Role of inoculum and mutant frequency on fosfomycin MIC discrepancies by agar dilution and broth microdilution methods in Enterobacteriaceae.

OBJECTIVES: Fosfomycin is re-evaluated as a treatment of multidrug-resistant Enterobacteriaceae infections. However, MIC differences have been described among the different susceptibility testing. The aim was to study the role of the different inoculum size used in agar dilution with respect to broth microdilution, according to CLSI, in the fosfomycin MIC discrepancies. METHODS: Fosfomycin MICs were determined using agar dilution (reference) and broth microdilution in 220 Escherichia coli (n=81) and Klebsiella pneumoniae (n=139) clinical isolates. Fosfomycin mutant frequencies were determined in 21 E. coli (MIC=1mg/L) and 21 K. pneumoniae (MIC=16mg/L). The emergence of resistant subpopulations of five E. coli strains (MIC=1mg/L) was monitored over the time by microdilution assay using 0, 4 and 8 mg/L of fosfomycin, and eight different inocula (5×105-3.91×103 CFU/well, 1 : 2 dilutions). RESULTS: For E. coli, 86.4% of categorical agreement (CA), 9.1% very major errors (VME), 3.3% major errors (ME) and 9.9% minor errors (mE) were found. For K. pneumoniae, CA was 51.1%, VME 15.7%, ME 28.4% and mE 25.2%. Essential agreement (±1-log2) was observed in 55.45%. By microdilution, 35.9% of the MICs showed discrepancies of ≥2 dilutions. Initial inoculum used was 5.63 times higher in the microdilution method, in range with CLSI methodology for both techniques. Fosfomycin mutant frequencies were 6.05×10-5 (4×MIC) to 5.59×10-7 (256×MIC) for E. coli, and 1.49×10-4 (4×MIC) to 1.58×10-5 (16×MIC) for K. pneumoniae. Resistant subpopulations arose mainly after 8 h of incubation with inocula >3.13×104 CFU/well. CONCLUSIONS: The higher inoculum used in the microdilution method enriched the initial inoculum with resistant subpopulations and could partially explain the fosfomycin MIC discrepancies with respect to the agar dilution method.

Pharmacodynamics of fosfomycin: insights into clinical use for antimicrobial resistance.

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.

Mobile genetic elements related to the diffusion of plasmid-mediated AmpC ß-lactamases or carbapenemases from Enterobacteriaceae: findings from a multicenter study in Spain.

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 bla AmpC 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 bla CMY-2-like genes were associated with ISEcp1; the surrounding bla DHA genes were similar to Klebsiella pneumoniae plasmid pTN60013 associated with IS26 and the psp and sap operons; and the bla ACC-1 genes were associated with IS26 elements inserted into ISEcp1. All of the carbapenemase genes (bla VIM-1, bla IMP-22, and bla IMP-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.

Lessons from an outbreak of metallo-ß-lactamase-producing Klebsiella oxytoca in an intensive care unit: the importance of time at risk and combination therapy.

BACKGROUND: Outbreaks of nosocomial infection due to carbapenem-resistant Enterobacteriaceae (CRE), mostly Klebsiella spp., have become a worldwide phenomenon. AIM: To investigate the risk factors for the acquisition of clonal multidrug-resistant Klebsiella oxytoca (MDRKO) producing the metallo-ß-lactamase IMP-8 and hyperproducing chromosomal OXY-2 ß-lactamase during a well-characterized outbreak, and to describe the clinical features of infections due to MDRKO. METHODS: A four-wave outbreak due to MDRKO occurred in the intensive care unit of a Spanish hospital between 2009 and 2011. The risk factors for acquisition of MDRKO during waves 1 and 2 (in which colonized patients served as the main reservoir for the epidemic strain) were analysed using a case-control study by Cox regression and logistic regression analysis. Clinical data and treatments of patients infected with MDRKO were also analysed. FINDINGS: For the study of risk factors, 26 cases and 45 controls were studied. None of the variables studied in the Cox regression analysis showed an association with MDRKO acquisition; time at risk was the only associated variable by logistic regression analysis. Colonization pressure was not associated with earlier acquisition. Overall, 14 patients were infected with MDRKO; ventilator-associated pneumonia (seven patients) was the most frequent type of infection. Monotherapy tended to be associated with higher mortality than combination therapy [60% (3/5) vs 16.6% (1/6); P = 0.07]. CONCLUSIONS: Time at risk was the most significant risk determinant for the acquisition of carbapenem-resistant Enterobacteriaceae (CRE) in this epidemiological context and should be included in any study of risk factors for the acquisition of multidrug-resistant bacteria. Combination therapy may be superior to monotherapy for the treatment of CRE infections.

Wastewater drainage system as an occult reservoir in a protracted clonal outbreak due to metallo-ß-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.

Prevalence and molecular epidemiology of acquired AmpC ß-lactamases and carbapenemases in Enterobacteriaceae isolates from 35 hospitals in Spain.

The purpose of this investigation was to determine the prevalence of plasmid-mediated AmpC (pAmpC) and carbapenemases in Enterobacteriaceae collected from 35 hospitals in Spain and to establish their epidemiological relationships. We conducted a prospective multi-centre study on pAmpC- or carbapenemase-producing Enterobacteriaceae isolates from clinical samples collected from February to July 2009. The strains suspected to carry pAmpC were resistant or showed intermediate susceptibility to co-amoxiclav and second- or third-generation cephalosporins. Strains suspected to carry a carbapenemase were selected because they showed a minimum inhibitory concentration (MIC) to imipenem >1 mg/L. Polymerase chain reaction (PCR) and a sequencing strategy were used to characterise the enzymes. The clonal relationships between isolates was analysed by pulsed field gel electrophoresis (PFGE). Among 100,132 Enterobacteriaceae isolates collected, 1,654 were compatible with the production of pAmpC or carbapenemases. We found a prevalence of 0.64 % of pAmpC (n = 635) and 0.04 % of carbapenemases (n = 43). The most prevalent pAmpC enzymes were CMY-type (78.3 %), DHA-type (19.5 %), ACC-type (1.6 %) and FOX-type (0.6 %). The CMY-type was the most frequent in Escherichia coli and Proteus mirabilis species, whereas the DHA-type was mainly found in Klebsiella spp. The enzymes involved in carbapenem resistance were VIM-1, IMP-22 and the new IMP-28. Nine new bla genes were described: bla (CMY-54), bla (CMY-55), bla (CMY-56), bla (CMY-57), bla (CMY-96), bla (DHA-6), bla (DHA-7), bla (FOX-8) and bla (IMP-28). The prevalence of pAmpC or carbapenemases found is not negligible. The CMY-types were the predominant pAmpC, whereas the VIM or IMP enzymes were the predominant carbapenemases. Furthermore, we observed a great genetic diversity among pAmpC-producing strains and a close clonal relationship between carbapenemase-producing strains.

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.

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.

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.

Actividad y permeabilidad de linezolid y vancomicina en biocapas de Staphylococcus epidermidis / Activity and penetration of linezolid and vancomycin against Staphylococcus epidermidis biofilms

Introducción. Se ha evaluado la actividad y capacidad de penetración de linezolid y vancomicina en biocapas de Staphylococcus epidermidis. Métodos. La actividad de linezolid en comparación con vancomicina se midió frente a biocapas bacterianas de S. epidermidis formadas sobre segmentos de silicona. La penetración de estos antimicrobianos se midió en las biocapas formadas sobre membranas microporosas de policarbonato. Ambos ensayos se realizaron comparativamente con una cepa de S. epidermidis productora de slime y otra no productora. Resultados. La actividad de linezolid frente a biocapas de S. epidermidis fue significativamente mayor que la de vancomicina tanto en la cepa productora de slime como en la no productora. Ninguno de los antimicrobianos erradicó las biocapas de 24 h. La penetración de linezolid en biocapas de S. epidermidis fue mayor que la de vancomicina en todos los tiempos estudiados para ambas cepas. Conclusiones. Linezolid ha mostrado mayor actividad in vitro que vancomicina en biocapas de S. epidermidis formadas sobre catéteres de silicona y este efecto podría ser debido a su capacidad para atravesar la biocapa bacteriana (AU)

Introduction. The activity and capacity for penetration of linezolid and vancomycin were comparatively evaluated against Staphylococcus epidermidis biofilms. Methods. The activity of linezolid versus vancomycin was assessed against 24-hour S. epidermidis biofilms developed on silicon catheters. Penetration of the two antimicrobial agents was measured in biofilms developed on polycarbonate membrane filters. Penetration and activity were comparatively tested using S. epidermidis, slime-producing and non-slime-producing strains. Results. The activity of linezolid against S. epidermidis biofilms was significantly greater than that of vancomycin for both strains. Neither antimicrobial completely eradicated bacterial survival in 24-hour biofilms. Linezolid penetration in biofilms was greater than that of vancomycin for both S. epidermidis strains. Conclusions. Linezolid showed higher in vitro activity than vancomycin against S. epidermidis biofilms on silicone catheters. This effect may be due to the capability of linezolid to cross the bacterial biofilm (AU)

Evaluation of antimicrobial susceptibility of bacteria containing the qnr gene and FOX-5 beta-lactamase by four automated systems.

The accuracy and performance of four automated instruments (BD Phoenix, MicroScan WalkAway, VITEK-2 and Wider) were evaluated for susceptibility testing of fluoroquinolones and beta-lactams with four clinical isolates of Klebsiella pneumoniae and the corresponding Escherichia coli transconjugants containing a plasmid carrying the qnr gene and coding for FOX-5 production. No major or very major errors were detected with the MicroScan system. Many of the minor errors for both quinolones and beta-lactams clustered around the intermediate breakpoints.

Fisioterapia y esclerosis lateral amiotrófica / Physioterapy and esclerosis lateral amiotrofica

La esclerosis lateral amiotrófica (ELA) es una enfermedad de la motoneurona superior e inferior, con afectación bulbar variable, sin alteraciones mentales, sensitivas, sensoriales ni esfinterianas. El objetivo de este trabajo es aportar nuestra experiencia profesional como fisioterapeutas, proponiendo un protocolo de exploración y tratamiento de estos pacientes, aunque destacando la importancia del tratamiento individualizado, que debe ser revisado y adaptado periódicamente según la situación clínica del sujeto (AU)

Activities of imipenem and cephalosporins against clonally related strains of Escherichia coli hyperproducing chromosomal beta-lactamase and showing altered porin profiles.

Forty clonally related clinical isolates of Escherichia coli from hospitalized patients were resistant to cefoxitin (MICs, >256 microg/ml) and ceftazidime (MICs, 32 to 256 microg/ml) and were intermediate or resistant to cefotaxime (MICs, 16 to 128 microg/ml) but susceptible to both cefepime (MICs, 0.5 to 2 microg/ml) and imipenem (MICs, 0.125 to 0.25 microg/ml). Resistance to beta-lactams was related to high-level production of AmpC beta-lactamase and loss of OmpF porin.

Resistance of Pseudomonas aeruginosa to imipenem induced by eluates from siliconized latex urinary catheters is related to outer membrane protein alterations.

The activity of imipenem against Pseudomonas aeruginosa HUS-3 decreased by 16 times in the presence of substances eluted from siliconized latex urinary catheters (SLUCs). SLUCs did not inactivate imipenem or increase beta-lactamase activity. The outer membrane of P. aeruginosa HUS-3 grown in the presence of eluate lacked an OprD-like protein and expressed a new 50-kDa protein. The decreased activity of imipenem against P. aeruginosa in the presence of SLUCs is related to the loss of an OprD-like protein and the expression of a new outer membrane protein.

Effect of paclitaxel alone or in combination on the intracellular penetration and activity of quinolones in human neutrophils.

We evaluated the effect of paclitaxel alone or in combination with cisplatin or doxorubicin, on the intracellular penetration and activity of ciprofloxacin, ofloxacin, levofloxacin and sparfloxacin in human polymorphonuclear leukocytes (PMN). In general, paclitaxel either alone or in combination did not affect the intracellular concentrations of the quinolones evaluated (intracellular to extracellular concentration ratio (C/E > 4). However, at high concentrations (20 mg/L) paclitaxel decreased the penetration of ofloxacin and levofloxacin although the C/E ratios remained higher than 3. Paclitaxel alone or in combination affected neither the production of oxygen radicals by PMN nor the intracellular activity of the quinolones against Staphylococcus aureus.

Evolution of the antimicrobial susceptibility of B. fragilis group at the university hospital of Seville (Spain) between 1977 and 1995.

A susceptibility survey of the B. fragilis group divided into three periods was carried out between 1977 and 1995 at the University Hospital of Seville (Spain) using the agar dilution method. No chloramphenicol, imipenem or meropenem-resistant strains were found. Metronidazole-resistant strains (2%) were isolated only in the first period. The most active beta-lactam drugs were piperacillin and ceftizoxime (resistance rate 16%), followed by ticarcillin mezlocillin and azlocillin (25%) and cefotaxime, cefotetam, and cefmetazol (around 40%). All strains tested were resistant to ampicillin and 4% to ampicillin/sulbactam. Cefoxitin resistance increased from 10% in the first two periods to 21% in the third and that of clindamycin from 12% in 1982 to 29% in 1987 and 50% in 1995.

[Intracellular penetration of 5 quinolones into non-phagocytic cells]. / Penetración intracelular de cinco quinolonas en células no fagocíticas.

BACKGROUND: The intracellular penetration and activity of antimicrobial agents can be an important factor in the treatment of infections caused by intracellular pathogens. The new quinolones are able to penetrate into phagocytes, but only a few studies have evaluated other types of cells. The purpose of this study is to evaluate the intracellular penetration of ofloxacin, levofloxacin, lomefloxacin, sparfloxacin and BAY Y 3118 into human polymorphonuclear leukocytes and tissue cultured epithelial cells. METHODS: Intracellular penetration was evaluated by a fluorometric assay for all the quinolones evaluated except for sparfloxacin, which was evaluated by a radiometric assay. RESULTS: All the quinolones evaluated reached intracellular concentrations higher than extracellular ones. The penetration of sparfloxacin and BAY Y 3118 into the epithelial cells was similar to those observed for PMNs (cellular to extracellular concentration ratio; C/E > or = 4). The C/E values of ofloxacin, levofloxacin and lomefloxacin for epithelial cells were lower than those observed in PMN, but still yielding C/E values > or = 2.

Factors affecting the intracellular accumulation and activity of azithromycin.

Azithromycin is a new macrolide which accumulates in high concentrations in human phagocytes. The cellular to extracellular ratio (C/E) of azithromycin concentrations (fixed extracellular concentration 1 mg/L) in human polymorphonuclear leucocytes (PMN) were significantly affected by small increases in the environmental temperature (C/E 20.3 +/- 2 and 59.4 +/- 6 at 37 degrees C and 40 degrees C, respectively). PMN-associated azithromycin was not affected by the presence of different concentrations of human serum. The intracellular accumulation of azithromycin decreased slightly (C/E approximately 5) when cells were activated with PMA or opsonized with zymosan. The phagocytosis of opsonized Staphylococcus aureus or Haemophilus influenzae, however, slightly increased the intracellular concentrations of azithromycin. At different extracellular concentrations, azithromycin did not affect the production of hydrogen peroxide and superoxide radicals by PMN. The intracellular survival of H. influenzae in human PMN was abolished in the presence of concentrations higher than 0.125 mg/L of azithromycin. Under the same experimental conditions, however, azithromycin did not show any intracellular activity against S. aureus.