a Department of Paediatrics b Centre for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178 c Childrens Hospital, San Diego, CA 92123, USA
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Abstract |
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Introduction |
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Moxifloxacin (BAY 12-8039) is an 8-methoxyquinolone with increased activity against Gram-positive bacteria.6,7,8,9,10,11,12 The purpose of this study was to determine (i) the in-vitro activity of moxifloxacin against bacteria with varying levels of resistance to ciprofloxacin; (ii) the potential of moxifloxacin to select mutational resistance; (iii) the degree of cross resistance of mutants selected with various fluoroquinolones; and (iv) the bactericidal activity of moxifloxacin, ciprofloxacin and ofloxacin against selected isolates of Staphylococcus aureus and Streptococcus pneumoniae.
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Materials and methods |
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A total of 279 clinical isolates with varying susceptibility to ciprofloxacin, ß-lactams and aminoglycosides were tested. The isolates were recovered from patients throughout Australia, Czechoslovakia, Hungary, Spain, Sweden, the UK and the USA. Some isolates were obtained from patients treated unsuccessfully with currently available antibiotics. They were not random clinical isolates.
Susceptibility testing
Antimicrobial agents tested were moxifloxacin, ciprofloxacin (Bayer, Inc., West haven, CT, USA), ofloxacin (R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ, USA), imipenem (Merck Sharp & Dohme, West Point, PA, USA), piperacillintazobactam (Wyeth-Ayerst, Pearl River, NY, USA), nalidixic acid, tetracycline, oxacillin, penicillin (Sigma Chemical Co., St Louis, MO, USA), ceftazidime (Glaxo Pharmaceuticals, Research Triangle Park, NC, USA), gentamicin (Schering Corporation, Bloomfield, NJ, USA) and vancomycin (Eli Lilly, Indianapolis, IN, USA), The quinolones were tested against all clinical isolates and mutants. Non-quinolone agents were tested only against parent and mutant strains to determine whether mutational cross resistance emerged to other classes of agents following exposure to quinolones.
Antibiotic susceptibilities were determined by agar dilution methodology using a multipoint inoculator (manufactured by the Physics Machine Shop at Creighton University, Omaha, NE, USA) and an inoculum of 104 cfu/spot on MuellerHinton agar (CM337, Oxoid Ltd., Basingstoke, UK).13 The MuellerHinton agar was supplemented with 4% NaCl for tests of staphylococci with ß-lactams, 5% sheep blood for S. pneumoniae, and Haemophilus Test medium supplementation for Haemophilus influenzae. Cultures were incubated for 1824 h in air, except for S. pneumoniae and H. influenzae which were incubated in 5% CO2. Quality control strains were Escherichia coli ATCC 25922, E. coli ATCC 35218, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, H. influenzae ATCC 49247 and S. pneumoniae ATCC 49619.
The staphylococci were separated into three phenotypic groups on the basis of their
susceptibility to ciprofloxacin in agar dilution tests: susceptible (MIC 0.5 mg/L),
moderately
resistant (14 mg/L) and highly resistant (
8 mg/L).
Mutational frequencies
Mutants were selected from eight representative strains of S. pneumoniae, E. faecalis, Enterococcus faecium, S. aureus, Staphylococcus epidermidis, E. coli, Klebsiella pneumoniae and Moraxella catarrhalis. Parent strains were grown in MuellerHinton broth until mid-logarithmic phase. S. pneumoniae was grown on blood agar overnight and suspended in sterile physiological saline. Inocula of 107109 cfu were added to MuellerHinton agar or MuellerHinton agar with 5% sheep blood (S. pneumoniae) containing quinolones at superinhibitory concentrations of two, four and eight times the MIC. The actual inoculum used was determined by serial dilution plate counts. Plates were incubated for 4872 h at 35°C in air (5% CO2 for S. pneumoniae) and colony counts were performed. Mutational frequencies were calculated from the results obtained for plates containing the highest drug concentration on which colonies were obtained. The procedure was repeated with first step mutants to select second and subsequent step mutants in tests with staphylococci. Antimicrobial susceptibilities of parents and mutants were determined as described above with the non-quinolone agents included to evaluate the development of resistance to other drug classes.
Bactericidal activity
Investigations into bactericidal activity were performed with S. aureus strain 41 (oxacillin susceptible), S. aureus strain 27 (oxacillin resistant) and S. pneumoniae strain 212. Stationary-phase cultures were exposed to each fluoroquinolone at multiples of 0.5 x, 1 x, and 4 x MIC and incubated at 37°C in air or 5% CO2 (S. pneumoniae). Samples were subcultured at intervals of 0, 2, 4, 6 and 24 h for viable counts on MuellerHinton or sheep blood (S. pneumoniae only) agar containing 5 mM FeCl3 to overcome drug carryover.14 Agar dilution MICs were determined for organisms that showed an increase in numbers after initial killing. Viable counts were compared with drug free control cultures. The lowest countable number obtainable with this procedure was 300 cfu/mL.
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Results |
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Moxifloxacin was eight- to 32-fold more potent than ciprofloxacin and ofloxacin against staphylococci and S. pneumoniae, and up to eight-fold more potent against enterococci (Table I). Moxifloxacin was two- to eight-fold more potent against oxacillin-susceptible S. aureus than against oxacillin-resistant S. aureus. Against Enterobacteriaceae, M. catarrhalis and H. influenzae, moxifloxacin was generally up to four-fold less potent than ciprofloxacin, and up to two-fold more potent than ofloxacin. The percentages of isolates susceptible to ciprofloxacin, ofloxacin, nalidixic acid or to 1 mg/L of moxifloxacin are shown in Table I. For all Gram-positive isolates these percentages were 77, 48 and 62% for moxifloxacin, ciprofloxacin and ofloxacin, respectively, while the comparable respective percentages for all Gram-negative isolates were 78, 80 and 76%. Resistance to 2 mg/L of moxifloxacin occurred in S. aureus (nine isolates), coagulase-negative staphylococci (eight isolates), enterococci (four isolates), Serratia marcescens (five isolates), Citrobacter freundii (four isolates), Klebsiella spp. (three isolates), E. coli (two isolates), Enterobacter aerogenes (two isolates) and E. cloacae (one isolate). All S. pneumoniae isolates were inhibited by 0.25 mg/L of moxifloxacin.
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The ability of the quinolones to select less susceptible single-step mutants was determined by exposing eightisolates to superinhibitory concentrations of each agent. Mutants were selected from all eight strains exposed to ciprofloxacin, and from six and five strains exposed to moxifloxacin and ofloxacin, respectively. A total of 32 mutants were selected. Of these, 69% and 63% were susceptible to 1 mg/L of moxifloxacin and ciprofloxacin, respectively, and 31% were susceptible to 2 mg/L of ofloxacin. When only mutants selected at the highest concentration of each quinolone were considered, mutational frequencies ranging from 10-6 to 10-8 were obtained for 17 of the 20 (85%) single-step mutants recovered.
Concurrent decreases in susceptibility to non-quinolone agents were detected in mutants selected from two Gram-negative organisms. Two of five mutants selected from K. pneumoniae strain 130 exhibited 16-fold increases in MIC of imipenem (from 0.12 to 2 mg/L). These mutants were selected with moxifloxacin and ciprofloxacin. Two out of eight mutants selected with ofloxacin from M. catarrhalis BT84 exhibited four-fold increases in the MIC of tetracycline (from 1 to 4 mg/L).
Effect of de-novo quinolone resistance on subsequent development of quinolone resistance
Attempts were made to select successive single-step mutants from a single strain each of S.
aureus and S. epidermidis. Using moxifloxacin and ciprofloxacin as selecting
agents it was not possible to select less susceptible mutants of S. aureus strain 255 with
moxifloxacin. First-, second- and third-step mutants however, were selected with ciprofloxacin,
each at a frequency of 10-7. After three mutational steps, the MICs of
ciprofloxacin and ofloxacin were 128 mg/L, whereas the MIC of moxifloxacin was only 4
mg/L (Table III).
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A first-step mutant of S. pneumoniae strain 212 was selected only with exposure to ciprofloxacin. No subsequent-step mutants were selected with either ofloxacin, moxifloxacin or ciprofloxacin.
Bactericidal activity against S. aureus and S. pneumoniae
Initial inocula were 4.4 x 105 cfu/mL, 5.5 x 104
cfu/mL and 5.6 x 105 cfu/mL for S. aureus strain 41
(oxacillin-susceptible), S. aureus strain 27 (oxacillin-resistant) and S. pneumoniae
strain 212, respectively. All quinolones tested were bactericidal ( 3 log10
decrease in viable count) against each strain at concentrations four-fold above the MIC, as shown
in Figures 1, 2 and 3.
Ofloxacin was also bactericidal at its MIC against S. aureus strain
41. Less susceptible mutants of S. aureus strain 41 were selected at the MIC and half the
MIC of ciprofloxacin, and at half the MIC of moxifloxacin. Less susceptible mutants of S.
aureus strain 27 were selected at the MIC of each agent. The concentrations of each drug to
inhibit all staphylococcal mutants were 0.25 mg/L of moxifloxacin and 2 mg/L of ciprofloxacin
and ofloxacin. No mutational decreases in susceptibility were detected in tests with S.
pneumoniae strain 212.
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Discussion |
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The reason for moxifloxacin being less affected than ciprofloxacin by mechanisms responsible for increasing quinolone resistance in staphylococci were not investigated. However, this finding was consistent with other reports in which ciprofloxacin was noted to be more affected than some other quinolones by quinolone resistance mechanisms in staphylococci and in other types of bacteria.5,14,1516 This feature of moxifloxacin, coupled with its enhanced potency against a variety of Gram-positive pathogens, suggests that it should be a suitable candidate for clinical evaluation.
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Acknowledgments |
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Notes |
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References |
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Received 5 February 1999; returned 21 April 1999; revised 28 May 1999; accepted 28 June 1999