (MHA) was inoculated in 500 mL of nutrient broth (NB), incubated at 37°C with shaking for
5 h and then adjusted to a turbidity matching that of a 1 McFarland standard. The cells were then harvested by centrifuging at 12 000 g for 30 min at 4°C and resuspending in 3 mL of NB. About 200 µL of this bacterial suspension (containing >1010 cfu) was spread onto MHA plates containing one of the six fluoroquinolones at serial 2-fold concentrations starting from 0.5x MIC. After 48 h of incubation at 37°C, colonies growing on the fluoroquinolone-containing plate were checked for resistance and identity by subculturing on MacConkey agar containing the same fluoroquinolone at the MIC followed by identification by standard microbiological procedures. The parent was used as a susceptible control on the fluoroquinolone-containing MacConkey plates. MPC was taken as the lowest concentration that inhibited all bacterial growth. The value of Cmax/MPC was calculated, where Cmax was the maximum peak serum concentration achievable 1–3 h after an oral dose of a fluoroquinolone.4,5 The MICs of the six fluoroquinolones for the strains tested (shown to be clonally unrelated by PFGE) are shown in Table 1. All were highly susceptible to the six fluoroquinolones tested with MICs as much as 64-fold lower than the susceptible breakpoints recommended by the CLSI6 or the BSAC.4 However, when the strains were subcultured in the presence of a fluoroquinolone, resistant strains developed at 1x–32x MIC at a frequency ranging from 10–11 to 10–6. Gemifloxacin could select resistant strains at the highest frequencies (10–10 – 10–6), indicating that both Salmonella Typhimurium and Salmonella Hadar could develop fluoroquinolone resistance more readily in the presence of gemifloxacin than any of the other fluoroquinolones.
The MPCs were compared with corresponding MIC and Cmax values (Table 1). In general, results for the standard strain ATCC 13311 were similar to those for clinical isolates of Salmonella Typhimurium except that the MICs and MPCs were 2- to 4-fold lower. The MPC/MIC value of moxifloxacin was the lowest for both Salmonella Typhimurium and Salmonella Hadar, i.e. the MSW was the narrowest, and that of gemifloxacin was the highest, indicating that moxifloxacin could efficiently inhibit the growth of resistant mutants. The Cmax/MPC of both gemifloxacin and norfloxacin was the lowest while that of ciprofloxacin and levofloxacin was the highest. The MPC of a drug must be below the Cmax and tissue concentration to effectively restrict growth of resistant mutants clinically. Since the fluoroquinolone concentration in serum is usually lower than that in tissues, an MPC that is lower than Cmax should also be lower than the concentration achievable in tissues. This is especially important for intracellular organisms such as salmonellae.
Our studies showed that ciprofloxacin was active against salmonellae and was most capable amongst the fluoroquinolones tested to restrict development of resistant strains because of its low MICs and MPCs but high Cmax/MPC ratio. In contrast, gemifloxacin had the widest MSW and the lowest Cmax/MPC ratio, indicating that this drug could readily select for resistance. More strains and different Salmonella serotypes should be tested to confirm such findings. Whether these in vitro studies can be reproduced in vivo requires further investigation.
Transparency declarations
None to declare.
Footnotes
Present address. Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China 
Acknowledgements
This study forms part of the Master of Philosophy thesis submitted to The Chinese University of Hong Kong. Y. J. was a recipient of a postgraduate studentship from The Chinese University of Hong Kong.
References
1 Zhao X, Drlica K. Restricting the selection of antibiotic-resistant mutants: a general strategy derived from fluoroquinolone studies. Clin Infect Dis 2001; 3 Suppl 3: 147–56.
2 Wain J, Hoa NT, Chinh NT et al. Quinolone-resistant Salmonella typhi in Viet Nam: molecular basis of resistance and clinical response to treatment. Clin Infect Dis 1997; 25: 1404–10.[Web of Science][Medline]
3 National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically—Sixth Edition: Approved Standards M7-A6. NCCLS, Wayne, PA, USA, 2003.
4
Andrews JM. BSAC standardized disk susceptibility testing method (version 3). J Antimicrob Chemother 2004; 53: 713–28.
5 Amsden GW, Schentag JJ. Tables of antimicrobial agent pharmacology. In: Mandell GL, Bennett JE, Dolin R, eds. Principles and Practice of Infectious Diseases. Volume 1. Fourth edition. New York: Churchill Livingstone, 1995; 492–528.
6 Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: Fifteenth Informational Supplement M100-S15. CLSI, Wayne, PA, USA, 2005.
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