First-step Mutants Research Articles

Fluoroquinolone-resistant Brucella melitensis mutants obtained in vitro

Three fluoroquinolone-resistant Brucella melitensis mutants were obtained after successive passages on norfloxacin-containing agar plates. The original strain was B. melitensis biovar 2 ATCC 23457. Mutants emerged at frequencies ranging between 1 × 10 −6 and 3 × 10 −8. The three mutants showed a gyrA Ala71 → Ser mutation as well as insertion of Ala340. No mutations were observed in the parC and gyrB genes. Minimum inhibitory concentrations (MICs) increased by four- to eight-fold from the wild-type to the first-step mutant for norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin. MICs of the second-step mutant increased significantly only for norfloxacin and those for the third-step mutant for norfloxacin and ciprofloxacin, suggesting the involvement of some efflux mechanism.

Dual-targeting properties of the 3-aminopyrrolidyl quinolones, DC-159a and sitafloxacin, against DNA gyrase and topoisomerase IV: contribution to reducing in vitro emergence of quinolone-resistant Streptococcus pneumoniae

DC-159a (a novel quinolone) and sitafloxacin (DU-6859a) are structurally related quinolones, bearing a 3-aminopyrrolidyl substitution. We investigated the relationship between the target preferences of these 3-aminopyrrolidyl quinolones, in vitro potencies and emergence of quinolone-resistant mutants in Streptococcus pneumoniae, compared with other quinolones. MICs, resistance frequencies and mutant prevention concentrations (MPCs) were determined using quinolone-susceptible strains and first-step parC mutant strains of S. pneumoniae. Target preferences were tested by the following two methods: antibacterial activities against gyrA or parC mutants and in vitro enzyme assays for the determination of 50% inhibition (IC(50)) values. DC-159a and sitafloxacin exhibited potent antibacterial activities, low frequencies of mutant selection, low MPCs and narrow mutant selection windows against both quinolone-susceptible strains and first-step parC mutants of S. pneumoniae, compared with gatifloxacin, moxifloxacin and other quinolones tested. DC-159a and sitafloxacin showed relatively low MIC ratios against single gyrA or parC mutants relative to the wild-type strain and low IC(50) ratios against DNA gyrase and topoisomerase IV. DC-159a and sitafloxacin demonstrated a more balanced dual-targeting activity than gatifloxacin, moxifloxacin and other quinolones tested. In addition, DC-159a and sitafloxacin have a lower propensity for selecting first- and second-step resistant mutants.

Fatal Levofloxacin Failure in Treatment of a Bacteremic Patient Infected with Streptococcus pneumoniae with a Preexisting parC Mutation

The fatal outcome of levofloxacin treatment in a patient with bacteremic pneumonia caused by Streptococcus pneumoniae with a preexisting parC mutation is reported. Failure was due to the emergence of a gyrA mutation after 4 days of therapy. Problems encountered in detecting first-step mutation isolates are discussed.

Fluoroquinolone-Resistant Group B Streptococci in Acute Exacerbation of Chronic Bronchitis

To the Editor: Fluoroquinolones (FQs) that are active against streptococcal species (e.g., levofloxacin and moxifloxacin) have been recommended by numerous national health authorities and international organizations for treating acute exacerbations of chronic bronchitis and pneumonia in adults (1). However, use of these antimicrobial drugs for treating community-acquired infections has led to an increase in FQ-resistant strains in bacteria such as Streptococcus pneumoniae. Group B streptococci (GBS, e.g., S. agalactiae) are the leading cause of invasive infections (pneumonia, septicemia, and meningitis) in neonates. GBS are also associated with bacteremia, endocarditis, and arthritis, and are responsible for deaths and illness in nonpregnant women with underlying diseases and in elderly adults (2). We describe, to our knowledge, the first GBS clinical isolate in France resistant to FQ; the isolate was from a patient treated with levofloxacin. GBS CNR0717 strain was isolated as the predominant bacterium in a culture (>107 CFU/mL) from 2 purulent sputum samples from an 80-year-old man (leukocytes >25, epithelial cells 64 mg/L, and showed increased MICs for ciprofloxacin, sparfloxacin, levofloxacin, and moxifloxacin. No reduction of FQ MICs was observed with reserpine (10 mg/L), which indicated that resistance to FQ was not caused by an active efflux pump system. Table MICs of fluoroquinolones for strains of group B streptococci (GBS), France Three major mutations have been reported for FQ resistance in streptococci at codon positions 81 in gyrA and 79 or 83 in parC (4). DNA sequence analysis of these regions showed a mutation in parC (Ser 79 → Tyr) but not in the wild-type susceptible strain (NEM316). No mutation was detected in the gyrA gene. FQ resistance in streptococci is acquired through a stepwise process and has been extensively studied in S. pneumoniae. First-step mutants conferring low-level resistance generally result from mutations in either gyrA or parC. There is also a molecule-dependent target specificity: mutations in parC are generally selected by pefloxacin, ciprofloxacin, and levofloxacin, and those in gyrA are selected by sparfloxacin, gatifloxacin, moxifloxacin, gemifloxacin, and garenoxacin (5). In second-step mutants, mutations are present in both parC and gyrA and confer resistance to the antistreptococcal FQs levofloxacin, moxifloxacin, and gatifloxacin. FQ resistance in GBS has been reported in Japan, the United States, and Spain (6–8). Up to now, all FQ-resistant GBS strains described were highly resistant because of point mutations in gyrA and parC QRDR; a parC mutation at position 79 was present in all strains. These strains were isolated from elderly adults who, in some cases, had received quinolone therapy. Low-level resistance to FQ in GBS CNR0717 was associated with a Ser 79 → Tyr mutation in parC. Therefore, although the FQ sensitivity of this strain is unknown, a first-step mutant could have been selected in vivo as our patient was treated with levofloxacin for 2 weeks. GBS is an unusual cause of acute bacterial exacerbation of chronic bronchitis compared with other respiratory pathogens such as S. pneumoniae, but pathologies associated with this bacterium are changing. Clinical microbiologists should be aware of these changes and test isolates of Streptococcus spp. for susceptibility to FQs. This report indicates that FQ resistance among streptococci is a growing concern and that levofloxacin can select in vivo parC first-step mutants that will facilitate emergence of high-level FQ-resistant GBS strains, as demonstrated in vitro for S. pneumoniae (9). Finally, although FQ treatment is recommended for high-risk groups with acute exacerbations of chronic bronchitis, these antimicrobial drugs must be reserved for situations in which there are no effective alternative drugs to treat infections caused by multidrug-resistant bacteria. For susceptible strains, β-lactams, which still constitute the first-line recommended antimicrobial drugs, should be used for treatment of these patients (10).

In vitro pharmacodynamics of moxifloxacin versus levofloxacin against 4 strains of Streptococcus pneumoniae: 1 wild type, 2 first-step parC mutants, and 1 pump mutant

Levofloxacin binds topoisomerase IV, whereas moxifloxacin preferentially binds DNA gyrase. Most 1st-step pneumococcal mutants have alterations in the parC gene of topoisomerase IV. Because of differential binding affinity, moxifloxacin may have superior activity against 1st-step mutants compared with levofloxacin. The purpose of this work was to compare rates and extent of bacterial killing of genetically characterized Streptococcus pneumoniae with moxifloxacin and levofloxacin. Four strains of S. pneumoniae were used: a wild type, 2 first-step parC mutants, and a pump mutant. Using an in vitro pharmacodynamic model run in duplicate, we exposed bacteria to unbound moxifloxacin and levofloxacin peaks of 2 and 4.5 mg/L, respectively, which emulated clinical dosing. Additional experiments were done in which the area under the curve (AUC)/MIC ratio of 1 agent was matched to the competing drug's clinical dose AUC/MIC ratio. Time kill curves were analyzed for rate and extent of bacterial kill and regrowth. Pre- and postexposure MIC and polymerase chain reaction (PCR) testing were done. Moxifloxacin and levofloxacin displayed similar rates and extent of bacterial kill for the wild type, efflux pump type, and parC mutant 27-1361B. Moxifloxacin initially achieved a faster rate of kill, regardless of the AUC/MIC ratio, against parC mutant 7362 ( P < 0.05) but not an advantage in time to 3 log kill. Postexposure MIC values were elevated for strain 7362 in 2 moxifloxacin experiments and 1 levofloxacin experiment. Post-PCR analysis revealed new gyrA mutations for all 3 isolates. Both moxifloxacin and levofloxacin are effective against multiple strains of S. pneumoniae.

Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Heteroaryl Isothiazolones in Staphylococcus aureus

Heteroaryl isothiazolones (HITZs) are antibacterial agents that display excellent in vitro activity against Staphylococcus aureus. We recently identified a series of these compounds that show potent bactericidal activities against methicillin-resistant Staphylococcus aureus (MRSA). We report here the results of in vitro resistance studies that reveal potential underlying mechanisms of action. HITZs selected gyrA mutations exclusively in first-step mutants of wild-type S. aureus, indicating that in contrast to the case with most quinolones, DNA gyrase is the primary target. The compounds displayed low mutation frequencies (10(-9) to 10(-10)) at concentrations close to the MICs and maintained low MICs (< or =0.016 microg/ml) against mutants with single mutations in either gyrA or grlA (parC). These data suggested that HITZs possess significant inhibitory activities against target enzymes, DNA gyrase and topoisomerase IV. This dual-target inhibition was supported by low 50% inhibitory concentrations against topoisomerase IV as measured in a decatenation activity assay and against DNA gyrase as measured in a supercoiling activity assay. Good antibacterial activities (< or =1 microg/ml) against staphylococcal gyrA grlA double mutants, as well as low frequencies (10(-9) to 10(-10)) of selection of still higher-level mutants, also suggested that HITZs remained active against mutant enzymes. We further demonstrated that HITZs exhibit good inhibition of both S. aureus mutant enzymes and thus continue to possess a novel dual-targeting mode of action against these mutant strains. In stepwise acquisition of mutations, HITZs selected quinolone resistance determining region mutations gyrA(Ser84Leu), grlA(Ser80Phe), grlA(Ala116Val), and gyrA(Glu88Lys) sequentially, suggesting that the corresponding amino acids are key amino acids involved in the binding of HITZs to topoisomerases. The overall profile of these compounds suggests the potential utility of HITZs in combating infections caused by S. aureus, including multidrug-resistant MRSA.

Streptococcus pneumoniae

Genetic analyses of Streptococcus pneumoniae clinical isolates causing invasive pneumococcal disease (IPD) have revealed within the "susceptible population" a significant proportion of strains harboring a single, principally parC mutation within the quinolone-resistance determining region. The occurrence of first-step mutants predisposes S. pneumoniae to a subsequent and more rapid second-step mutation resulting in elevated levels of fluoroquinolone resistance and clinical failure. Fluoroquinolones are a common therapeutic choice for residents of long-term care facilities and/or elderly patients in the community at higher risk of IPD. Despite the advent of the pneumococcal conjugate vaccine, levofloxacin resistance among vaccine serotypes is increasing, and the majority of these isolates are closely related to widespread antibiotic-resistant clones. Use of fluoroquinolones with activity against levofloxacin-nonsusceptible isolates of S. pneumoniae should be considered in the management of IPD among elderly patients in the community and long-term care residents and among at-risk patients with chronic pulmonary infections.

Selection of a gyrA Mutation and Treatment Failure with Gatifloxacin in a Patient with Streptococcus pneumoniae with a Preexisting parC Mutation

An 81-year-old woman had pneumonia caused by Streptococcus pneumoniae (levofloxacin Etest minimum inhibitory concentration [MIC] 1.5 microg/ml) and was treated with intravenous gatifloxacin 200 mg/day. After 3 days of therapy, repeat sputum cultures were positive for S. pneumoniae, which was resistant to levofloxacin (Etest MIC > 32 microg/ml). The isolate obtained before therapy showed a preexisting parC mutation of aspartic acid-83 to asparagine (Asp83-->Asn), and the isolate obtained during therapy showed an acquired gyrA mutation from serine-81 to phenylalanine (Ser81-->Phe) and a second parC mutation from lysine-137 to Asn (Lys137-->Asn). Both isolates were the same strain, as determined with pulsed-field gel electrophoresis. This case demonstrates the potential for resistance to emerge during 8-methoxy fluoroquinolone therapy for fluoroquinolone-susceptible S. pneumoniae with a preexisting parC mutation. Additional clinical failures with a fluoroquinolone may occur unless these first-step parC mutants can be identified to assist clinicians in selecting appropriate antimicrobial therapy.

In vivo selection during ofloxacin therapy of Escherichia coli with combined topoisomerase mutations that confer high resistance to ofloxacin but susceptibility to nalidixic acid

To investigate quinolone resistance mechanisms in an Escherichia coli clinical isolate (Ar2) resistant to ofloxacin but susceptible to nalidixic acid selected after 10 days of ofloxacin therapy in a patient with prostatitis. Molecular typing (ERIC-PCR and RAPD), antibiotic susceptibility and gyrA, gyrB, parC and parE QRDR sequences were compared for E. coli Ar2 and a wild-type E. coli (Ar1) isolated 2 months earlier in the same patient. Ofloxacin-resistant mutants were selected in vitro in order to reproduce the mutations observed and the original phenotype. The two strains were similar with regard to antibiotic susceptibility except quinolones and for ERIC-PCR and RAPD patterns, suggesting a clonal relationship and acquisition of quinolone resistance by chromosomal mutation. Quinolone MICs were 3, 0.12, 0.05 and 0.02 mg/L of nalidixic acid, ofloxacin, levofloxacin and ciprofloxacin, respectively, for E. coli Ar1 and 6, 32, 8 and 1 mg/L, respectively, for E. coli Ar2. The strain Ar2 harboured two substitutions, Gly-81-->Asp in GyrA and Ser-80-->Arg in ParC. Introduction into E. coli Ar2 of the wild-type gyrA fully complemented fluoroquinolone resistance. Although the strain was not a hypermutator, ofloxacin first-step resistant mutants with gyrA mutations were easily obtained from E. coli Ar1 and 25% of them were at codon 81. In vitro stepwise combination of Gly-81-->Asp in GyrA and Ser-80-->Arg in ParC reproduced the original phenotype in E. coli KL16. A double topoisomerase mutant was selected in vivo by 10 days ofloxacin. The mutations were originally combined for a result of ofloxacin resistance but nalidixic acid susceptibility.

Call for the international adoption of microbiological breakpoints for fluoroquinolones and Streptococcus pneumoniae

The use of current Clinical and Laboratory Standards Institute levofloxacin breakpoints for assessing fluoroquinolone resistance in Streptococcus pneumoniae is inadequate for detecting isolates possessing first-step parC mutations. Consequently, the risk for development of fluoroquinolone resistance is greatly underestimated. Adopting microbiological breakpoints for fluoroquinolones and S. pneumoniae, where parC mutations are rare in susceptible isolates, more accurately describes the emergence of resistance and may help to prevent a number of future fluoroquinolone treatment failures. Additionally, we propose that the use of a second fluoroquinolone marker, such as ciprofloxacin, offers the best prediction for detecting an isolate possessing a first-step parC mutation.

Optimizing Antibiotic Therapy

I am in full agreement with Dr. Hibbard and his recommendation that antimicrobials should be used in combination to reduce the likelihood of resistance selection (Microbe, May 2006, p. 209). His explanation of bacterial population genetics clearly highlights the issue. Indeed this concept was originally proposed by Paul Ehrlich as early as 1913 (P. Ehrlich, Lancet 1913, p. 455–453) and has been the backbone of antituberculosis therapy for almost 50 years. However the rationale behind the single drug use, often at inappropriate doses for long durations, is not based on science, but most probably on cost or adverse event savings with the logic that in many infections the antibacterials are used to “support” the host defense in combating infections. In many cases this works well to resolve infections but also serves to select the first-step resistant mutants as Dr. Hibbard describes. Karl Drlica's group have espoused a new concept to tackle this issue, the mutant prevention concept wherein they test bacterial populations to determine how much drug it would take to prevent resistance developing by selection of first–step mutants (Y. Dong, X. Zhao, B. N. Kreiswirth, and K. Drlica, Antimicrob. Agents Chemother. 44:2581–2584, 2000). Today, I suspect cost savings and a wary eye towards adverse events are preventing a more complete embrace of the two-or-more-drugs approach. Moreover, if the most pharmacodynamically effective members of a drug class are used (W. M. Scheld, Emerg. Infect. Dis. 9:1–9, 2003) and shown not to be antagonistic to each other then shorter courses maybe possible and overall costs maybe reduced as fewer relapses and follow-up therapy will be required as resistance is kept at bay. As we have few new antibacterials in development we should recall the advice of our predecessors and use this valuable resource in a more thoughtful way.

Evaluation of bacterial kill when modelling the bronchopulmonary pharmacokinetic profile of moxifloxacin and levofloxacin against parC-containing isolates of Streptococcus pneumoniae

The increasingly recognized prevalence of first-step parC mutants in Streptococcus pneumoniae and the development of de novo resistance while on fluoroquinolone therapy are of concern. Previous work by our group demonstrated the ability of moxifloxacin, but not levofloxacin, to eradicate parC mutants. The objective of this experiment was to determine whether these fluoroquinolone antibiotics provided equivalent bacterial kill when similar AUC/MICs were examined. An in vitro pharmacodynamic model was used to simulate the epithelial lining fluid (ELF) concentrations following oral administration of levofloxacin 500 mg once daily and moxifloxacin 400 mg once daily in older adults. In addition, a range of AUC/MICs were also modelled, including levofloxacin 750 mg once daily. Five different S. pneumoniae containing first-step parC mutations and one isolate without mutations were tested for 48 h and time-kill curves were constructed. Samples at 0, 24 and 48 h were collected for phenotypic and genotypic profiling. HPLC was used to verify that target exposures were achieved. The isolate without a parC mutation displayed a 4 log reduction in cfu after treatment with levofloxacin 500 mg and did not select for resistance. In all five isolates containing first-step parC mutations, resistance emerged within 48 h with a > or =16-fold increase in MIC and the acquisition of a gyrA mutant. Increasing the exposure of levofloxacin to approximately 750 mg dose still led to > or =16-fold increase in MIC at 48 h in two of the four isolates containing parC mutations. On the other hand, moxifloxacin 400 mg sustained bacterial killing against the two isolates tested without the selection of resistant mutants. It appears that the critical AUC/MIC necessary to prevent the acquisition of resistance for levofloxacin is 200 and approximately 400 for moxifloxacin. Due to suboptimal exposures, once-daily oral regimens of levofloxacin at both 500 and 750 mg inconsistently led to bactericidal activity and the frequent acquisition of a second-step gyrA mutation in S. pneumoniae isolates already containing a first-step parC mutation. Conversely, once-daily moxifloxacin 400 mg provides exposures that vastly exceed the apparent efficacy breakpoint and did not select for second-step mutants until exposures were decreased 4-fold. As a result of these data and the emerging literature involving mutations in the pneumococcus, caution should be exercised when the respiratory fluoroquinolones are used to treat patients infected with S. pneumoniae suspected of having parC mutations.

Combination of known and unknown mechanisms confers high-level resistance to fluoroquinolones in Enterococcus faecium

In order to elucidate the mechanisms of fluoroquinolone resistance in Enterococcus faecium, spontaneous mutants isolated from Ent. faecium ATCC 19434 by stepwise selection with sparfloxacin (SPX) or norfloxacin (NOR) and 13 clinical isolates of Ent. faecium were characterized by analysing quinolone-resistance-determining regions (QRDRs) of the gyrA, gyrB, parC and parE genes and examining changes in MICs of SPX and NOR in the presence of efflux pump inhibitors. The SPX-selected first-step mutant had a point mutation only in gyrA, and the mutants QR7-18 and QR7-39, and clinical isolates that had point mutations in parC, showed NOR resistance. These results indicate that the primary targets of SPX and NOR are DNA gyrase and topoisomerase IV, respectively, and therefore that the primary target of fluoroquinolones in Ent. faecium differs depending on the structure of the compound used. The characterization of the spontaneous mutants and the clinical isolates demonstrates that in addition to the previously reported alterations in GyrA and ParC, an alteration in GyrB, a NorA-like pump, an unknown efflux pump, which excretes both SPX and NOR from bacterial cells, and probably other unknown mechanism(s) all contribute to fluoroquinolone resistance in Ent. faecium.

Activity of Gatifloxacin in an In Vitro Pharmacokinetic-Pharmacodynamic Model against Staphylococcus aureus Strains either Susceptible to Ciprofloxacin or Exhibiting Various Levels and Mechanisms of Ciprofloxacin Resistance

Gatifloxacin (GAT) is a new 8-methoxy fluoroquinolone with enhanced activity against gram-positive cocci. Its activity was studied in an in vitro pharmacokinetic-pharmacodynamic model against five Staphylococcus aureus strains, either susceptible to ciprofloxacin or exhibiting various levels and mechanisms of ciprofloxacin (CIP) resistance: the ATCC 25923 reference strain (MICs of CIP and GAT: 0.5 and 0.1 microg/ml, respectively), its efflux mutant SA-1 (16 and 0.5 microg/ml; mutation in the norA promoter region), and three clinical strains, Sa2102 (2 and 0.2 microg/ml), Sa2667 (4 and 0.5 microg/ml), and Sa2669 (16 and 1 microg/ml), carrying mutations in the grlA (Ser80Tyr or Phe) and gyrA (Ser84Ala) quinolone resistance-determining regions (QRDRs) for Sa2669. Plasmatic pharmacokinetic profiles after daily 1-h perfusion of 400 mg for 48 h were accurately simulated. Thus, mean maximum concentration of drug in serum values for the two administration intervals were 5.36 and 5.80 microg/ml, respectively, and the corresponding half-life at beta-phase values were 8.68 and 7.80 h (goodness of fit coefficient, >0.98). Therapeutic concentrations of GAT allowed the complete eradication of the susceptible strain within 12 h (difference between the bacterial counts at the beginning of the treatment and at a defined time: -2.18 at the 1-h time point [t(1)] and -6.80 at t(24) and t(48); the bacterial killing and regrowth curve from 0 to 48 h was 30.2 h x log CFU/milliliter). However, mutants (M) with GAT MICs increased by 4- to 40-fold were selected from the other strains. They acquired mutations either supplementary (MSa2102 and MSa2667) or different (Ala84Val for MSa2669) in gyrA or in both gyrA and grlA QRDRs (MSA-1). MSa2667 additionally overproduced efflux system(s) without norA promoter modification. Thus, GAT properties should allow the total elimination of ciprofloxacin-susceptible S. aureus, but resistant mutants might emerge from strains showing reduced susceptibility to older fluoroquinolones independently of the first-step mutation(s).

Prevalence and antimicrobial susceptibility patterns among gastroenteritis-causing pathogens recovered in Europe and Latin America and Salmonella isolates recovered from bloodstream infections in North America and Latin America: report from the SENTRY Antimicrobial Surveillance Program (2003)

Gastroenteritis-causing pathogens are the second leading cause of morbidity and mortality worldwide. Complicating the clinical diarrhoea syndrome is the emergence of antimicrobial resistance among the responsible bacterial pathogens. The reported increases in fluoroquinolone resistance in Salmonella, Shigella and Campylobacter have been extremely worrisome considering the primary role of ciprofloxacin as a treatment. In this study, 1479 bacterial isolates from gastroenteritis infections were collected in Europe and Latin America, which included Salmonella spp. (834; 56%), Shigella spp. (311; 21%), Campylobacter spp. (182; 12%) and Aeromonas spp. (72; 5%). The fluoroquinolones displayed the greatest activity against these pathogens, with only three non- Campylobacter spp. strains being non-susceptible using current Clinical and Laboratory Standards Institute (CLSI) breakpoint criteria. Whilst ciprofloxacin resistance in European and Latin American Salmonella was only 0.2% and 0.0%, respectively, a total of 16.2% and 12.9% of isolates were resistant to nalidixic acid, indicating possible first-step gyrA mutations. Among confirmed extended-spectrum β-lactamase-producing Salmonella strains, CTX-M genes were detected in 15 originating from Russia. Erythromycin and azithromycin were the most potent agents tested against Campylobacter spp. (values of minimum inhibitory concentration for 90% of the organisms, 0.5 mg/L and 0.12 mg/L, respectively), with erythromycin displaying the highest susceptibility (91.1%). Salmonella isolates from bloodstream infections displayed antibiograms that were nearly identical to strains causing gastroenteritis. Considering the role that antimicrobial therapy plays in the management of moderate to severe bacterial gastroenteritis, global surveillance and local/national public health programmes can provide critical data illuminating the dissemination of resistance and guidance for empirical therapy.

Selection of ciprofloxacin resistance in Escherichia coli in an in vitro kinetic model: relation between drug exposure and mutant prevention concentration

To evaluate the mutant prevention concentrations (MPCs) of ciprofloxacin for two susceptible and one first-step gyrA resistant mutant Escherichia coli strains in an in vitro kinetic model and to identify the pharmacodynamic index that best predicts prevention of resistance emergence. An in vitro kinetic model was used to measure MPC with static antibiotic concentrations and to test different dosing profiles to study pharmacokinetics/pharmacodynamics indices important to prevent the growth of resistant mutants. In one set of kinetic experiments the starting concentration was equal to the MPC and the T > MPC was varied before antibiotic dilution was begun. In a second set of kinetic experiments C(max) was varied and dilution of the antibiotic was started at time zero. From the static experiments we calculated MPC values of 0.128 mg/L for both the susceptible strains (16x MIC) and 0.188 mg/L (4x MIC) for the first-step resistant (gyrA) strain. The kinetic experiments showed that the T > MPC needed to prevent the growth of resistant bacteria was shorter with an increased C(max). When resistance was selected, several subpopulations with different levels of susceptibility to ciprofloxacin emerged. Neither T > MPC nor C(max) proved to be single correlates for preventing resistance development. For the two investigated wild-type strains, an AUC/MPC ratio of > or =22 was the single pharmacodynamic index that predicted prevention of resistant mutant development.

Prevalence of First-Step Mutants among Levofloxacin-Susceptible Invasive Isolates of Streptococcus pneumoniae in the United States

By use of a PCR-restriction fragment length polymorphism assay, we screened 496 levofloxacin-susceptible invasive pneumococcal strains (MIC<or=2 mg/liter) for quinolone resistance-determining region mutations known to confer fluoroquinolone resistance. Among those with a levofloxacin MIC of 2 mg/liter, 16.2% of isolates recovered from nursing home residents and 6.4% from non-nursing home residents had first-step mutations.

Prevalence of pneumococci with single mutations within fluoroquinolone resistance genes

Background: Discordant antibiotic treatment of fluoroquinolone (FQ) resistant pneumococci causes treatment failure in patients with pneumonia. Resistance to FQs occurs in a stepwise fashion by the acquisition of mutations within the FQ binding sites (QRDR) in the target enzymes DNA gyrase (gyrA and gyrB), and topoisomerase IV (parC and parE). Whereas complete resistance to FQs mainly occurs if mutations in both target enzymes are present, strains containing single mutations in only one of the enzymes frequently have susceptible phenotypes and cannot be detected by microbiological routine testing. These first-step mutants have an increased likelihood for the acquisition of mutations in the second enzyme leading to complete resistance. Data about the prevalence of first-step mutants among susceptible isolates are rare.

Nonmolecular Test for Detection of Low-Level Resistance to Fluoroquinolones in Streptococcus pneumoniae

With respect to pneumococci, there is a need to detect first-step mutants with reduced fluoroquinolone (FQ) susceptibility from which second-step, resistant mutants are likely to be selected in the presence of antipneumococcal FQs. Here, we describe an interpretative disk diffusion test, of which three options are presented, that allows the distinction between first- and second-step mutants. Using five FQ disks (pefloxacin, norfloxacin, levofloxacin, ciprofloxacin, and sparfloxacin, option 1), all known mechanisms of altered FQ susceptibility found in first-step mutants (ParC, ParE, GyrA, or efflux) and in second-step mutants (ParC and GyrA or ParE and GyrA) can be accurately detected, making this option a useful epidemiological tool. Using three FQ disks (pefloxacin, norfloxacin, and levofloxacin, option 2), the most prevalent FQ-resistant mutants, but not the first-step GyrA mutants, can be detected. With only two FQ disks (norfloxacin and levofloxacin) in the third and simplest option, first-step mutants can be distinguished from second-step mutants, however, without differentiation of ParC, ParE, or efflux alterations.

Comparison of the Minimum Inhibitory, Mutant Prevention and Minimum Bactericidal Concentrations of Ciprofloxacin, Levofloxacin and Garenoxacin Against Enteric Gram-negative Urinary Tract Infection Pathogens

Acute, uncomplicated urinary tract infections (UTIs) are among the most commonly encountered bacterial infections and management has been made more complicated in the last decade due to the trend toward increasing antimicrobial resistance to ampicillin and trimethoprim/sulfamethoxazole (TMP/SMX). Fluoroquinolones are suggested as alternative antimicrobials for the treatment of UTIs in communities for which TMP/SMX resistance is ≥10%. The mutant-prevention concentration (MPC) is a novel susceptibility parameter designed to minimize the selection of first-step resistant mutants present in large, ≥1010 CFU/mL, heterogeneous bacterial populations and is a distinct measurement from minimum inhibitory concentration testing. We measured MPC results for 80 enteric Gramnegative and 20 Pseudomonas aeruginosa urinary isolates against ciprofloxacin, levofloxacin and garenoxacin. Ciprofloxacin, levofloxacin and garenoxacin MPC results for Escherichia coli, Citrobacter freundii, Enterobacter cloacae, Klebsiella pneumoniae and P. aeruginosa respectively were 0.5, 1, 1, 1 and 4 mg/L; 1, 2, 4, 2 and 16 mg/L; 1, 8, >8, 4 and ≥32 mg/L. By comparison, minimum inhibitory concentration (MIC)90 results for the Enterobacteriaceae organisms ranged from ≤0.06-4 mg/L for the three drugs and 1-4 mg/L against P. aeruginosa. Similarly, MBC90 results ranged from ≤0.06-4 mg/L and 2-8 mg/L respectively. For ciprofloxacin against E.coli, E. cloacae and K. pneumoniae and for levofloxacin against E.coli, C. freundii and K. pneumoniae, MPC results were below susceptible breakpoints and within clinically achievable and sustainable drug concentrations for >24 hours of the dosing interval against. For garenoxacin, urine drug concentrations are expected to be in excess of MPC results for the entire length of the dosing interval for E.coli. Application of MPCs to fluoroquinolones and management of UTIs represents a situation where high levels of in vitro activity, based on low MICs, is reflected in correspondingly low MPC values for most of the organisms tested. Incorporation of MPC strategies into current fluoroquinolone dosing in UTI represents a realistic approach for preventing the further selection of resistant organisms associated with UTIs.