Glen T. Hansen

Mutant Prevention Concentrations of Fluoroquinolones for Clinical Isolates of Streptococcus pneumoniae

ABSTRACT The mutant prevention concentration (MPC) represents a threshold above which the selective proliferation of resistant mutants is expected to occur only rarely. A provisional MPC (MPCpr) was defined and measured for five fluoroquinolones with clinical isolates of Streptococcus pneumoniae. Based on their potential for restricting the selection of resistant mutants, the five fluoroquinolones, in descending order, were found to be moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of about 90 clinical isolates that lacked a known resistance mutation had a value of MPCpr that was close to or below the serum levels that could be attained with a dosing regimen recommended by the manufacturers. Since MPCpr overestimates MPC, these data identify moxifloxacin and gatifloxacin as good candidates for determining whether MPCpr can be used as a guide for choosing and eventually administering fluoroquinolones to significantly reduce the development of resistance.

Abrupt Emergence of a Single Dominant Multidrug-Resistant Strain of Escherichia coli

BACKGROUND Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins--potentially critical for control efforts--remain undefined. METHODS Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967-2009) E. coli isolates representing sequence type ST131 and 853 recent (2010-2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. RESULTS Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%-52%). CONCLUSIONS Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.

Molecular epidemiological analysis of Escherichia coli sequence type ST131 (O25:H4) and bla CTX-M-15among extended-spectrum-β- lactamase-producing E. coli from the United States, 2000 to 2009

ABSTRACT Escherichia coli sequence type ST131 (from phylogenetic group B2), often carrying the extended-spectrum-β-lactamase (ESBL) gene blaCTX-M-15, is an emerging globally disseminated pathogen that has received comparatively little attention in the United States. Accordingly, a convenience sample of 351 ESBL-producing E. coli isolates from 15 U.S. centers (collected in 2000 to 2009) underwent PCR-based phylotyping and detection of ST131 and blaCTX-M-15. A total of 200 isolates, comprising 4 groups of 50 isolates each that were (i) blaCTX-M-15 negative non-ST131, (ii) blaCTX-M-15 positive non-ST131, (iii) blaCTX-M-15 negative ST131, or (iv) blaCTX-M-15 positive ST131, also underwent virulence genotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). Overall, 201 (57%) isolates exhibited blaCTX-M-15, whereas 165 (47%) were ST131. ST131 accounted for 56% of blaCTX-M-15-positive- versus 35% of blaCTX-M-15-negative isolates (P < 0.001). Whereas ST131 accounted for 94% of the 175 total group B2 isolates, non-ST131 isolates were phylogenetically distributed by blaCTX-M-15 status, with groups A (blaCTX-M-15-positive isolates) and D (blaCTX-M-15-negative isolates) predominating. Both blaCTX-M-15 and ST131 occurred at all participating centers, were recovered from children and adults, increased significantly in prevalence post-2003, and were associated with molecularly inferred virulence. Compared with non-ST131 isolates, ST131 isolates had higher virulence scores, distinctive virulence profiles, and more-homogeneous PFGE profiles. blaCTX-M-15 was associated with extensive antimicrobial resistance and ST131 with fluoroquinolone resistance. Thus, E. coli ST131 and blaCTX-M-15 are emergent, widely distributed, and predominant among ESBL-positive E. coli strains in the United States, among children and adults alike. Enhanced virulence and antimicrobial resistance have likely promoted the epidemiological success of these emerging public health threats.

Comparison of Escherichia coli ST131 pulsotypes, by epidemiologic traits, 1967-2009

Certain high-prevalence pulsed-field gel electrophoresis types exhibited distinctive temporal patterns and epidemiologic associations.

Comparison of 2 Blood Culture Media Shows Significant Differences in Bacterial Recovery for Patients on Antimicrobial Therapy

BACKGROUND Antimicrobial removal devices in blood culture media are designed to remove antibiotics from the blood culture solution, thereby facilitating bacterial growth. How well these devices function clinically has not been established. METHODS All blood drawn for culture from adult inpatients and emergency department visitors in a level I trauma center was placed in paired BACTEC Plus and BacT/Alert FAN culture media and studied simultaneously, consecutively, and prospectively between 1 February and 30 September 2011. All cultures were processed per standard laboratory protocols. RESULTS Of 9395 total cultures collected, 1219 (13%) were positive, 831 were included, and 524 (33%) contained pathogens. BACTEC had a 4.5-hour faster detection time (P < .0001), and isolated exclusively 182 of 524 (35%; P < .001) pathogens, 136 of 345 (39%) of the gram-positive cocci (P < .001), 48 of 175 (27%; P = .02) of the gram-negative rods, 101 of 195 (52%) of Staphylococcus aureus (P < .001), and 59 of 120 (49%; P = .004) septic events. If active antibiotics had been dosed 0-4 or 4-48 hours prior to culture collection, the odds of that culture growing in BACTEC were 4.8- and 5.2-fold greater, respectively, than of growing in BacT/Alert (P < .0001). Both were equivalent in the recovery of yeast and when no antimicrobials were dosed. CONCLUSIONS BACTEC media has faster time to detection and increased bacterial recovery over the BacT/Alert media in the following categories: overall growth, pathogens, septic events, gram-positive cocci, gram-negative rods, Staphylococcus aureus, and cultures where antimicrobials were dosed up to 48 hours before culture collection.

Identification of Gram-Negative Bacteria and Genetic Resistance Determinants from Positive Blood Culture Broths by Use of the Verigene Gram-Negative Blood Culture Multiplex Microarray-Based Molecular Assay

ABSTRACT Bloodstream infection is a serious condition associated with significant morbidity and mortality. The outcome of these infections can be positively affected by the early implementation of effective antibiotic therapy based on the identification of the infecting organism and genetic markers associated with antibiotic resistance. In this study, we evaluated the microarray-based Verigene Gram-negative blood culture (BC-GN) assay in the identification of 8 genus or species targets and 6 genetic resistance determinants in positive blood culture broths. A total of 1,847 blood cultures containing Gram-negative organisms were tested using the BC-GN assay. This comprised 729 prospective fresh, 781 prospective or retrospective frozen, and 337 simulated cultures representing 7 types of aerobic culture media. The results were compared to those with standard bacterial culture and biochemical identification with nucleic acid sequence confirmation of the resistance determinants. Among monomicrobial cultures, the positive percent agreement (PPA) of the BC-GN assay with the reference method was as follows; Escherichia coli, 100%; Klebsiella pneumoniae, 92.9%; Klebsiella oxytoca, 95.5%; Enterobacter spp., 99.3%; Pseudomonas aeruginosa, 98.9%; Proteus spp., 100%; Acinetobacter spp., 98.4%; and Citrobacter spp., 100%. All organism identification targets demonstrated >99.5% negative percent agreement (NPA) with the reference method. Of note, 25/26 cultures containing K. pneumoniae that were reported as not detected by the BC-GN assay were subsequently identified as Klebsiella variicola. The PPA for identification of resistance determinants was as follows; bla CTX-M, 98.9%; bla KPC, 100%; bla NDM, 96.2%; bla OXA, 94.3%; bla VIM, 100%; and bla IMP, 100%. All resistance determinant targets demonstrated >99.9% NPA. Among polymicrobial specimens, the BC-GN assay correctly identified at least one organism in 95.4% of the broths and correctly identified all organisms present in 54.5% of the broths. The sample-to-result processing and automated reading of the detection microarray results enables results within 2 h of culture positivity.

Mutant Prevention Concentration of Gemifloxacin for Clinical Isolates of Streptococcus pneumoniae

Fluoroquinolone resistance is beginning to appear among isolates of Streptococcus pneumoniae (4, 7, 8, 10). We have argued that resistance arises as a consequence of dosing that places tissue concentrations between the MIC and the mutant prevention concentration (MPC), a new measure of activity related to the MIC of the least susceptible, single-step mutant (15, 16). If this is true, MPC can be used to identify fluoroquinolones that are least likely to selectively enrich resistant subpopulations. We previously estimated MPC for several fluoroquinolones with about 100 clinical isolates of S. pneumoniae obtained from the Royal University Hospital, Saskatoon, Canada (2). We now add gemifloxacin to the list of compounds compared and increase the number of isolates tested to 146 for all of the compounds. Table ​Table11 lists MICs and MPCs for gemifloxacin, moxifloxacin, gatifloxacin, and levofloxacin determined as described previously (2) using the same set of isolates for each compound. Fluoroquinolone-resistant isolates were excluded. Gemifloxacin had the lowest modal MPC (0.25 μg/ml), followed by moxifloxacin (0.5 μg/ml), gatifloxacin (1 μg/ml), and levofloxacin (2 μg/ml). The same rank order was observed when MPC was determined for 90% of the isolates. These data are consistent with gemifloxacin having more activity than the other compounds against resistant mutants (9, 14). When the MIC at which 90% of the susceptible isolates are inhibited (MIC90) was determined, gemifloxacin was also more active than moxifloxacin, gatifloxacin, and levofloxacin in these comparisons by 2, 3, and 4 dilutions, respectively. TABLE 1. Fluoroquinolone activity with clinical isolates of S. pneumoniaea Since the effectiveness of an antibacterial agent is likely to be a function of both activity (MIC and MPC) and pathogen exposure (5, 11), comparision of compounds requires consideration of drug pharmacokinetics in human tissues. From published values of concentrations in serum, we calculated the time above MPC for each compound when dosed as recommended by the manufacturer. Moxifloxacin is expected to have a concentration in serum above the MPC at which 90% of the isolates tested are prevented (MPC90) for 18 h. For gemifloxacin, gatifloxacin, and levofloxacin, those times are 4, 1 to 2, and 0 h, respectively. This suggests that moxifloxacin may be the most effective at restricting the development of resistance, even though gemifloxacin has the lowest MIC and MPC. Table ​Table11 also lists values of the area under the concentration-time curve from 0 to 24 h/MIC and the maximum concentration of drug in serum (Cmax)/MIC for recommended doses. For both parameters gemifloxacin exhibits higher values than moxifloxacin. If these two parameters are inversely related to the selection of resistant mutants (1, 6, 13), resistance should develop less often from treatment with gemifloxacin than with moxifloxacin. But time above MPC (Table ​(Table1)1) and low-concentration cycling (12) predict the opposite outcome. A clinical comparison of these two compounds may help distinguish between MPC-based ideas (15) and empirical pharmacodynamics (6, 13) for predicting the development of resistance. Such a comparison is important because neither method can be easily tested: MPC is an in vitro measure that does not take into account compartments in patients where drug concentrations and bacterial growth properties are poorly defined, and pharmacodynamic methods require examining very large numbers of patients to identify the point at which the overall prevalence of resistance does not increase.

Moxifloxacin: a review of the microbiological, pharmacological, clinical and safety features

Antimicrobial agents are used to treat patients with infectious diseases. Initial antimicrobial compounds originated from natural sources and were generally deemed to be narrow in spectrum. Today, we are in the era of designer drugs that have been specifically developed with current issues in infectious diseases in mind. For example, some new compounds require once daily dosing, have minimal side effects, are active against resistant pathogens and, for some, have a lower propensity for selecting for antimicrobial resistance during patient therapy.

Canadian practice guidelines for surgical intra-abdominal infections

IAIs remain a major challenge in clinical practice. They are the main cause of postoperative morbidity following abdominal surgery and the most frequent cause for admission to a surgical intensive care unit (2,3). IAIs differ from infections encountered elsewhere in several respects. First, the clinical spectrum of IAI is extremely wide, ranging from uncomplicated acute appendicitis with a relatively benign course to diffuse peritonitis from perforated viscus or ischemic bowel with high morbidity and mortality. While both scenarios comprise of IAIs, they require different approaches to diagnosis and treatment. Additionally, the role of surgery in the management of patients with IAIs is pivotal and generally considered to be a decisive factor in the outcome. The clinical and microbiological diagnosis is also often problematic: IAIs are typically polymicrobial, and not every microorganism involved can be identified in the clinical microbiology laboratory by routine cultures; the pathogenicity of certain microorganisms cultured from IAIs is not considered to be the same for every patient and often relates more directly to the severity of underlying disease or comorbid conditions of the host; and the clinical signs and symptoms do not often match the severity of disease and may lead to substantial delays in appropriate diagnosis and management (3–8). Additionally, antibiotic resistance among enteric pathogens has evolved globally and at an alarming rate, while very few newer agents have emerged to replace older therapeutic regimens. The current clinical practice guideline was jointly developed by the CSS and AMMI Canada. The primary goal was to develop updated recommendations for the medical and surgical management of complicated IAIs since publication of the 2003 antimicrobial treatment guideline by the IDSA (1). Particular focus is directed at risk stratification for poor outcomes based on epidemiological studies, current status of antimicrobial susceptibility and resistance profiles among enteric pathogens, therapeutic efficacy of antimicrobial regimens based on randomized clinical trials, operative versus percutaneous approaches for source control, the role of IAH and ACS in IAIs, and infection control and preventive measures for postoperative IAIs and surgical site infections. An additional objective is to categorize the recommendations according to the strength and quality of the available evidence using a standardized grading system. Importantly, the current guideline provides recommendations for initial empirical antimicrobial management of complicated IAIs based on clinical settings and issues unique to the Canadian health care system (eg, publicly funded health care system and regionalization of health care delivery).

In vitro activity of fluoroquinolones against clinical isolates of Nocardia identified by partial 16S rRNA sequencing

Fluoroquinolones have several properties that make them potentially attractive candidates for the treatment of Nocardia infections, but information regarding their in vitro activity is limited. Minimum inhibitory concentrations (MIC) of five fluoroquinolones and other antimicrobials were determined by the reference broth dilution and E-test methods for 33 consecutive clinical isolates of Nocardia speciated by 16S rRNA gene sequences. The isolates included: Nocardia cyriacigeorgica (n = 6), N. nova (n = 8), N. farcinica (n = 8), N. brasiliensis (n = 3), N. asteroides (n = 4), and N. veterana (n = 4). MIC50/MIC90 results for ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin by broth dilution were 32/32, 2/4, 1/4, 32/32, and 2/2 μg/ml, respectively. The MICs by broth dilution and E-test were within a two-fold doubling dilution for 94%, 97%, 97%, 100%, and 100% of isolates for ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin, respectively. For ciprofloxacin, the E-test results showed either complete categorical agreement or minor error compared to the reference broth dilution method for 97% (32/33) of the isolates. For other fluoroquinolones, using Streptococcus pneumoniae breakpoints, 94% (124/132) of MIC results by E-test showed either complete agreement or minor error compared to the reference broth dilution method. Fluoroquinolones show variable in vitro activity against clinical isolates of Nocardia spp., and MICs determined by the E-test show reasonable agreement with those determined by the reference broth dilution method.