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There has been renewed interest in combining traditional small-molecule antimicrobial agents with nontraditional therapies to potentiate antimicrobial effects. Apotransferrin, which decreases iron availability to microbes, is one such approach. We conducted a 48-h one-compartment infection model to explore the impact of apotransferrin on the bactericidal activity of ciprofloxacin. The challenge panel included four isolates with ciprofloxacin MIC values ranging from 0.08 to 32 mg/liter. Each challenge isolate was subjected to an ineffective ciprofloxacin monotherapy exposure (free-drug area under the concentration-time curve over 24 h divided by the MIC [AUC/MIC ratio] ranging from 0.19 to 96.6) with and without apotransferrin. As expected, the no-treatment and apotransferrin control arms showed unaltered prototypical logarithmic bacterial growth. We identified relationships between exposure and change in bacterial density for ciprofloxacin alone ( = 0.64) and ciprofloxacin in combination with apotransferrin ( = 0.84). Addition of apotransferrin to ciprofloxacin enabled a remarkable reduction in bacterial density across a wide range of ciprofloxacin exposures. For instance, at a ciprofloxacin AUC/MIC ratio of 20, ciprofloxacin monotherapy resulted in nearly 2 log CFU increase in bacterial density, while the combination of apotransferrin and ciprofloxacin resulted in 2 log CFU reduction in bacterial density. Furthermore, addition of apotransferrin significantly reduced the emergence of ciprofloxacin-resistant subpopulations compared to monotherapy. These data demonstrate that decreasing the rate of bacterial replication with apotransferrin in combination with antimicrobial therapy represents an opportunity to increase the magnitude of the bactericidal effect and to suppress the growth rate of drug-resistant subpopulations.
Copyright © 2019 American Society for Microbiology.
Although fluoroquinolones (FQs) play an important role in the treatment of multidrug-resistant tuberculosis (MDR-TB), there are several issues that need to be addressed to optimize their effectiveness and minimize toxicity. This includes identification of the optimal dose of FQs such as levofloxacin (LVX) and moxifloxacin, and the optimal role of FQs in combination with other anti-tuberculosis drugs, particularly those with overlapping toxicity, such as QT prolongation. While the ability of FQs to penetrate into cavities and granulomas is likely beneficial, suboptimal sensitivity of genotypic tests to detect FQ resistance could negatively affect treatment outcomes of FQ-containing regimens. Several trials are underway to evaluate the safety and effectiveness of FQs as part of combination MDR-TB therapy; there are also two planned studies of LVX to prevent tuberculosis among close contacts of MDR-TB.
BACKGROUND/PURPOSE - Escherichia coli is a common pathogen to cause clinical and subclinical mastitis in cows. A total of 57 E. coli isolates from raw milk from cows were characterized genetically and biochemically.
METHODS - Extended-spectrum β-lactamase (ESBL) genes, the mechanism for fluoroquinolone resistance, and variations in virulence genes and genomes of these E. coli isolates were investigated by the antimicrobial susceptibility test, simplex and multiplex polymerase chain reaction (PCR), and pulsed-field gel electrophoresis (PFGE).
RESULTS - All E. coli isolates were resistant to cloxacillin (100%) and to a lesser extent (50%) to tetracycline, neomycin, gentamycin, ampicillin, ceftriaxone, cefotaxime (CTX), and ceftazidime (CAZ). Nearly 70% of the isolates were resistant to at least two antimicrobials and 28.1% carried AmpA and AmpC genes simultaneously. The predominant bla gene was bla, followed by bla, bla, bla, and bla Among the six (10.5%) ESBL-producing E. coli carrying bla, bla, or bla, two isolates 31 of ST410 in the ST23 complex and 58 of ST167 in the ST10 complex were also resistant to ciprofloxacin, enrofloxacin, and levofloxacin, with mutations at codon 83 from serine to leucine and codon 87 from aspartic acid to asparagine in GyrA and at codon 80 from serine to isoleucine in ParC. These isolates were genetically diverse in pulsotype analysis, lacked toxin genes of human pathogenic E. coli and carried mostly the prevalent virulence genes fimH, papGII, and α-hemolysin.
CONCLUSION - Lacking virulence genes examined, genetic diverse E. coli isolates are unrelated to human pathogenic E. coli. Enhancing sanitation in milk processing and transportation is needed to eliminate multidrug-resistant (MDR), fluoroquinolone-resistant, and ESBL-producing E. coli isolates.
Copyright © 2014. Published by Elsevier B.V.
SUMMARY Fluoroquinolone use before tuberculosis (TB) diagnosis delays the time to diagnosis and treatment, and increases the risk of fluoroquinolone-resistant TB and death. Ascertainment of fluoroquinolone exposure could identify such high-risk patients. We compared four methods of ascertaining fluoroquinolone exposure in the 6 months prior to TB diagnosis in culture-confirmed TB patients in Tennessee from January 2007 to December 2009. The four methods included a simple questionnaire administered to all TB suspects by health department personnel (FQ-Form), an in-home interview conducted by research staff, outpatient and inpatient medical record review, and TennCare pharmacy database review. Of 177 TB patients included, 72 (41%) received fluoroquinolones during the 6 months before TB diagnosis. Fluoroquinolone exposure determined by review of inpatient and outpatient medical records was considered the gold standard for comparison. The FQ-Form had 61% [95% confidence interval (CI) 48-73] sensitivity and 93% (95% CI 85-98) specificity (agreement 79%, kappa = 0.56) while the in-home interview had 28% (95% CI 18-40) sensitivity and 99% (94-100%) specificity (agreement 68%, kappa = 0.29). A simple questionnaire administered by health department personnel identified fluoroquinolone exposure before TB diagnosis with moderate reliability.
Heteroresistance is the coexistence of populations with differing nucleotides at a drug resistance locus within a sample of organisms. Although Sanger sequencing is the gold standard for sequencing, it may be less sensitive than deep sequencing for detecting fluoroquinolone heteroresistance in Mycobacterium tuberculosis. Twenty-seven fluoroquinolone monoresistant and 11 fluoroquinolone-susceptible M. tuberculosis isolates were analyzed by Sanger and Illumina deep sequencing. Individual sequencing reads were analyzed to detect heteroresistance in the gyrA and gyrB genes. Heteroresistance to fluoroquinolones was identified in 10/26 (38%) phenotypically fluoroquinolone-resistant samples and 0/11 (P = 0.02) fluoroquinolone-susceptible controls. One resistant sample was excluded because of contamination with the laboratory strain M. tuberculosis H37Rv. Sanger sequencing revealed resistance-conferring mutations in 15 isolates, while deep sequencing revealed mutations in 20 isolates. Isolates with fluoroquinolone resistance-conferring mutations by Sanger sequencing all had at least those same mutations identified by deep sequencing. By deep sequencing, 10 isolates had a single fixed (defined as >95% frequency) mutation, while 10 were heteroresistant, 5 of which had a single unfixed (defined as <95% frequency) mutation and 5 had multiple unfixed mutations. Illumina deep sequencing identified a higher proportion of fluoroquinolone-resistant M. tuberculosis isolates with heteroresistance than did Sanger sequencing. The heteroresistant isolates frequently demonstrated multiple mutations, but resistant isolates with fixed mutations each had only a single mutation.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
The need for new antibiotics that address serious Gram-negative infections is well recognized. Our efforts with a series of novel bacterial type II topoisomerase inhibitors (NBTIs) led to the discovery of NBTI 5463, an agent with improved activity over other NBTIs against Gram-negative bacteria, in particular against Pseudomonas aeruginosa (F. Reck, D. E. Ehmann, T. J. Dougherty, J. V. Newman, S. Hopkins, G. Stone, N. Agrawal, P. Ciaccio, J. McNulty, H. Barthlow, J. O'Donnell, K. Goteti, J. Breen, J. Comita-Prevoir, M. Cornebise, M. Cronin, C. J. Eyermann, B. Geng, G. R. Carr, L. Pandarinathan, X. Tang, A. Cottone, L. Zhao, N. Bezdenejnih-Snyder, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections.
Fluoroquinolone exposure before tuberculosis (TB) diagnosis is common. We anticipated that exposure to older-generation fluoroquinolones is associated with greater fluoroquinolone MICs in Mycobacterium tuberculosis than exposure to newer agents. A nested case-control study was performed among newly diagnosed TB patients reported to the Tennessee Department of Health (January 2002-December 2009). Each fluoroquinolone-resistant case (n=25) was matched to two fluoroquinolone-susceptible controls (n=50). Ciprofloxacin and ofloxacin were classified as older-generation fluoroquinolones; levofloxacin, moxifloxacin and gatifloxacin were considered newer agents. There was no difference between median ofloxacin MIC for isolates from 9 patients exposed only to older fluoroquinolones, 25 exposed only to newer fluoroquinolones, 6 exposed to both and 35 fluoroquinolone-unexposed patients (Kruskal-Wallis, P=0.35). Using multivariate proportional odds logistic regression adjusting for age and sex, duration of exposure to newer fluoroquinolones was independently associated with higher MIC (OR=1.79, 95% CI 1.22-2.64), but duration of exposure to older fluoroquinolones was not (OR=0.94, 95% CI 0.50-1.78). Isolates from patients exposed only to newer fluoroquinolones tended to have mutations at gyrA codons 90, 91 or 94 more frequently than those exposed only to older fluoroquinolones (44% vs. 11%). We were surprised to find that duration of exposure to newer fluoroquinolones, but not older ones, was independently associated with higher ofloxacin MIC. This suggests that the mutant selection window lower boundary is likely to have clinical relevance; caution is warranted when newer fluoroquinolones are prescribed to patients with TB risk factors.
Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
SETTING - Fluoroquinolone (FQ) exposure before tuberculosis (TB) diagnosis is common, but its effect on outcomes, including mortality, is unclear.
DESIGN - Among TB patients reported to the Tennessee Department of Health from 2007 to 2009, we assessed FQ exposure within 6 months before TB diagnosis. The primary outcome was the combined endpoint of death at the time of TB diagnosis and during anti-tuberculosis treatment.
RESULTS - Among 609 TB cases, 214 (35%) received FQs within 6 months before TB diagnosis. A total of 71 (12%) persons died; 10 (2%) were dead at TB diagnosis and 61 (10%) died during anti-tuberculosis treatment. In multivariable logistic regression analysis, factors independently associated with death were older age (OR 1.05 per year, 95%CI 1.04-1.07), human immunodeficiency virus infection (OR 8.08, 95%CI 3.83-17.06), US birth (OR 3.03, 95%CI 1.03-9.09), and any FQ exposure before TB diagnosis (OR 1.82, 95%CI 1.05-3.15). Persons with FQ exposure before TB diagnosis were more likely to have culture- and smear-positive disease than unexposed persons.
CONCLUSIONS - Among this patient population, FQ exposure before TB diagnosis was associated with an increased risk of death. These findings underscore the need for cautious use of FQs in persons with possible TB.
Microcolony growth of Mycobacterium tuberculosis on agar proportion susceptibility testing is neither well-defined nor previously reported with fluoroquinolone susceptibility testing. We describe here M. tuberculosis microcolony growth with fluoroquinolones, and assess its clinical significance. We screened 797 M. tuberculosis isolates for ofloxacin resistance (2.0 μg/mL) by agar proportion; 19 ofloxacin-resistant and 38 ofloxacin-susceptible isolates were selected for more detailed susceptibility testing with ofloxacin, ciprofloxacin, levofloxacin (all at 2.0 μg/mL) and moxifloxacin (0.5 μg/mL). The 57 isolates were also tested at two concentrations both above and below the critical concentrations. Microcolonies were defined as colonies 0.2-0.4 mm in diameter; confirmed microcolonies were present on repeat testing. Of the 57 isolates tested in detail, 7 grew microcolonies, of which 2 (0.3% of all isolates tested) had confirmed microcolonies on repeat testing (6 tests performed, and microcolonies were present on at least 4). Both M. tuberculosis isolates were ofloxacin-resistant on screening, and had ofloxacin minimum inhibitory concentration (MIC) >8 μg/mL. The five other isolates were ofloxacin-susceptible on screening, but had regular colony growth (i.e., resistance) at the drug concentration that initially resulted in microcolonies (ofloxacin 0.5 or 1.0 μg/mL). Microcolonies were observed infrequently with fluoroquinolone susceptibility testing, but when confirmed, they were associated with drug resistance.