Role of low-level quinolone resistance in generating tolerance in Escherichia coli under therapeutic concentrations of ciprofloxacin.

BACKGROUND: Tolerance (including persistence) and resistance result in increased survival under antibiotic pressure. OBJECTIVES: We evaluated the interplay between resistance and tolerance to ciprofloxacin under therapeutic and killing conditions to determine the contribution of low-level quinolone resistance (LLQR) mechanisms to tolerance. We also determined how the interaction between resistance (LLQR phenotypes) and tolerance was modified under SOS response suppression. METHODS: Twelve isogenic Escherichia coli strains harbouring quinolone resistance mechanisms combined with SOS response deficiency and six clinical E. coli isolates (LLQR or non-LLQR) were evaluated. Survival (tolerance or persistence) assays were used to measure surviving bacteria after a short period (up to 4 h) of bactericidal antibiotic treatment under therapeutic and killing concentrations of ciprofloxacin [1 mg/L, EUCAST/CLSI breakpoint for resistance; and 2.5 mg/L, peak serum concentration (Cmax) of this drug]. RESULTS: QRDR substitutions (S83L in GyrA alone or combined with S80R in ParC) significantly increased the fraction of tolerant bacteria (2-4 log10 cfu/mL) after exposure to ciprofloxacin at clinically relevant concentrations. The impact on tolerant bacteria due to SOS response suppression (including persistence mediated by the tisB gene) was reversed by LLQR mechanisms at therapeutic concentrations. Furthermore, no reduction in the fraction of tolerant bacteria due to SOS response suppression was observed when S83L in GyrA plus S80R in ParC were combined. CONCLUSIONS: Tolerance and quinolone resistance mutations interact synergistically, giving LLQR mechanisms an additional role in allowing bacterial survival and evasion of therapeutic antimicrobial conditions by a combination of the two strategies. At clinically relevant concentrations, LLQR mechanisms reverse further impact of SOS response suppression in reducing bacterial tolerance.

Increased Quinolone-Resistant Mutations of gyrA and parC Genes after Pouchitis Treatment with Ciprofloxacin.

BACKGROUND: Oral antibiotics, such as ciprofloxacin (CFX), are widely used for the treatment of acute and chronic pouchitis. Most bacterial mutations that confer quinolone resistance are at Ser-83 and Asp-87 in the gyrA gene and Ser-80 and Glu-84 in the parC gene. METHODS: We obtained 51 stool samples from 43 patients who were diagnosed with ulcerative colitis and underwent ileal pouch-anal anastomosis. Patients were divided into 2 groups: 13 patients with CFX treatment of pouchitis and 30 patients without pouchitis. After extraction of fecal DNA, the amount of Escherichia coli 16S rRNA, gyrA, and parC gene DNA were measured using real-time polymerase chain reaction (PCR). Possible mutations at gyrA 83 and 87 and at parC 80 and 84 were investigated by PCR cloning and sequencing, and mutation rates were quantified by rapid PCR-restriction fragment length polymorphism. RESULTS: Samples from both CFX-treated and -untreated patients had comparable levels of gyrA and parC gene DNA. Nucleic acid and amino acid mutations were identified at gyrA 83 and 87, and at parC 80 and 84. We successfully quantified mutation rates at gyrA 83 and 87, and at parC 84, all of which were significantly higher in samples from CFX-treated patients (70, 84, and 38%) than from CFX-untreated patients (13, 11, and 5%). CONCLUSION: E. coli in patient pouches may have mutations in their gyrA and parC genes that produce CFX resistance. Mutation rates of these genes were significantly higher in samples from CFX-treated patients. This study contributes to understanding the decrease and loss of CFX effectiveness against pouchitis.

Moxifloxacin and Sitafloxacin Treatment Failure in Mycoplasma genitalium Infection: Association with parC Mutation G248T (S83I) and Concurrent gyrA Mutations.

BACKGROUND: The basis of fluoroquinolone treatment failure for Mycoplasma genitalium is poorly understood. METHODS: To identify mutations associated with failure we sequenced key regions of the M. genitalium parC and gyrA genes for patients undergoing sequential therapy with doxycycline-moxifloxacin (201 patients, including 21 with failure) or doxycycline-sitafloxacin (126 patients, including 13 with failure). RESULTS: The parC G248T/S83I mutation was more common among patients with failed sequential doxycycline-moxifloxacin (present in 76.2% of failures vs 7.8% cures, P < .001) or doxycycline-sitafloxacin (50% vs 16.8%, respectively; P = .01) treatment. Doxycycline-sitafloxacin was more efficacious than doxycycline-moxifloxacin against infections carrying the parC mutation conferring S83I amino acid change. Treatment was more likely to fail in these infections if they had a concurrent gyrA mutation (M95I or D99N) (P = .07 for doxycycline-moxifloxacin group and P = .009 for doxycycline-sitafloxacin group), suggesting an additive effect. CONCLUSIONS: This study indicates that parC G248T/S83I mutations contribute to failure of moxifloxacin and sitafloxacin, and the findings will inform the development of quinolone resistance assays needed to ensure optimal selection of antimicrobials for M. genitalium.

Chromosomal and plasmid-mediated fluoroquinolone resistance in human Salmonella enterica infection in Ghana.

BACKGROUND: Salmonella infection poses significant public health threat globally, especially in resource-limited countries. Emergence and spread of antibiotic resistant strains to fluoroquinolones have led to treatment failures and increased mortality in Salmonella infection. However, there is dearth of information regarding mechanisms of resistance to fluoroquinolones in Ghana. This study therefore sought to identify chromosomal mutations and plasmid-mediated resistance as possible mechanisms of fluoroquinolone resistance from clinical isolates in Ghana. METHODS: This was a retrospective study of archived isolates biobanked at Kumasi Centre for Collaborative Research in Tropical Medicine, Ghana. Isolates were obtained from blood, stool and oropharynx samples at two hospitals, between May, 2016 and January, 2018. Salmonella identification was done using standard microbiological protocols and antibiotic susceptibility testing performed by Kirby-Bauer disc diffusion method. Isolates with intermediate susceptibility and/or resistance to nalidixic acid and/or ciprofloxacin were selected and examined for chromosomal mutations by Sanger sequencing and plasmid-mediated resistance by PCR. RESULTS: Of 133 biobanked isolates cultured, 68 (51.1%) and 16 (12%) were identified as Salmonella Typhi and non-typhoidal Salmonella (NTS), respectively. Sequence analysis of gyrA gene revealed the presence of 5 different nonsynonymous mutations, with the most frequent mutation (Ile203Ser) occurring in 12 out of 13 isolates tested. Gyrase B (gyrB) gene had 1 nonsynonymous mutation in 3 out of 13 isolates, substituting phenylalanine with leucine at codon 601 (Phe601Leu). No mutation was observed in parC and parE genes. Two NTS isolates were found to harbour qnrS plasmid-mediated resistant gene of molecular size 550 bp with high ciprofloxacin MIC of 0.5 µg/ml. CONCLUSION: This study reports for the first time in Ghana plasmid-mediated fluoroquinolone resistant gene qnrS in Salmonella clinical isolates. Nonsynonymous mutations of gyrA and gyrB genes likely to confer Salmonella reduced susceptibility to ciprofloxacin were also reported.

In Vitro and In Vivo Activity of Zabofloxacin and Other Fluoroquinolones Against MRSA Isolates from A University Hospital in Egypt.

The widespread of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), has necessitated the search for alternative therapies; introduction of new agents being a suggestion. This study compares the in vitro and in vivo activities of zabofloxacin, a novel fluoroquinolone, with moxifloxacin, levofloxacin and ciprofloxacin against clinical isolates of MRSA from patients hospitalized in the Alexandria Main University hospital; a tertiary hospital in Alexandria, Egypt, where zabofloxacin has not been yet introduced. The strains tested showed the highest percentage of susceptibility to zabofloxacin (61.2%) among the tested fluoroquinolones with the most effective MIC50 and MIC90 (0.25 and 2 µg/ml, respectively). Time-kill curve analysis revealed a rapid bactericidal activity of zabofloxacin after 6 h of incubation with a quinolone-resistant isolate and complete killing when tested against a quinolone-sensitive isolate with inhibition of regrowth in both cases. PCR amplification and sequencing of QRDRs in selected strains revealed the following amino acid substitutions: Ser-84→Leu in GyrA, Ser-80→Phe in GrlA and Pro-451→Ser in GrlB. The in vivo studies demonstrated that zabofloxacin possessed the most potent protective effect against systemic infection in mice (ED50: 29.05 mg/kg) with lowest count in the dissected lungs (3.66 log10 CFU/ml). The histopathological examination of lung specimens of mice treated with zabofloxacin displayed least congestion, inflammation, oedema and necrosis with clear alveolar spaces and normal vessels. In conclusion, zabofloxacin was proved to possess high in vitro and in vivo efficacy encompassing its comparators and could be considered as a possible candidate for the treatment of infections caused by MRSA. To our knowledge, this is the first study evaluating the in vitro and in vivo activity of zabofloxacin against Egyptian MRSA clinical isolates.The widespread of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), has necessitated the search for alternative therapies; introduction of new agents being a suggestion. This study compares the in vitro and in vivo activities of zabofloxacin, a novel fluoroquinolone, with moxifloxacin, levofloxacin and ciprofloxacin against clinical isolates of MRSA from patients hospitalized in the Alexandria Main University hospital; a tertiary hospital in Alexandria, Egypt, where zabofloxacin has not been yet introduced. The strains tested showed the highest percentage of susceptibility to zabofloxacin (61.2%) among the tested fluoroquinolones with the most effective MIC50 and MIC90 (0.25 and 2 µg/ml, respectively). Time-kill curve analysis revealed a rapid bactericidal activity of zabofloxacin after 6 h of incubation with a quinolone-resistant isolate and complete killing when tested against a quinolone-sensitive isolate with inhibition of regrowth in both cases. PCR amplification and sequencing of QRDRs in selected strains revealed the following amino acid substitutions: Ser-84→Leu in GyrA, Ser-80→Phe in GrlA and Pro-451→Ser in GrlB. The in vivo studies demonstrated that zabofloxacin possessed the most potent protective effect against systemic infection in mice (ED50: 29.05 mg/kg) with lowest count in the dissected lungs (3.66 log10 CFU/ml). The histopathological examination of lung specimens of mice treated with zabofloxacin displayed least congestion, inflammation, oedema and necrosis with clear alveolar spaces and normal vessels. In conclusion, zabofloxacin was proved to possess high in vitro and in vivo efficacy encompassing its comparators and could be considered as a possible candidate for the treatment of infections caused by MRSA. To our knowledge, this is the first study evaluating the in vitro and in vivo activity of zabofloxacin against Egyptian MRSA clinical isolates.

Microbiological Analysis from a Phase 2 Randomized Study in Adults Evaluating Single Oral Doses of Gepotidacin in the Treatment of Uncomplicated Urogenital Gonorrhea Caused by Neisseria gonorrhoeae.

We evaluated microbiological correlates for the successful treatment of Neisseria gonorrhoeae isolates from a phase 2 study of gepotidacin, a novel triazaacenaphthylene antibacterial, for therapy of uncomplicated urogenital gonorrhea. Culture, susceptibility testing, genotypic characterization, and frequency of resistance (FoR) were performed for selected isolates. Microbiological success was defined as culture-confirmed eradication of N. gonorrhoeae Against 69 baseline urogenital isolates, gepotidacin MICs ranged from ≤0.06 to 1 µg/ml (MIC90 = 0.5 µg/ml). For gepotidacin, the ratio of the area under the free-drug concentration-time curve to the MIC (fAUC/MIC) was associated with therapeutic success. Success was 100% (61/61) at fAUC/MICs of ≥48 and decreased to 63% (5/8) for fAUC/MICs of ≤25. All 3 isolates from microbiological failures were ciprofloxacin resistant, had a baseline gepotidacin MIC of 1 µg/ml, and carried a preexisting ParC D86N mutation, a critical residue for gepotidacin binding. In a test-of-cure analysis, the resistance to gepotidacin emerged in 2 isolates (MICs increased ≥32-fold) with additional GyrA A92T mutations, also implicated in gepotidacin binding. Test-of-cure isolates had the same sequence type as the corresponding baseline isolates. For 5 selected baseline isolates, all carrying a ParC D86N mutation, the in vitro FoR to gepotidacin was low (10-9 to 10-10); the resistant mutants had the same A92T mutation as the 2 isolates in which resistance emerged. Five participants with isolates harboring the ParC D86N mutation were treatment successes. In summary, fAUC/MICs of ≥48 predicted 100% microbiological success, including 3 isolates with the ParC D86N mutation (fAUC/MICs ≥ 97). Pharmacokinetic/pharmacodynamic determinations may help to evaluate new therapies for gonorrhea; further study of gepotidacin is warranted. (This study has been registered at ClinicalTrials.gov under identifier NCT02294682.).

Observation of a new pattern of mutations in gyrA and parC within Escherichia coli exhibiting fluroquinolone resistance.

Therapeutic options with quinolones are severely compromised in infections caused by members of Enterobacteriaceae family. Mutations in chromosomal region are one of the major reasons for bacterial resistance towards this group of antibiotic. The aim of the study is to detect the mutations in gyr A and par C responsible for quinolone resistance among clinical isolates of Escherichia coli. A total of 96 quinolone-resistant clinical isolates of E. coli were collected from a tertiary care hospital of North-east India during March 2015 to August 2015. All the quinolone-resistant E. coli strains were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the quinolone resistance determining regions. Among the 96 E. coli isolates, 83.3% were resistant to nalidixic acid and 80.2%, 66.6%, 23.9% and 50% to ciprofloxacin, norfloxacin, levofloxacin and ofloxacin, respectively. Several alterations were detected in gyrA and parC genes. Three new patterns of amino acid substitution are reported in E. coli isolates. The findings of this study warrant a review in quinolone-based therapy in this region of the world to stop or slow down the irrational use this drug.

Laboratory investigation of a suspected outbreak caused by Providencia stuartii with intermediate resistance to imipenem at a long-term care facility.

BACKGROUND: Providencia stuartii survives well in natural environment and often causes opportunistic infection in residents of long-term care facilities (LTCFs). Clinical isolates of P. stuartii are usually resistant to multiple antibiotics. The bacterium is also naturally resistant to colistin and tigecycline. Treatment of infections caused by carbapenem-resistant P. stuartii is challenging. METHODS: During a 15-month period in 2013-2014, four isolates (P1, P2, and P3B/P3U) of P. stuartii showing intermediate resistance to imipenem were identified at a regional hospital in southern Taiwan. They were identified from three patients (P1-P3) transferred from the same LTCF for the treatment of the infection. Pulsed-field gel electrophoresis was used to genotype the isolates. Resistance genes/plasmids and outer membrane proteins were investigated by polymerase chain reaction and sequence analysis. RESULTS: Isolates P1 and P3B/P3U demonstrated similar pulsotypes. All isolates were found to have resistance genes (blaCMY-2, qnrD1, aac(6')-Ib-cr) carried on nonconjugative IncA/C plasmids of different sizes. A single point mutation was identified in the chromosomal gyrA (Ser83Ile) and parC (Ser84Ile) genes of all isolates. Various point mutations and insertion/deletion changes were found in their major outer membrane protein gene ompPst1. CONCLUSIONS: Isolates of similar pulsotypes could appear after 15 months and caused urosepsis in another resident of the same LTCF. The bacterium may have persisted in the environment and caused opportunistic infection. As LTCF residents are usually vulnerable to infections, surveillance of multidrug-resistant organisms and infection control intervention that have been established in acute-care hospitals to control infections by resistant organisms are apparently as essential in LTCFs.

Clinical characteristics in adult patients with Salmonella bacteremia and analysis of ciprofloxacin-nonsusceptible isolates.

BACKGROUND/PURPOSE: The purpose of this study is to describe clinical characteristics of Salmonella bacteremia in adult patients and analyze ciprofloxacin-nonsusceptible isolates. METHODS: A total of 101 Salmonella blood isolates from adult patients were collected from January 2011 to December 2013 in MacKay Memorial Hospital. Eight ciprofloxacin-nonsusceptible Salmonella blood isolates were screened for carbapenemase and other ß lactamase genes. Isolates were examined by PCR for the quinolone resistance-determining region (QRDR) of all subunits for DNA gyrase (gyrA and gyrB) genes and topoisomerase IV (parC and parE) genes. RESULTS: There were 22 (21.78%) S. enterica serovar B, 5 (4.95%) S. enterica serovar C1, 7 (6.93%) S. enterica serovar C2, 65 (64.36%) S. enterica serovar D, and 2 (1.98%) S. enterica serovar Typhi (S. typhi) isolates. ß-lactamase gene screening and sequencing yielded only one blaCMY-2-positive isolate. In multivariate risk factor analysis, renal insufficiency [odds ratio (OR) 3.774; p = 0.020] and heart disease (OR 2.922; p = 0.027) were more common among elderly patients (≥65 years). Independent risk factors for ciprofloxacin-nonsusceptible strains included S. enterica serovar C2 (OR 28.430; p = 0.032), renal insufficiency (OR 13.927; p = 0.032), and immunosuppression agent usage (OR 60.082; p = 0.006). 87.50% (7/8) of isolates had gyrA mutation, 62.50% (5/8) had parC mutation, and none had gyrB and parE mutations. Isolates with both Ser83Phe/Asp87Asn gyrA and Thr57Ser/Ser80Ile parC mutation genes were highly ciprofloxacin-resistant (minimum inhibitory concentration ≥4 mg/L). CONCLUSIONS: Elderly patients with renal insufficiency and heart disease were at risk for Salmonella bacteremia. Those for ciprofloxacin-nonsusceptible strains included S. enterica serovar C2, renal insufficiency, and immunosuppression agent usage. The 8 ciprofloxacin-nonsusceptible isolates carried gyrA and parC mutations, which cause resistance that poses a major concern.

Quinolone resistance and ornithine decarboxylation activity in lactose-negative Escherichia coli.

Quinolones and fluoroquinolones are widely used to treat uropathogenic Escherichia coli infections. Bacterial resistance to these antimicrobials primarily involves mutations in gyrA and parC genes. To date, no studies have examined the potential relationship between biochemical characteristics and quinolone resistance in uropathogenic E. coli strains. The present work analyzed the quinolone sensitivity and biochemical activities of fifty-eight lactose-negative uropathogenic E. coli strains. A high percentage of the isolates (48.3%) was found to be resistant to at least one of the tested quinolones, and DNA sequencing revealed quinolone resistant determining region gyrA and parC mutations in the multi-resistant isolates. Statistical analyses suggested that the lack of ornithine decarboxylase (ODC) activity is correlated with quinolone resistance. Despite the low number of isolates examined, this is the first study correlating these characteristics in lactose-negative E. coli isolates.

Quinolone resistance and ornithine decarboxylation activity in lactose-negative Escherichia coli

Quinolones and fluoroquinolones are widely used to treat uropathogenic Escherichia coli infections. Bacterial resistance to these antimicrobials primarily involves mutations in gyrA and parC genes. To date, no studies have examined the potential relationship between biochemical characteristics and quinolone resistance in uropathogenic E. coli strains. The present work analyzed the quinolone sensitivity and biochemical activities of fifty-eight lactose-negative uropathogenic E. coli strains. A high percentage of the isolates (48.3%) was found to be resistant to at least one of the tested quinolones, and DNA sequencing revealed quinolone resistant determining region gyrA and parC mutations in the multi-resistant isolates. Statistical analyses suggested that the lack of ornithine decarboxylase (ODC) activity is correlated with quinolone resistance. Despite the low number of isolates examined, this is the first study correlating these characteristics in lactose-negative E. coli isolates.

Molecular characterization of fluoroquinolone resistance in nontypeable Haemophilus influenzae clinical isolates.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of respiratory infections in adults, who are frequently treated with fluoroquinolones. The aims of this study were to characterize the genotypes of fluoroquinolone-resistant NTHi isolates and their mechanisms of resistance. Among 7,267 H. influenzae isolates collected from adult patients from 2000 to 2013, 28 (0.39%) were ciprofloxacin resistant according to Clinical and Laboratory Standards Institute (CLSI) criteria. In addition, a nalidixic acid screening during 2010 to 2013 detected five (0.23%) isolates that were ciprofloxacin susceptible but nalidixic acid resistant. Sequencing of their quinolone resistance-determining regions and genotyping by pulse-field gel electrophoresis and multilocus sequence typing of the 25 ciprofloxacin-resistant isolates available and all 5 nalidixic acid-resistant isolates were performed. In the NTHi isolates studied, two mutations producing changes in two GyrA residues (Ser84, Asp88) and/or two ParC residues (Ser84, Glu88) were associated with increased fluoroquinolone MICs. Strains with one or two mutations (n = 15) had ciprofloxacin and levofloxacin MICs of 0.12 to 2 µg/ml, while those with three or more mutations (n = 15) had MICs of 4 to 16 µg/ml. Long persistence of fluoroquinolone-resistant strains was observed in three chronic obstructive pulmonary disease patients. High genetic diversity was observed among fluoroquinolone-resistant NTHi isolates. Although fluoroquinolones are commonly used to treat respiratory infections, the proportion of resistant NTHi isolates remains low. The nalidixic acid disk test is useful for detecting the first changes in GyrA or in GyrA plus ParC among fluoroquinolone-susceptible strains that are at a potential risk for the development of resistance under selective pressure by fluoroquinolone treatment.

Induction of resistant mutants of Salmonella enterica serotype Typhi under ciprofloxacin selective pressure.

BACKGROUND & OBJECTIVES: Infection with Salmonella enterica serovar Typhi (hereafter S. Typhi) is an important public health problem in India. There has been an increase in the number of reported clinical failures to ciprofloxacin treatment but the data on possible mechanism of failure are limited. One mechanism that has been widely reported and found associated with ciprofloxacin resistance, is the mutations in target genes in QRDR (quinolone resistance determining region). It is hypothesized that mutations in DNA gyrase or topoisomerase IV result in therapeutic failure under selective pressure of antibiotic while the patient is on treatment. We undertook in vitro sequential selection studies to expose the clinical isolates of S. Typhi to different concentration of ciprofloxacin to study the role of antibiotic selective pressure in the development of mutations in QRDR. METHODS: Total 26 clinical isolates were divided in to two parts: part I included six isolates obtained from three patients with relapse of enteric fever and part II included 20 isolates with different ciprofloxacin MIC levels. For in vitro induction of mutation experiment, five S. Typhi isolates were selected which included three NAS (nalidixic acid sensitive) and 2 NAR (nalidixic acid resistant) S. Typhi. These isolates were grown under increasing concentrations of ciprofloxacin and mutations acquired in QRDR of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) were investigated by sequencing. RESULTS: For the isolates included in the part I of the study, it was found that the MIC to ciprofloxacin increased in the isolates obtained during the relapse of enteric fever as compare to the first isolate. All isolates had single mutation in gyrA gene at S83 without additional mutation in the second isolate. In the second part of the study, the nine isolates with varying MICs to ciprofloxacin also had single mutation in gyrA gene at S83 and another six had triple mutations, two mutations in gyrA gene (at S83 and D87) and one mutation in parC gene (at S80). In in vitro induction of mutation experiment, all mutated isolates showed triple mutation (two mutation in gyrA and one in parC gene) while no mutations were found in wild isolates. INTERPRETATION & CONCLUSIONS: Upon exposure to the step-wise increased concentration of ciprofloxacin, isolates become more tolerant to the ciprofloxacin and showed 2-4 fold higher MICs without new mutation after 8 µg/ml. So the accumulation of mutations under continuous ciprofloxacin pressure and tolerance of the mutant isolates led to the clinical failure. These results also suggested that there could be another mechanism responsible for resistance.

Failure of moxifloxacin treatment in Mycoplasma genitalium infections due to macrolide and fluoroquinolone resistance.

Increasing azithromycin treatment failure in sexually transmitted Mycoplasma genitalium infection, is linked to macrolide resistance and second-line treatment relies on the fluoroquinolone, moxifloxacin. We recently detected fluoroquinolone and macrolide resistance-associated mutations in 15% and 43%, respectively, of 143 initial M. genitalium PCR-positive specimens. For a subset of 33 Western Sydney Sexual Health Centre patients, clinical information and results of sequence analysis of M. genitalium macrolide and fluoroquinolone target genes - the 23S rRNA gene, and parC and gyrA, respectively - were used to examine whether mutations were associated with treatment failure. Macrolide resistance-associated mutations correlated with microbiological (p = 0.013) and clinical (p = 0.024) treatment failure, and fluoroquinolone resistance-associated mutations with microbiological moxifloxacin treatment failure (p = 0.005). We describe the first reported cases of clinical and microbiological moxifloxacin treatment failure. Failure of first- and second-line antibiotic treatment of M. genitalium infection is occurring and likely to increase with current treatment strategies.

MIDGET connects COP1-dependent development with endoreduplication in Arabidopsis thaliana.

In Arabidopsis thaliana, loss of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) function leads to constitutive photomorphogenesis in the dark associated with inhibition of endoreduplication in the hypocotyl, and a post-germination growth arrest. MIDGET (MID), a component of the TOPOISOMERASE VI (TOPOVI) complex, is essential for endoreduplication and genome integrity in A. thaliana. Here we show that MID and COP1 interact in vitro and in vivo through the amino terminus of COP1. We further demonstrate that MID supports sub-nuclear accumulation of COP1. The MID protein is not degraded in a COP1-dependent fashion in darkness, and the phenotypes of single and double mutants prove that MID is not a target of COP1 but rather a necessary factor for proper COP1 activity with respect to both, control of COP1-dependent morphogenesis and regulation of endoreduplication. Our data provide evidence for a functional connection between COP1 and the TOPOVI in plants linking COP1-dependent development with the regulation of endoreduplication.

High prevalence of multiresistance in levofloxacin-nonsusceptible Streptococcus pneumoniae isolates in Korea.

Korea exhibits the highest rates of multidrug resistance among Streptococcus pneumoniae. The increasing use of levofloxacin has raised concern about the dissemination of levofloxacin resistance in dominant multidrug-resistant (MDR) clones of our pneumococcal population. A total of 50 levofloxacin-nonsusceptible S. pneumoniae (MIC, ≥4 µg/mL) collected from a multihospital network from 1996 to 2006 were analyzed for serotype, antibiotic resistance profile, quinolone resistance-determining region mutation, and multilocus sequence type. Most levofloxacin-nonsusceptible S. pneumoniae (94.0%) exhibited an MDR phenotype. This phenotype was closely associated with a limited number of epidemic MDR clones that are well-known key agents of the global spread of antimicrobial resistance in S. pneumoniae. However, the clonal dissemination of levofloxacin-nonsusceptible S. pneumoniae was rare. Levofloxacin-nonsusceptible clones with nonvaccine serotypes increased during the post-vaccine era in this study. This result suggests that Korean clinicians must be aware of the levofloxacin resistance trend and need to be more prudent for the first choice of fluoroquinolone for empiric treatment of respiratory tract infections in clinical setting. Moreover, the emergence of new clones and their variations may be more frequently associated with resistance under this selective pressure, such as the introduction of a 7-valent pneumococcal conjugate vaccine into our community.

Impact of drug-exposure intensity and duration of therapy on the emergence of Staphylococcus aureus resistance to a quinolone antimicrobial.

We have shown previously in animal model and in vitro systems that antimicrobial therapy intensity has a profound influence on subpopulations of resistant organisms. Little attention has been paid to the effect of therapy duration on resistant subpopulations. We examined the influence of therapy intensity (area under the concentration/time curve for 24 h:minimum inhibitory concentration [AUC24:MIC] ratio) and therapy duration on resistance emergence using an in vitro model of Staphylococcus aureus infection. AUC24:MIC ratios of>or=100 were necessary to kill a substantial portion of the total population. Importantly, we demonstrated that therapy duration is a critical parameter. As the duration increased beyond 5 days, the intensity needed to suppress the antibiotic-resistant subpopulations increased, even when the initial bacterial kill was>4 log10 (cfu/mL). These findings were prospectively validated in an independent experiment in which exposures were calculated from the results of fitting a large mathematical model to all data simultaneously. All of the prospectively determined predictions were fulfilled in this validation experiment.

Persistence of Mycoplasma hyopneumoniae in experimentally infected pigs after marbofloxacin treatment and detection of mutations in the parC gene.

The ability of Mycoplasma hyopneumoniae to persist despite fluoroquinolone treatments was investigated with pigs. Groups of specific-pathogen-free pigs were experimentally infected with M. hyopneumoniae strain 116 and treated with marbofloxacin at the therapeutic dose (TD) or half of the therapeutic dose (TD/2) for 3 days. Results showed that, despite tissue penetration of marbofloxacin, particularly in the trachea and the tracheal secretions, the treatments did not have any influence on M. hyopneumoniae recovery from tracheal swabs. Mycoplasmas were also isolated from inner organs and tissues such as liver, spleen, kidneys, and bronchial lymph nodes. Recontamination of pigs via environment could not explain mycoplasma persistence after medication, as decontamination of pigs and allocation to a new disinfected environment did not have any significant effect on the phenomenon. A significant decrease in the susceptibility level to marbofloxacin of 12 mycoplasma clones reisolated after the treatments (TD/2 and TD) was observed. Two point mutations were found in the ParC quinolone resistance-determining region (QRDR) of DNA topoisomerase IV (Ser80-->Phe and Asp84-->Asn), and one point mutation was observed just behind the QRDR of ParC (Ala116-->Glu). This is the first time that mutations in a gene coding for topoisomerase IV have been described for M. hyopneumoniae after in vivo marbofloxacin treatments in experimentally infected pigs. However, development of resistance is not sufficient to explain M. hyopneumoniae persistence in vivo since (i) marbofloxacin concentrations were above the marbofloxacin MIC of the wild-type strain and (ii) mycoplasmas reisolated after a single injection of marbofloxacin did not display an increased marbofloxacin MIC.

Short-course therapy of gemifloxacin effective against pneumococcal pneumonia in mice.

Standard 7-14 day (d) courses of antimicrobial therapy for community-acquired pneumonia (CAP) are thought to have contributed to the emergence of resistant pneumoccoci. Consequently, short-course fluoroquinolone regimens have been proposed to minimize resistance. To test this, we examined 2-day versus 5-day regimens of gemifloxacin and levofloxacin for treatment of pneumonia in a murine model. In doing so, we also investigated whether the enhanced potency of gemifloxacin would influence outcomes. CD1 Swiss mice were infected intratracheally with 10(5)-CFU of a virulent Streptococcus pneumoniae strain. Drugs were administered every 8 h for 2 d and 5 d, starting at 24 h postinfection. Temperature was used to assess disease progression. Gemifloxacin remained effective for 2 d and 5 d, with survival rates of 100%-83% compared with 40%-58% for levofloxacin. Eighty-nine to 100% of gemifloxacin-treated mice were clear of pulmonary bacteria compared with only 0%-20% for levofloxacin. For levofloxacin-treated mice, 2 of 7 (29%) isolates with a levofloxacin minimum inhibitory concentration (MIC) 4 times that of the infecting parent strain had ParC mutations. By contrast, no isolates recovered from gemifloxacin-treated mice were reduced in susceptibility. Gemifloxacin could be effective in shortening duration of therapy for CAP treatment as well as minimize resistance development.