Mar Solé

First Outbreak of a Plasmid-Mediated Carbapenem-Hydrolyzing OXA-48 β-Lactamase in Klebsiella pneumoniae in Spain

ABSTRACT Twenty Klebsiella pneumoniae isolates producing OXA-48 were collected from April 2009 to September 2010. Strains were clonally related and coproduced a CTX-M-15 β-lactamase. A conjugative plasmid of circa 70 kb carrying blaOXA-48 was identified. Eleven isolates showed low-level resistance to carbapenems, whereas nine showed high-level resistance. Decreased expression of OmpK36 was related to high-level resistance to carbapenems. The isolates belonged to sequence type 101 (ST101). This is the first outbreak caused by an OXA-48-producing K. pneumoniae strain in Spain.

First Description of an Escherichia coli Strain Producing NDM-1 Carbapenemase in Spain

ABSTRACT A carbapenem-resistant Escherichia coli strain (DVR22) was recovered from a stool specimen from a patient with travelers diarrhea who had traveled to India. Molecular screening led to the first identification of NDM-1 in Spain. The blaNDM-1 gene was located in a conjugative plasmid of ca. 300 kb that also contained the blaCTX-M-15, blaTEM-1, ΔblaDHA-1, and armA genes. In addition, blaNDM-1 was preceded by an ISAba125 insertion element only found in Acinetobacter spp.

Pseudomonas aeruginosa isolates in severe chronic obstructive pulmonary disease: characterization and risk factors

BackgroundPatients with severe chronic obstructive pulmonary disease (COPD) are at increased risk of infection by P. aeruginosa. The specific role of bronchiectasis in both infection and chronic colonization by this microorganism in COPD, however, remains ill defined.To evaluate the prevalence and risk factors for P. aeruginosa recovery from sputum in outpatients with severe COPD, characterizing P. aeruginosa isolates by pulsed-field gel electrophoresis (PFGE) and focusing on the influence of bronchiectasis on chronic colonization in these patients.MethodsA case-cohort study of 118 patients with severe COPD attended at a Respiratory Day Unit for an acute infectious exacerbation and followed up over one year. High-resolution CT scans were performed during stability for bronchiectasis assessment and sputum cultures were obtained during exacerbation and stability in all patients. P. aeruginosa isolates were genotyped by PFGE. Determinants of the recovery of P. aeruginosa in sputum and chronic colonization by this microorganism were assessed by multivariate analysis.ResultsP. aeruginosa was isolated from 41 of the 118 patients studied (34.7%). Five of these 41 patients (12.2%) with P. aeruginosa recovery fulfilled criteria for chronic colonization. In the multivariate analysis, the extent of bronchiectasis (OR 9.8, 95% CI: 1.7 to 54.8) and the number of antibiotic courses (OR 1.7, 95% CI: 1.1 to 2.5) were independently associated with an increased risk of P. aeruginosa isolation. Chronic colonization was unrelated to the presence of bronchiectasis (p=0.75). In patients with chronic colonization the isolates of P. aeruginosa retrieved corresponded to the same clones during the follow-up, and most of the multidrug resistant isolates (19/21) were harbored by these patients.ConclusionsThe main risk factors for P. aeruginosa isolation in severe COPD were the extent of bronchiectasis and exposure to antibiotics. Over 10% of these patients fulfilled criteria for chronic colonization by P. aeruginosa and showed clonal persistence, independently of the presence of bronchiectasis.

Molecular Characterization of blaNDM-5 Carried on an IncFII Plasmid in an Escherichia coli Isolate from a Nontraveler Patient in Spain

ABSTRACT A carbapenem-resistant Escherichia coli isolate (sequence type 448 [ST448]) was recovered from a urine culture of a female patient with no recent record of traveling. PCR screening identified the presence of blaNDM-5, blaTEM-1, blaOXA-1, blaCMY-42, and rmtB. blaNDM-5 was carried in a conjugative IncFII-type plasmid (90 kb) together with blaTEM-1 and rmtB. The genetic environment of blaNDM-5 showed a structure similar to those of pMC-NDM and pGUE-NDM, identified in Poland and France in E. coli of African and Indian origin, respectively.

Molecular study of quinolone resistance mechanisms and clonal relationship of Salmonella enterica clinical isolates

In the last few years, the number of Salmonella enterica strains resistant to nalidixic acid has steadily increased. In a previous study, the quinolone susceptibility phenotype and genotype of 38 S. enterica clinical isolates (19 S. enterica serovar Typhimurium and 19 S. enterica serovar Enteritidis) were determined. Forty-two percent of the isolates showed nalidixic acid resistance associated with a mutation in gyrA together with putative overexpression of efflux pump(s). In this study, mutations in the quinolone resistance-determining region (QRDR) of parE and the regulators of AcrAB (acrR, marRAB, soxRS and ramR) were analysed. Intracellular accumulation of ciprofloxacin and nalidixic acid was determined. Gene expression of the efflux pump components acrB, tolC, acrF and emrB was also assessed. In addition, an epidemiological study of the isolates by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) was performed. No mutations were detected in parE, whereas two amino acid substitutions were found in two susceptible strains in MarR (I84L) and AcrR (N214T) in one strain each, although both were suggested to be polymorphisms. No changes in the gene expression of acrB, tolC, acrF and emrB were detected between nalidixic-acid-resistant and -susceptible strains. Intracellular accumulation was not useful to reveal differences. Epidemiological analysis showed an important clonal relatedness among the S. Enteritidis isolates, whereas major divergence was seen for S. Typhimurium. Altogether, these results suggest the presence of previously undiscovered drug efflux pump(s) and confirm the high clonality of S. Enteritidis and the genetic divergence of S. Typhimurium.

In vivo evolution of resistance of Pseudomonas aeruginosa strains isolated from patients admitted to an intensive care unit: mechanisms of resistance and antimicrobial exposure

OBJECTIVES The main objective of this study was to investigate the relationship among the in vivo acquisition of antimicrobial resistance in Pseudomonas aeruginosa clinical isolates, the underlying molecular mechanisms and previous exposure to antipseudomonal agents. METHODS PFGE was used to study the molecular relatedness of the strains. The MICs of ceftazidime, cefepime, piperacillin/tazobactam, imipenem, meropenem, ciprofloxacin and amikacin were determined. Outer membrane protein profiles were assessed to study OprD expression. RT-PCR was performed to analyse ampC, mexB, mexD, mexF and mexY expression. The presence of mutations was analysed through DNA sequencing. RESULTS We collected 17 clonally related paired isolates [including first positive samples (A) and those with MICs increased ≥4-fold (B)]. Most B isolates with increased MICs of imipenem, meropenem and ceftazidime became resistant to these drugs. The most prevalent resistance mechanisms detected were OprD loss (65%), mexB overexpression (53%), ampC derepression (29%), quinolone target gene mutations (24%) and increased mexY expression (24%). Five (29%) B isolates developed multidrug resistance. Meropenem was the most frequently (71%) received treatment, explaining the high prevalence of oprD mutations and likely mexB overexpression. Previous exposure to ceftazidime showed a higher impact on selection of increased MICs than previous exposure to piperacillin/tazobactam. CONCLUSIONS Stepwise acquisition of resistance has a critical impact on the resistance phenotypes of P. aeruginosa, leading to a complex scenario for finding effective antimicrobial regimens. In the clinical setting, meropenem seems to be the most frequent driver of multidrug resistance development, while piperacillin/tazobactam, in contrast to ceftazidime, seems to be the β-lactam least associated with the selection of resistance mechanisms.

SoxS-dependent coregulation of ompN and ydbK in a multidrug-resistant Escherichia coli strain

SoxS, MarA, and Rob are homologous transcriptional activators of numerous superoxide- and antibiotic resistance genes but many of the regulated genes are yet to be characterized. In this study, microarrays and RT-PCR analysis were used to show the overexpression of the ompN porin and its upstream gene, ydbK, in an Escherichia coli multidrug-resistant mutant and in a strain constitutive for SoxS. However, transcriptional fusions revealed that SoxS (not MarA or Rob) only activated the ydbK promoter but not the ompN upstream region. RT-PCR experiments showed the overexpression of a combined ydbK - ompN transcript in the SoxS-overexpressing strain. Surprisingly, a bioinformatic approach revealed no soxbox upstream of the ydbK promoter. Thus, the ydbK and ompN genes are coexpressed in an operon and are likely activated by SoxS indirectly. It is known that YdbK is involved in superoxide resistance. Thus, individual ompN and ydbK mutants were tested for superoxide susceptibility. Nonetheless, only the ydbK mutant was susceptible to paraquat, a superoxide generator. These mutants, as well as an OmpN-overproducing strain, were further tested for antibiotic resistance. No significant decreased susceptibility was observed. Thus, ydbK plays a role in superoxide resistance but no role for either gene is found in resistance to the antibiotics tested.

Evaluation of a Mixing versus a Cycling Strategy of Antibiotic Use in Critically-Ill Medical Patients: Impact on Acquisition of Resistant Microorganisms and Clinical Outcomes

Objective To compare the effect of two strategies of antibiotic use (mixing vs. cycling) on the acquisition of resistant microorganisms, infections and other clinical outcomes. Methods Prospective cohort study in an 8-bed intensive care unit during 35- months in which a mixing-cycling policy of antipseudomonal beta-lactams (meropenem, ceftazidime/piperacillin-tazobactam) and fluoroquinolones was operative. Nasopharyngeal and rectal swabs and respiratory secretions were obtained within 48h of admission and thrice weekly thereafter. Target microorganisms included methicillin-resistant S. aureus, vancomycin-resistant enterococci, third-generation cephalosporin-resistant Enterobacteriaceae and non-fermenters. Results A total of 409 (42%) patients were included in mixing and 560 (58%) in cycling. Exposure to ceftazidime/piperacillin-tazobactam and fluoroquinolones was significantly higher in mixing while exposure to meropenem was higher in cycling, although overall use of antipseudomonals was not significantly different (37.5/100 patient-days vs. 38.1/100 patient-days). There was a barely higher acquisition rate of microorganisms during mixing, but this difference lost its significance when the cases due to an exogenous Burkholderia cepacia outbreak were excluded (19.3% vs. 15.4%, OR 0.8, CI 0.5–1.1). Acquisition of Pseudomonas aeruginosa resistant to the intervention antibiotics or with multiple-drug resistance was similar. There were no significant differences between mixing and cycling in the proportion of patients acquiring any infection (16.6% vs. 14.5%, OR 0.9, CI 0.6–1.2), any infection due to target microorganisms (5.9% vs. 5.2%, OR 0.9, CI 0.5–1.5), length of stay (median 5 d for both groups) or mortality (13.9 vs. 14.3%, OR 1.03, CI 0.7–1.3). Conclusions A cycling strategy of antibiotic use with a 6-week cycle duration is similar to mixing in terms of acquisition of resistant microorganisms, infections, length of stay and mortality.