In vitro and in vivo antipseudomonal activity, acute toxicity, and mode of action of a newly synthesized fluoroquinolonyl ampicillin derivative WEN-PO LIN, DAR-DER JI, CHIA-YANG SHIAU, TSE-CHUN YANG, YUNG-WEN YANG, TAI-LI TSOU, SHANG-TAO TANG, CHI-HSING CHEN, and YU-TIEN LIU TAIPEI, TAIWAN, REPUBLIC OF CHINA
Compounds N-(6,7-difluoroquinolonyl)-ampicillin (AU-1) and N-(6-fluoroquinolonyl)-ampicillin (FQ-1), synthesized by coupling of the carboxyl group of 6,7-difluoroquinolone (FP-3) and 6-fluoroquinolone (FP4), respectively, with the ␣-aminogroup of ampicillin side chain, exhibit antipseudomonal activity similar to and lower acute toxicity than that of norfloxacin, whereas neither ampicillin nor the fluoroquinolone moieties, compound FP-3 or FP4, alone have such activity. Also, AU-1 and FQ-1 are active against tested clinical isolates of Pseudomonas aeruginosa that are highly resistant to norfloxacin, gentamicin, or both. The therapeutic efficacies of FQ-1 and norfloxacin were assessed and compared in neutropenic mice infected with a 90% lethal dose of P aeruginosa. Mice intraperitoneally administered FQ-1 (10 mg/kg) 4, 8, 24, and 48 hours after infection had survival rates as high as 80%, comparable to those of mice treated with norfloxacin at the same dosage and dosing schedule. The study of protoplast formation revealed that FQ-1 did not inhibit cell-wall biosynthesis but did induce cell filamentation of Bacillus subtilis at a level close to its minimal inhibition concentration. Both AU-1 and FQ-1 were able to intercalate into the double-stranded DNA. However, that FQ-1 lost such activity after it was treated with penicillinase suggests that the lactam-ring structure in ampicillin moiety of FQ-1 was hydrolyzed by penicillinase and that the hydrolyzed structure of FQ-1 does not own DNA-intercalation activity. (J Lab Clin Med 2003;142:158-65) Abbreviations: AU-1 ⫽ N-(6,7-difluoroquinolonyl)-ampicillin; ccc DNA ⫽ covalently closed circular DNA; CFU ⫽ colony-forming unit; DMSO ⫽ dimethylsulfoxide; FP-3 ⫽ 6,7-difluoroquinolonic acid; FP-4 ⫽ 6-fluoroquinolonic acid; FQ-1 ⫽ N-(6-fluoroquinolonyl)-ampicillin; LD50 ⫽ lethal dose for 50% of population; LD90 ⫽ lethal dose for 90% of population; MIC ⫽ minimal inhibition concentration; PBPs ⫽ penicillin-binding proteins; PBS ⫽ phosphate-buffered saline solution; TSGH ⫽ Tri-Service General Hospital.
seudomonas aeruginosa is a major problem as a multidrug-resistant nosocomial pathogen; especially in burn patients and other immunocompromised subjects in hospitals,1 and most infections
caused by this bacterium are associated with high rates of morbidity and mortality.2-4 Ampicillin and fluoroquinolones are antibacterial agents with potent action against a broad spectrum of
From the Institutes of Microbiology and Immunology, Preventive Medicine, and Medical Science, and the Section of Bacteriology, Division of Clinical Pathology, Tri-Service General Hospital, National Defense Medical Center. Supported by grants NSC 88-2314-B-016-051, DOD-89-16, and NSC 89-2320-B-016 from the National Science Council, ROC, and the Department of National Defense, ROC. Submitted for publication July 30, 2002; accepted May 21, 2003.
Reprint requests: Yu-Tien Liu, PhD, Institute of Microbiology and Immunology, National Defense Medical Center, PO Box 90048-505, Neihu, Taipei 114, Taiwan, Republic of China; e-mail: [email protected]
Copyright © 2003 by Mosby, Inc. All rights reserved. 0022-2143/2003/$30.00 ⫹ 0 doi:10.1016/S0022-2143(03)00112-4
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Fig 1. Structure of compounds AU-1, FQ-1, FP-3, and FP-4 and ampicillin, as well as the process for synthesizing AU-1 and FQ-1.
bacterial species, of which fluoroquinolone derivatives (eg, norfloxacin and ciprofloxacin) have been popular prescriptions for the treatment of bacterial infection– caused diseases. However, the intensive use of these antibiotics has led to the emergence of multidrug-resistant strains of P aeruginosa.5-12 It has therefore become important to develop new antibiotics for the treatment of multidrug-resistant P aeruginosa infections. Compounds consisting of a cephalosporin covalently linked at the 3'-position to a quinolone by way of an ester, carbamate, or tertiary amine bond have been developed.13-16 These compounds have a dual mechanism of action, reflecting characteristics of both the ␤-lactam and quinolone components by binding to penicillinbinding proteins and inhibiting DNA gyrase.13-15 In recent years, our group has taken a different approach by synthesizing several new compounds consisting of an ampicillin covalently linked to a fluoroquinolone by way of an amide bond.17,18 These compounds exhibit antibacterial activity against all pyocin types of P aeruginosa strains highly resistant to ampicillin.17,18 However, the in vivo antipseudomonal
activity, toxicity, and mode of action of the newly synthesized fluoroquinolonyl ampicillin–adduct derivatives are still unknown. In this article we report the in vivo antipseudomonal activities, toxicities, and modes of action of AU-1 and FQ-1. METHODS Bacteria and plasmid. P aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 25922, Proteus mirabilis ATCC 7002, and Salmonella typhi ATCC 6539 were obtained from American Type Culture Collection (Manassas, Va). Plasmid pUC18 DNA was purchased from Amersham Pharmacia Biotech Inc (Piscataway, NJ). Clinical isolates of P aeruginosa, Bacillus subtilis, Serratia marcescens, and Klebsiella pneumoniae were obtained from the culture collection of Tri-Service General Hospital. All bacteria were stored in trypticase soy broth (Difco Laboratories, Detroit, Mich) with 15% glycerol (vol/vol) at ⫺70°C. Preparation of fluoroquinolonyl ampicillin. Ampicillin was obtained from Sigma Chemical Co (St Louis, Mo). We synthesized the starting materials, compounds FP-3 and FP-4 (Fig 1), in our laboratory. The methods described by Chen et
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Table I. Antibacterial spectrum of AU-1, FQ-1, FP3, FP4, ampicillin, and norfloxacin MIC (g/mL) Test organism
S aureus ATCC 25923 S pyogenes ATCC 19615 B subtilis TSGH 743 S marcescens TSGH 1105 P mirabilis ATCC 7002 E coli ATCC 25922 S typhi ATCC 6539 K pneumoniae TSGH 902 P aeruginosa ATCC 27853
0.25 0.75 4 0.5 4 1 0.5 16 4
0.25 0.75 4 1 4 4 2 16 4
0.5 1 1 8 1 2 ⬎128 ⬎128 ⬎128
2 ⬍0.06 ⬍0.06 0.13 ⬍0.06 ⬍0.06 0.13 ⬍0.06 4
⬎128 ⬎128 ⬎128 ⬎128 ⬎128 128 ⬎128 ⬎128 ⬎128
⬎128 ⬎128 ⬎128 ⬎128 ⬎128 128 ⬎128 ⬎128 ⬎128
Table II. Antibacterial activities of AU-1 and FQ-1 against clinical isolates of P aeruginosa resistant to norfloxacin, ciprofloxacin, and gentamicin MIC (g/mL) Clinical isolates of P aeruginosa
TSGHa 2002 (sputumb) TSGH 218 (urine) TSGH 1003 (wound discharge) TSGH 217 (urine) TSGH 601 (sputum) TSGH 3008 (urine) TSGH 414 (urine) TSGH 501 (urine) TSGH 6003 (sputum) TSGH 5630 (wound discharge) P aeruginosa ATCC 27853
4 4 8 4 4 8 8 4 8 4 4
4 8 16 4 8 16 8 8 8 4 4
8 32 64 32 4 64 32 64 8 64 4
16 1 ⬎128 1 ⬎128 ⬎128 ⬎128 1 ⬎128 ⬎128 4
al17 and Tsou et al18 were used to prepare fluoroquinolonyl ampicillin adduct derivatives AU-1 and FQ-1. Thus the ␣-amino group of ampicillin side chain was coupled with the carboxyl groups of compounds FP-3 and FP-4 (Fig 1). The final product was purified by means of liquid column chromatography on Diaion HP-20 (Mitshbishi Kasei Co, Tokyo, Japan), eluted with acetone and aqueous methanol.18 After purification, the product was subjected to instrumental analyses previously described,17,18 and only those compounds with purity showing a single spot on the thin-layer chromatography plate were used in this study. Determination of antibacterial activity. To determine the efficacies of antibacterial activities of AU-1 and FQ-1, a wide range of Gram-positive and Gram-negative bacterial species were used in the study. In addition to the test drugs (ie, AU-1, FQ-1, FP-3, FP-4; Fig 1), the prevailing antibiotics, including ampicillin, norfloxacin, and gentamicin (Sigma Chemical Co) were included for comparison. Determination of antibacterial activity was carried out in accordance with our previously published method.17 Stock solutions of test compounds were diluted in Mueller Hinton agar immediately before use. The MIC of each compound for each test organism was determined by means of serial agar dilution with an inoculum size of 105 CFUs of the test organism, dispersed with a Steer
replicator (AutoMed Inc, Arden Falls, Minn). Cultures were incubated at 35°C for 18 hours. The in vitro test for antibacterial activity was performed in triplicate. LD90 of P aeruginosa.We determined LD90 using the method of Zuravleff et al.19 In brief, 10-fold dilutions of the 48-hour culture for P aeruginosa ATCC 27853, containing 7.5 ⫻ 109 CFU/mL, in brain-heart infusion broth (Difco Laboratories, Detroit, Mich) were performed in equal volumes of the same broth and 5% gastric mucin (Sigma), pH 7.4. Two hundred microliters of each dilution was inoculated intraperitoneally into each of 10 female ICR mice, weighing 20 to 25 gm, 48 hours after neutropenia induction by means of intraperitoneal administration of cyclophosphamide at a dose of 200 mg/kg. Mice were then observed for 120 hours. The reproducible LD90 was demonstrated by the repeated experiments in a group of 10 mice. The count of CFUs per mouse for the LD90 inoculum was 1.5 ⫻ 106 for strain ATCC 27853. Infection and treatment. Female ICR mice (20 – to 25 gm; National Laboratory of Animal Breeding and Research Center, Taipei, Taiwan) were intraperitoneally infected, 48 hours after administration of cyclophosphamide, with 0.20 mL of the LD90 bacteria suspension prepared in an equal volume of
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Table III. Therapeutic activity of FQ-1, norfloxacin, ampicillin, and FP-4 in mice infected with LD90 of P aeruginosa ATCC 27853
Each dose (mg/kg)
% of Mice surviving 120 hours after infection
10 5 2.5 10 5 2.5 10 10
0 (0/10)* 80 (8/10) 30 (3/10) 0 (0/10) 90 (10/10) 40 (4/10) 0 (0/10) 0 (0/10) 0 (0/10)
Dosing schedule for each dose was 2, 8, 24, and 48 hours after infection. *Number of mice surviving to total number of mice studied is indicated in parentheses.
Table IV. Acute toxicity of AU-1, FQ-1, and norfloxacin in mice Drug
AU-1 FQ-1 Norfloxacin
523 631 250
agarose gel as described in our previous work.21 DNA bands were stained with ethidium bromide and visualized under UV light. In another experiment, FQ-1 was treated with penicillinase (Sigma Chemical Co) before reaction with DNA. In brief, 10 L of FQ-1 solution (156 g/mL) was mixed with an equal volume of penicillinase solution containing 4 U of enzyme activity, after which the mixture was incubated at 37°C for 1 hour. Next, the mixture was heated in a boiling-water bath for 20 minutes to inactivate the enzyme. Finally the penicillinase-treated FQ-1 was used to react with DNA. RESULTS
broth and 5% gastric mucin. One hundred microliters of the antibiotic solution(s) containing each dose was intraperitoneally administered 2, 8, 24, and 48 hours after infection, in accordance with the method described by Zuravleff et al.19 Each group in this experiment comprised 10 mice. All mice were observed for 120 hours. The end point was mortality 120 hours after infection. We compared mortality among mice administered FQ-1 with that in each of the other treatment groups using unpaired Student t tests, considering P values of less than .01 statistically significant. Determination of acute toxicity. Female ICR mice were purchased from NLABRC and used at 4 weeks of age, weighing 18 to 20 gm. Stock solutions of AU-1, FQ-1, and norfloxacin (200 mg/mL) were made in 50% DMSO. This was followed by a twofold dilution with 5% glucose solution. The resultant solution was then serially diluted twofold in sterile 5% glucose solution. The volume of each drug tested, intraperitoneally injected, was 0.2 mL. The injected mice were observed every day for 1 week. We computed acute toxicity, referred to as LD50, using the method of Reed and Muench.20 Protoplast formation. One hundred microliters of Bacillus subtilis TSGH 732 culture grown in LB broth for 18 hours at 37°C was used to inoculate 1 mL of LB broth containing various test drugs at half-MIC. After incubation for 24 hours at 37°C, an aliquot of each culture was mixed with an equal volume of PBS containing crystal violet (0.5 %, wt/vol), then the cells were washed with PBS 3 times and examined under a microscope for protoplast formation. Interaction of AU-1 and FQ-1 with DNA. Ladder DNA (1 kb) purchased from New England BioLabs, Inc (Beverly, Mass) was used as DNA size markers. The reaction mixtures (20 L) containing 0.25 g of plasmid pUC18 DNA and an appropriate amount of drug in 20 mmol/L Tris-HCl buffer (pH 8.0) were incubated at 37°C for 1 hour. Samples of each mixture were analyzed by means of electrophoresis on 0.8%
Antibacterial activities of AU-1 and FQ-1. In tests against S aureus, S pyogenes, S marcescens, B subtilis, P mirabilis, E coli, and S typhi, both AU-1 and FQ-1 exhibited significant effectiveness (MIC 0.25– 4 g/mL) (Table I). They were as good as ampicillin (MIC 0.5– 8 g/ mL) but not as active as norfloxacin (MIC 0.06 – 0.13 g/mL), except against S aureus (MIC 2 g/mL). However, when tested against P aeruginosa, ampicillin (MIC ⬎ 128 g/mL) was inferior to both AU-1 (MIC 4 g/mL) and FQ-1 (MIC 4 g/mL). The latter approached the efficacy of norfloxacin against Pseudomonas (MIC 8 g/mL). When tested against K pneumoniae, both AU-1 and FQ-1 exhibited better effectiveness (MIC 16 g/mL) than ampicillin, which was inactive (MIC ⬎ 28 g/mL). The adduct derivatives showed enhanced antibacterial activities of AU-1 and FQ-1 against P aeruginosa and K pneumoniae compared with the parent molecule of ampicillin, FP-4, and FP-3. The results set forth in Table II show that some clinical isolates of P aeruginosa tested in this study were highly resistant to norfloxacin, gentamicin (MIC 64 g/mL and ⬎128 g/mL, respectively), or both but susceptible to either AU-1 or FQ-1 (MIC 4 – 8 and 4 –16 g/mL, respectively). Infection and treatment. Survival of mice treated with 4 compounds (FQ-1, norfloxacin, ampicillin, and FP-4) individually is seen in Table III. The efficacy of FQ-1 was comparable to that of norfloxacin in mice infected with P aeruginosa ATCC 27853. No significant difference was noted between the survival of mice treated with FQ-1 (10 mg/kg) and those given norfloxacin (10 mg/kg) (P ⬎ .05). On the other hand, the failure of treatment with either ampicillin or FP-4 at the same
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Fig 2. Protoplast formation of B subtilis. A, Drug-free control; B-D, B subtilis grown in the presence of ampicillin, FQ-1, and norfloxacin at a level close to the MIC of each. In B, protoplasts of B subtilis formed in the presence of ampicillin are indicated by arrows. In D, cell filamentation of B subtilis is shown. In C, no structural changes occurred in the presence of norfloxacin. All cells were stained with 1.25% crystal violet and examined under a photomicroscope at a magnification of 1000⫻.
dose (10 mg/kg) and dosing schedule as those of FQ-1 (Table III) confirmed the in vitro results showing that FQ-1 was superior to both FP-4 and ampicillin against P aeruginosa (Table I). No mortality was observed in any of the groups of noninfected mice treated with cyclophosphamide, FQ-1, norfloxacin, ampicillin, and FP-4 (data not shown). Acute toxicities.To compare the acute toxicities of AU-1, FQ-1, and norfloxacin, we administered single intraperitoneal injections of the drugs in question to mice (female ICR strain). Both AU-1 and FQ-1 exhibited much lower acute intraperitoneal toxicity (LD50 523 and 631 mg/kg, respectively) than norfloxacin (LD50 250 mg/kg) (Table IV). Protoplast formation. As expected, protoplast of B subtilis was formed in the presence of ampicillin, but no protoplast was observed in the cultures containing FQ-1 or norfloxacin (Fig 2). In the drug-free control, most of the vegetative cells had changed into spores after 24 hours’ incubation (data not shown). Surprisingly, FQ-1 induced filamentation of B subtilis at a level close to its MIC, whereas norfloxacin and ampicillin did not have such effect under the same condition (Fig 2). Intercalation of ampicillin derivatives in DNA. The results shown in Fig 3 indicate that AU-1 and FQ-1 can induce
a dramatic electrophoretic mobility shift on plasmid DNA, suggesting the binding of AU-1 and FQ-1 to supercoiled DNA, whereas neither ampicillin and norfloxacin nor the compounds FP-3 and FP-4 have such an effect. In addition, when the concentration of AU-1 or FQ-1 was increased, the migration rate of pUC18 DNA was gradually slowed (lanes C and D). At a critical concentration of drug, the rate of migration of pUC18 DNA reached its minimal value (lane E). As the concentration of drugs tested was increased, the mobility of pUC18 DNA started to increase again (Fig 4, lanes F and G). We found it interesting that FQ-1 lost its DNAbinding activity after treatment with penicillinase (Fig 5). DISCUSSION
Two fluoroquinolonyl ampicillin adduct derivatives, compounds AU-1 and FQ-1, are active against a broad spectrum of microorganisms (Table I). However, compounds FP-3 and FP-4 were inactive against all bacterial species tested in the study (Table I), suggesting that they failed to inhibit bacterial gyrase, an essential bacterial enzyme in the replication of bacterial chromosomal DNA, which is the common target of quinolone
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Fig 3. Binding of drugs with DNA. The reaction mixture (20 L) containing 0.25 g of pUC18 DNA and the drug at 0.72 mmol/L in 20 mmol/L Tris-HCl buffer (pH 8.0) were incubated at 37°C for 1 hour and analyzed by means of agarose-gel electrophoresis. Lane M: 1-kb ladder DNA as DNA molecular-size markers; lane A: drug-free control; lane B: ampicillin; lane C: norfloxacin; lane D: FQ-1; lane E: FP-3; lane F: FP-4; lane G: AU-1.
drugs, and that they might be unable to penetrate the cell envelopes of the test organisms. On the other hand, as a result of the special cell-wall structures containing outer membrane and capsule, both P aeruginosa and K pneumoniae are more resistant to the polar ␤-lactam antibiotics, such as ampicillin (Table I). Usually the cleavage of the ␤-lactam ring of ampicillin by penicillinase is one of the major mechanisms of resistance to ampicillin. However, Bonfiglio et al22 have reported that the most common mechanism of resistance to ␤-lactam antibiotics was probably ␤-lactamase–independent, namely, the so called intrinsic resistance resulting from impermeability, the efflux mechanism, or both. In this study, both fluoroquinolonyl ampicillin adduct derivatives were active against P aeruginosa strains that were highly resistant to ampicillin (Table I). This finding may have been a result of reduced polarity of the adduct derivatives.17 Thus AU-1 and FQ-1 may exhibit not only a more lipophilic character but also a greater affinity and penetrating power for the cell wall of P aeruginosa and, as a result, render them more effective against these bacteria. Furthermore, the finding that the antibacterial activities exhibited by AU-1 were almost the same as those of FQ-1 (Tables I and II) indicates that the addition of one more fluoro-group at position 7 of quinolone moiety does not affect the antibacterial activity of the fluoroquinolonyl ampicillin adduct derivatives against bacteria. The clinical isolates of the P aeruginosa strain that was highly resistant to norfloxacin and gentamicin (MIC 25-100 g/mL) were susceptible to both AU-1
Fig 4. Intercalation of increasing concentration of compounds AU-1 (A) and FQ-1 (B) in supercoiled DNA. The reaction mixtures (20 L), containing 0.25 g of supercoiled pUC18 DNA and the drug in question at the indicated concentration, were incubated at 37°C for 1 hour. Lane A: 1-kb ladder DNA; lane B: drug-free control. Lanes C–G, drug concentrations in millimoles per liter: C, 0.18; D, 0.36; E, 0.72; F, 1.45; G, 2.9.
and FQ-1 (MIC 4-8 and 4-16 g/mL, respectively) (Table II), demonstrating that the mode of action of compounds AU-1 and FQ-1 was not necessarily the same as those of norfloxacin and gentamicin. The mice made neutropenic by intraperitoneal injections of a sublethal dose of cyclophosphamide had a 100-fold increase in their susceptibility to intraperitoneal infection of Gram-negative bacteria, specifically P aeruginosa.23 Zuravleff et al19 reported that after the administration of cyclophosphamide (200 mg/kg), total blood leukocytes in mice decreased to 40% of baseline level at 48 hours and reached a nadir at 120 hours. The tested mice, 48 hours after administration of cyclophosphamide, were therefore considered profoundly neutropenic during antibiotic therapy. The initial step in ampicillin action is binding of the drug to cell receptors (PBPs). After ampicillin molecules adhere to the PBP-2 (transpeptidase), peptidoglycan synthesis is inhibited because final transpeptidation is blocked, resulting in the formation of protoplast in an isotonic solution such as LB broth. The protoplastformation study revealed that FQ-1 did not inhibit cell-wall biosynthesis of B subtilis to an obvious extent, although it contained an intact ampicillin moiety (Fig 2). Furthermore, FQ-1 induced obvious cell filamenta-
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structure of FQ-1 does not own DNA-intercalation activity. We appreciate the assistance of Professor Chei S. Wang in reviewing the manuscript.
Fig 5. Effect of penicillinase treatment on the DNA-binding activity of FQ-1. The reaction mixtures (20 L), containing 0.25 g of supercoiled pUC18 plasmid DNA and the drug (0.72 mmol/L) were incubated at 37°C for 1 hour. Lane M: 1-kb ladder DNA marker; lane A: drug-free control; lane B: FQ-1 without penicillinase treatment; lane C: FQ-1 treated with penicillinase before reaction with DNA; lane D: FQ-1 treated with inactivated penicillinase (by heating at 100°C for 20 minutes) before reaction with DNA.
tion of B subtilis (Fig 2), suggesting that FQ-1 blocks septum formation and cell division by binding to those PBPs involved in septum formation and inhibiting the cell-division gene of B subtilis.24,25 This result is similar to that previously reported for cell filamentation of P aeruginosa induced by AU-1.17 On the other hand, the results of electrophoretic mobility shift studies of AU-1– or FQ-1–treated DNA strongly suggest that both drugs can bind to DNA (Fig 3). However, ampicillin, FP-3, FP-4, and norfloxacin do not have such properties (Fig 3). It is interesting that a ccc DNA molecule such as plasmid pUC18 has no free ends and can only unwind to a limited extent, thus limiting the amount of drug bound. As the concentration of AU-1 or FQ-1 is increased, more of the drug becomes bound to DNA. The negative superhelical turns in the ccc molecule are progressively removed, and their rate of migration is slowed. At the critical drug concentration, where no superhelical turns remain, the rate of migration of ccc DNA reaches its minimal value. As concentration of the drug tested is increased, positive superhelical turns start to occur, and the mobility of ccc DNA increases rapidly (Fig 4). This result is similar to the interaction of ethidium bromide with ccc-form plasmid DNA described by Sambrook et al.26 That FQ-1 lost its DNA-binding activity after the treatment with penicillinase (Fig 5) strongly suggests that the lactamring structure in ampicillin moiety of FQ-1 was hydrolyzed by penicillinase and that the hydrolyzed
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Susceptibility of Pseudomonas aeruginosa of various pyocin types to the newly synthesized ampicillin derivative N-(6,7-difluoroquinolonyl)-ampicillin. J Antimicrob Chemother 1997;39:325-30. Tsou TL, Tang ST, Wu JR, Hung YW, Liu YT. Synthesis and antipseudomonal activity of fluoroquinolonyl-penicillin derivatives. Eur J Med Chem 1999;34:255-9. Zuravleff JJ, Chervenick P, Yu VL, Muder RR, Diven WF. Addition of rifampin to ticarcillin-tobramycin combination for the treatment of Pseudomonas aeruginosa infections: assessment in a neutropenic mouse model. J Lab Clin Med 1984; 103:878-85. Reed LJ, Muench H. A simple method of estimating fifty per cent end points. Am J Hyg 1938;27:493-7. Liu YT, Yeh SJ, Liao CL, Chou CF, Shou L. Mechanism of action of chounghwamycin A. Proc Natl Sci Coun R O C 1989;13(part B):262-6.
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