M. C. Conejo

Qnr-like pentapeptide repeat proteins in Gram-positive bacteria

OBJECTIVES To study the role of Qnr-like pentapeptide repeat proteins (PRPs) from several gram-positive species with quinolone resistance in vitro. METHODS A PCR-based strategy was used to clone and express genes coding for Qnr-like PRPs in Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Clostridium perfringens, C. difficile, Bacillus cereus and B. subtilis in Escherichia coli DH10B. MIC values of nalidixic acid and fluoroquinolones were determined for reference strains and E. coli DH10B harbouring recombinant plasmids containing genes coding for PRPs. RESULTS Amino acid identity of Qnr-like PRPs in gram-positive strains compared with that of the plasmid-mediated quinolone resistance determinants QnrA1, QnrB1 and QnrS1 was in the range of 16% to 22%. Recombinant plasmids coding for Qnr-like PRPs conferred reduced susceptibility to fluoroquinolones (in the range of 0.016 to 0.064 mg/L for ciprofloxacin) and nalidixic acid (from 6 to 12 mg/L), depending on the antimicrobial agent and PRP. The PRP from B. subtilis showed no protective effect. CONCLUSIONS The PRPs analysed conferred a reduced susceptibility phenotype in E. coli; the data provide further evidence of the possible roles in quinolone resistance of PRPs from different gram-positive species. These gram-positive species may constitute a reservoir for Qnr-like quinolone resistance proteins.

Wastewater drainage system as an occult reservoir in a protracted clonal outbreak due to metallo-β-lactamase-producing Klebsiella oxytoca

We describe the epidemiology of a protracted nosocomial clonal outbreak due to multidrug-resistant IMP-8 producing Klebsiella oxytoca (MDRKO) that was finally eradicated by removing an environmental reservoir. The outbreak occurred in the ICU of a Spanish hospital from March 2009 to November 2011 and evolved over four waves. Forty-two patients were affected. First basic (active surveillance, contact precautions and reinforcement of surface cleaning) and later additional control measures (nurse cohorting and establishment of a minimum patient/nurse ratio) were implemented. Screening of ICU staff was repeatedly negative. Initial environmental cultures, including dry surfaces, were also negative. The above measures temporarily controlled cross-transmission but failed to eradicate the epidemic MDRKO strain that reappeared two weeks after the last colonized patients in waves 2 and 3 had been discharged. Therefore, an occult environmental reservoir was suspected. Samples from the drainpipes and traps of a sink were positive; removal of the sink reduced the rate number but did not stop new cases that clustered in a cubicle whose horizontal drainage system was connected with the eliminated sink. The elimination of the horizontal drainage system finally eradicated the outbreak. In conclusion, damp environmental reservoirs (mainly sink drains, traps and the horizontal drainage system) could explain why standard cross-transmission control measures failed to control the outbreak; such reservoirs should be considered even when environmental cultures of surfaces are negative.

Mutational analysis of quinolone resistance in the plasmid-encoded pentapeptide repeat proteins QnrA, QnrB and QnrS

OBJECTIVES Pentapeptide repeat proteins (PRPs) QnrA, QnrB and QnrS confer reduced susceptibility to quinolones. This study presents an in vitro analysis of the genetic evolution of quinolone resistance mediated by changes in the coding sequences and promoter regions of qnrA1, qnrS1 and qnrB1 genes. METHODS A random mutagenesis technique was used to predict the evolutionary potential of these PRPs against nalidixic acid and fluoroquinolones. After comparing the amino acid sequences of these and other PRPs protecting bacteria from quinolone activity, several conserved positions were found. The role of these residues in their effect against quinolones was evaluated by site-directed mutagenesis. RESULTS Three different phenotypes (similar resistance, higher resistance or lower resistance to quinolones) were obtained in the random mutagenesis assays when compared with wild-type phenotypes. Only one mutant increased quinolone resistance: QnrS1 containing D185Y substitution (4-fold for ciprofloxacin). Using site-directed mutagenesis, residues G56, C72, C92, G96, F114, C115, S116, A117 and L159, according to the sequence of QnrA1, were analysed and despite the wide amino acid variability of the PRPs, most conserved residues analysed were critical to QnrA1, QnrB1 and QnrS1. CONCLUSIONS Amino acid sequences of PRPs QnrA1, QnrB1 and QnrS1 could be already optimized for quinolone resistance. One or several changes appear to be insufficient to obtain variants producing fluoroquinolone clinical resistance (MIC > 1 mg/L). Critical residues for quinolone resistance in PRPs were described. Interestingly, different effects were observed for QnrA1, QnrB1 and QnrS1 with the same substitution in several positions.

Pharmacodynamics of Fosfomycin: Insights into Clinical Use for Antimicrobial Resistance

ABSTRACT The aim of this study was to improve the understanding of the pharmacokinetic-pharmacodynamic relationships of fosfomycin against extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains that have different fosfomycin MICs. Our methods included the use of a hollow fiber infection model with three clinical ESBL-producing E. coli strains. Human fosfomycin pharmacokinetic profiles were simulated over 4 days. Preliminary studies conducted to determine the dose ranges, including the dose ranges that suppressed the development of drug-resistant mutants, were conducted with regimens from 12 g/day to 36 g/day. The combination of fosfomycin at 4 g every 8 h (q8h) and meropenem at 1 g/q8h was selected for further assessment. The total bacterial population and the resistant subpopulations were determined. No efficacy was observed against the Ec42444 strain (fosfomycin MIC, 64 mg/liter) at doses of 12, 24, or 36 g/day. All dosages induced at least initial bacterial killing against Ec46 (fosfomycin MIC, 1 mg/liter). High-level drug-resistant mutants appeared in this strain in response to 12, 15, and 18 g/day. In the study arms that included 24 g/day, once or in a divided dose, a complete extinction of the bacterial inoculum was observed. The combination of meropenem with fosfomycin was synergistic for bacterial killing and also suppressed all fosfomycin-resistant clones of Ec2974 (fosfomycin MIC, 1 mg/liter). We conclude that fosfomycin susceptibility breakpoints (≤64 mg/liter according to CLSI [for E. coli urinary tract infections only]) should be revised for the treatment of serious systemic infections. Fosfomycin can be used to treat infections caused by organisms that demonstrate lower MICs and lower bacterial densities, although relatively high daily dosages (i.e., 24 g/day) are required to prevent the emergence of bacterial resistance. The ratio of the area under the concentration-time curve for the free, unbound fraction of fosfomycin versus the MIC (fAUC/MIC) appears to be the dynamically linked index of suppression of bacterial resistance. Fosfomycin with meropenem can act synergistically against E. coli strains in preventing the emergence of fosfomycin resistance.

Fluorometric and high-performance liquid chromatographic measurement of quinolone uptake by human neutrophils

A fluorometric assay, based on the natural fluorescence of the quinolone nucleus, and high-performance liquid chromatography (HPLC) were compared to determine the penetration of six quinolones (norfloxacin, ciprofloxacin, ofloxacin, fleroxacin, lomefloxacin and temafloxacin) into human polymorphonuclear neutrophils (PMN). Intracellular concentrations after 20 min incubation of the cells with 10 and 25 mg/l were three or more times higher than the extracellular ones. There were no statistical differences between the two methods. At lower extracellular concentrations (2 and 5 mg/l) the intracellular levels of all the quinolones tested were undetectable by HPLC. Fluorometry is a useful method for determining the intracellular penetration of quinolones. The usefulness of HPLC, however, is questionable since it requires great volumes of cells and extracellular concentrations much higher than the therapeutic ones.

Comparative activities of eight quinolones against members of the Bacteroides fragilis group.

The in vitro activities of five new quinolones (clinafloxacin [CI-960 or PD-127391], BAY Y 3118, E-4868, E-5065, and E-5068) against 100 Bacteroides fragilis group bacterial isolates were compared with those of ciprofloxacin, ofloxacin, and sparfloxacin. Overall, E-5068 was the most active in vitro (MIC for 90% of isolates tested [MIC90], 0.25 microgram/ml); this was followed by clinafloxacin and BAY Y 3118 (MIC90, 0.5 microgram/ml). Ciprofloxacin, sparfloxacin, and ofloxacin were the least active (MIC90s, 64, 16, and 16 micrograms/ml, respectively). B. fragilis and Bacteroides caccae were more susceptible than the other members of the B. fragilis group to all of the quinolones tested.

Mobile Genetic Elements Related to the Diffusion of Plasmid-Mediated AmpC β-Lactamases or Carbapenemases from Enterobacteriaceae: Findings from a Multicenter Study in Spain

ABSTRACT We examined the genetic context of 74 acquired ampC genes and 17 carbapenemase genes from 85 of 640 Enterobacteriaceae isolates collected in 2009. Using S1 pulsed-field gel electrophoresis and Southern hybridization, 37 of 74 blaAmpC genes were located on large plasmids of different sizes belonging to six incompatibility groups. We used sequencing and PCR mapping to investigate the regions flanking the acquired ampC genes. The blaCMY-2-like genes were associated with ISEcp1; the surrounding blaDHA genes were similar to Klebsiella pneumoniae plasmid pTN60013 associated with IS26 and the psp and sap operons; and the blaACC-1 genes were associated with IS26 elements inserted into ISEcp1. All of the carbapenemase genes (blaVIM-1, blaIMP-22, and blaIMP-28) were located in class 1 integrons. Therefore, although plasmids are the main cause of the rapid dissemination of ampC genes among Enterobacteriaceae, we need to be aware that other mobile genetic elements, such as insertion sequences, transposons, or integrons, can be involved in the mobilization of these genes of chromosomal origin. Additionally, three new integrons (In846 to In848) are described in this study.

Epidemiological and clinical complexity of amoxicillin-clavulanate-resistant Escherichia coli.

ABSTRACT Two hundred twelve patients with colonization/infection due to amoxicillin-clavulanate (AMC)-resistant Escherichia coli were studied. OXA-1- and inhibitor-resistant TEM (IRT)-producing strains were associated with urinary tract infections, while OXA-1 producers and chromosomal AmpC hyperproducers were associated with bacteremic infections. AMC resistance in E. coli is a complex phenomenon with heterogeneous clinical implications.

A dynamic in vitro model for evaluating antimicrobial activity against bacterial biofilms using a new device and clinical-used catheters

The activity of daptomycin compared to vancomycin against Staphylococcus epidermidis-biofilms on intravascular catheters has been evaluated using the new Sevilla device that enables to use medical grade-catheters, in an in vitro model that simulates the in vivo conditions. S. epidermidis-biofilms were obtained on polyurethane catheter segments using the Sevilla device linked to a continuous culture system for 24 h. To assess the antimicrobial activity, at this time the continuous culture system was changed to therapeutic antimicrobial concentration solutions for 48 h. At each 24 h interval time, catheter segments were taken out, washed and sonicated. Viable adherent bacteria were determined by agar plating. Data of surviving bacteria numbers attached to the catheter surface obtained with the Sevilla device showed a very good reproducibility. Daptomycin showed a good activity against S. epidermidis-biofilm on polyurethane catheter surface. After 48 h exposure to daptomycin, surviving adherent bacteria were reduced by 4 log compared to the control with no antimicrobial. Using the same model, vancomycin reduced bacterial survival by only 1.3 log. The Sevilla device enables antimicrobial agent activity against bacterial biofilms grown on the external surface of catheters used in clinical practice to be evaluated. The model used replicates as closely as possible the biofilm formed in a highly standardized way. Using this model, daptomycin demonstrates potent in vitro activity against S. epidermidis-biofilm on a polyurethane catheter; this activity was greater than that showed by vancomycin.

Local imipenem activity against Pseudomonas aeruginosa decreases in vivo in the presence of siliconized latex

Zinc eluted from siliconized latex (SL) increases resistance of Pseudomonas aeruginosa to imipenem in vitro. A foreign body peritonitis model was used to evaluate the activity of imipenem using SL or silicone (S) implants. No differences were observed in mortality, positive blood cultures and tissue bacterial counts between SL and S implants. Implant-associated counts, however, were significantly higher in the SL group. It is concluded that SL decreases the activity of imipenem against P. aeruginosa.