Antimicrobial susceptibility testing in biofilm-growing bacteria.

Biofilms are organized bacterial communities embedded in an extracellular polymeric matrix attached to living or abiotic surfaces. The development of biofilms is currently recognized as one of the most relevant drivers of persistent infections. Among them, chronic respiratory infection by Pseudomonas aeruginosa in cystic fibrosis patients is probably the most intensively studied. The lack of correlation between conventional susceptibility test results and therapeutic success in chronic infections is probably a consequence of the use of planktonically growing instead of biofilm-growing bacteria. Therefore, several in vitro models to evaluate antimicrobial activity on biofilms have been implemented over the last decade. Microtitre plate-based assays, the Calgary device, substratum suspending reactors and the flow cell system are some of the most used in vitro biofilm models for susceptibility studies. Likewise, new pharmacodynamic parameters, including minimal biofilm inhibitory concentration, minimal biofilm-eradication concentration, biofilm bactericidal concentration, and biofilm-prevention concentration, have been defined in recent years to quantify antibiotic activity in biofilms. Using these parameters, several studies have shown very significant quantitative and qualitative differences for the effects of most antibiotics when acting on planktonic or biofilm bacteria. Nevertheless, standardization of the procedures, parameters and breakpoints, by official agencies, is needed before they are implemented in clinical microbiology laboratories for routine susceptibility testing. Research efforts should also be directed to obtaining a deeper understanding of biofilm resistance mechanisms, the evaluation of optimal pharmacokinetic/pharmacodynamic models for biofilm growth, and correlation with clinical outcome.

First report of an OXA-23 carbapenemase-producing Acinetobacter baumannii clinical isolate related to Tn2006 in Spain.

A carbapenem-resistant Acinetobacter baumannii clinical isolate belonging to European clone II and sequence type 2 was recovered from a patient in the Son Espases hospital in Mallorca, Spain. Genetic analysis showed the presence of the bla(OXA-23) gene in association with the widely disseminated transposon Tn2006. This is the first reported identification of A. baumannii carrying bla(OXA-23) in Spain.

Características clínicas y epidemiológicas de los pacientes pediátricos con neumonía causada por los virus gripales / Clinical and epidemiological characteristics of the pediatric patients with influenza virus-induced pneumonia

Estudio retrospectivo sobre características clínicas y epidemiológicas de los pacientes pediátricos con neumonía y aislamiento de virus gripales (influenza A y B) durante el período 1995-2005. A las muestras respiratorias se les realizó la detección antigénica frente a los virus gripales y el aislamiento viral en cultivo celular. Durante el período de estudio se han diagnosticado 598 casos de infección respiratoria aguda causada por los virus gripales A y B, de los cuales 414 (69,2%) correspondían a pacientes pediátricos. Se diagnosticó neumonía gripal en 28 casos (6,7%). El virus gripal tipo A fue aislado en 22 casos (78,5%) y el virus gripal tipo B en 6 casos (21,5%). Los 22 virus gripales A fueron subtipados como pertenecientes 17 al subtipo H3 (77,2%) y 5 al subtipo H1 (22,8%). Las principales manifestaciones clínicas fueron: fiebre (96,4%), tos (78,5%), rinorrea (28,5%), cefalea (28,5%), otitis media aguada (14,2%) y mialgias, diarrea y conjuntivitis (3,5%). La edad media de nuestros pacientes fue de 4,4 años. El 75% de los pacientes precisó del ingreso hospitalario debido a su corta edad o complicaciones patológicas. En los pacientes ingresados se observó una estancia media de 8,3 días (intervalo de 3-17 días). Las manifestaciones clínicas son muy similares a las observadas en el resto de infecciones respiratorias agudas, precisando el estudio virológico para establecer su etiología definitiva (AU)

We performed a retrospective study of the clinical and epidemiological characteristics of pediatric patients with pneumonia and isolation of influenza viruses A and B during the 1995-2005 period. The specific antigenic detection and viral isolation in cell culture were the methods used in the etiological diagnosis. In the study period we detected 598 cases of acute respiratory infection caused by influenza ciruses A and B; of them 414 (69.2%) in pediatric patients. We could diagnosticated 28 patients (6.7%) with influenza pneumonia. Influenza A virus was isolated in 22 (78.5%) patients and Influenza B virus in 6 (21.5%) patients. The 22 influenza A viruses were subtyped as 17 (77,2%) H3 and 5 (22,8%) H1 subtypes. The most frequent clinical manifestations detected in the patients with pneumonia were: fever (96.4%), cough (78.5%), rhinorrea (28.5%), headache (28.5%), acute otitis (14,2%) and mialgia, diarrhea and conjunctivitis (3.5%). The mean age of patients was 4.4 years old; 75% of patients were hospitalized with a media of 8.3 days (3-17 days). The clinical manifestations of this pathology were very similar to detected in other acute viral respiratory infections, so only the virological methods could established the definitive etiological diagnosis (AU)

Influence of high mutation rates on the mechanisms and dynamics of in vitro and in vivo resistance development to single or combined antipseudomonal agents.

We studied the mechanisms and dynamics of the development of resistance to ceftazidime (CAZ) alone or combined with tobramycin (TOB) or ciprofloxacin (CIP) in vitro and in vivo (using a mouse model of lung infection with human antibiotic regimens). Pseudomonas aeruginosa strain PAO1 and its hypermutable derivative PAODeltamutS were used, and the results were compared with those previously obtained with CIP, TOB, and CIP plus TOB (CIP-TOB) under the same conditions. An important (200-fold) amplification of the number of resistant mutant cells was documented for PAODeltamutS-infected mice that were under CAZ treatment compared to the number for mice that received placebo, whereas the median number of resistant mutant cells was below the detection limits for mice infected by PAO1. These results were intermediate between the high amplification with CIP (50,000-fold) and the low amplification with TOB (10-fold). All CAZ-resistant single mutant cells selected in vitro or in vivo hyperproduced AmpC. On the other hand, the three combinations studied were found to be highly effective in the prevention of in vivo resistance development in mice infected with PAODeltamutS, although the highest therapeutic efficacy (in terms of mortality and total bacterial load reduction) compared to those of the individual regimens was obtained with CIP-TOB and the lowest was with CAZ-CIP. Nevertheless, mutant cells that were resistant to the three combinations tested were readily selected in vitro for PAODeltamutS (mutation rates from 1.2 x 10(-9) to 5.8 x 10(-11)) but not for PAO1, highlighting the potential risk for antimicrobial resistance development associated with the presence of hypermutable strains, even when combined therapy was used. All five independent CAZ-TOB-resistant PAODeltamutS double mutants studied presented the same resistance mechanism (AmpC hyperproduction plus an aminoglycoside resistance mechanism not related to MexXY), whereas four different combinations of resistance mechanisms were documented for the five CAZ-CIP-resistant double mutants.

Efficacy and potential for resistance selection of antipseudomonal treatments in a mouse model of lung infection by hypermutable Pseudomonas aeruginosa.

Hypermutable Pseudomonas aeruginosa strains are found with high frequency in the lungs of patients with chronic infections and are associated with high antibiotic resistance rates. The in vivo consequences of hypermutation for treatment in a mouse model of lung infection using strain PAO1 and its hypermutable derivative PAOdeltamutS are investigated. Groups of 30 mice were treated for 3 days with humanized regimens of ciprofloxacin (CIP), tobramycin (TOB), CIP plus TOB, or placebo, and mortality, total lung bacterial load, and 4x- and 16x-MIC mutants were recorded. The rates of mutation and the initial in vivo frequencies of mutants (at the onset of treatment) were also estimated and the in vitro- and in vivo-selected mutants characterized. Since both strains had identical MICs, the same pharmacokinetic/pharmacodynamic (PK/PD) parameters were obtained: area under the 24-h concentration-time curve (fAUC)/MIC = 385 for CIP and maximum concentration of drug in serum (fC(max))/MIC = 19 for TOB. Despite adequate PK/PD parameters, persistence of high bacterial numbers and amplification (50,000-fold) of resistant mutants (MexCD-OprJ hyperexpression) were documented with CIP treatment for PAOdeltamutS, in contrast to complete resistance suppression for PAO1 (P < 0.01), showing that conventional PK/PD parameters may not be applicable to infections by hypermutable strains. On the other hand, the efficacy of TOB monotherapy in terms of mortality reduction and bacterial load was very low regardless of the strain but not due to resistance development, since mutants were not selected for PAO1 and were only modestly amplified for PAOdeltamutS. Finally, the CIP-plus-TOB combination was synergistic, further reducing mortality and bacterial load and completely preventing resistance even for PAOdeltamutS (P < 0.01 compared to monotherapy), showing that it is possible to suppress resistance selection in infections by hypermutable P. aeruginosa using appropriate combined regimens.