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ABSTRACTThe aim of this study was to determine the impact of carbapenemase-producing Enterobacteriaceae(CPE) in Spain in 2013 by describing the prevalence, dissemination, and geographic distribution of CPE clones, and their population structure and antibiotic susceptibility. From February 2013 to May 2013, 83 hospitals (about 40,000 hospital beds) prospectively collected nonduplicate Enterobacteriaceaeusing the screening cutoff recommended by EUCAST. Carbapenemase characterization was performed by phenotypic methods and confirmed by PCR and sequencing. Multilocus sequencing types (MLST) were determined for Klebsiella pneumoniaeand Escherichia coli. A total of 702 Enterobacteriaceaeisolates met the inclusion criteria; 379 (54%) were CPE. OXA-48 (71.5%) and VIM-1 (25.3%) were the most frequent carbapenemases, and K. pneumoniae(74.4%), Enterobacter cloacae(10.3%), and E. coli(8.4%) were the species most affected. Susceptibility to colistin, amikacin, and meropenem was 95.5%, 81.3%, and 74.7%, respectively. The most prevalent sequence types (STs) were ST11 and ST405 for K. pneumoniaeand ST131 for E. coli. Forty-five (54.1%) of the hospitals had at least one CPE case. For K. pneumoniae, ST11/OXA-48, ST15/OXA-48, ST405/OXA-48, and ST11/VIM-1 were detected in two or more Spanish provinces. ST11 isolates carried four carbapenemases (VIM-1, OXA-48, KPC-2, and OXA-245), but ST405 isolates carried OXA-48 only. A wide interregional spread of CPE in Spain was observed, mainly due to a few successful clones of OXA-48-producing K. pneumoniae(e.g., ST11 and ST405). The dissemination of OXA-48-producing E. coliis a new finding of public health concern. According to the susceptibilities determined in vitro, most of the CPE (94.5%) had three or more options for antibiotic treatment.

ABSTRACTIn a previous prospective multicenter study in Spain, we found that OXA-1 and inhibitor-resistant TEM (IRT) β-lactamases constitute the most common plasmid-borne mechanisms of genuine amoxicillin-clavulanate (AMC) resistance in Escherichia coli. In the present study, we investigated the population structure and virulence traits of clinical AMC-resistant E. colistrains expressing OXA-1 or IRT and compared these traits to those in a control group of clinical AMC-susceptible E. coliisolates. All OXA-1-producing (n= 67) and IRT-producing (n= 45) isolates were matched by geographical and temporal origin to the AMC-susceptible control set (n= 56). We performed multilocus sequence typing and phylogenetic group characterization for each isolate and then studied the isolates for the presence of 49 virulence factors (VFs) by PCR and sequencing. The most prevalent clone detected was distinct for each group: group C isolates of sequence type (ST) 88 (C/ST88) were the most common in OXA-1 producers, B2/ST131 isolates were the most common in IRT producers, and B2/ST73 isolates were the most common in AMC-susceptible isolates. The median numbers of isolates per ST were 3.72 in OXA-1 producers, 2.04 in IRT producers, and 1.69 in AMC-susceptible isolates; the proportions of STs represented by one unique isolate in each group were 19.4%, 31.1%, and 48.2%, respectively. The sum of all VFs detected, calculated as a virulence score, was significantly higher in AMC-susceptible isolates than OXA-1 and IRT producers (means, 12.5 versus 8.3 and 8.2, respectively). Our findings suggest that IRT- and OXA-1-producing E. coliisolates resistant to AMC have a different and less diverse population structure than AMC-susceptible clinical E. coliisolates. The AMC-susceptible population also contains more VFs than AMC-resistant isolates.

ABSTRACTA group of antibiotic resistance genes (ARGs) (blaTEM, blaCTX-M-1, mecA, armA, qnrA, and qnrS) were analyzed by real-time quantitative PCR (qPCR) in bacteriophage DNA isolated from feces from 80 healthy humans. Seventy-seven percent of the samples were positive in phage DNA for one or more ARGs. blaTEM, qnrA, and, blaCTX-M-1were the most abundant, and armA, qnrS, and mecAwere less prevalent. Free bacteriophages carrying ARGs may contribute to the mobilization of ARGs in intra- and extraintestinal environments.

ABSTRACTAn isolate of Klebsiella oxytocacarrying a novel IMP metallo-β-lactamase was discovered in Madrid, Spain. The blaIMP-28gene is part of a chromosomally located class I integron. The IMP-28 kcat/Kmvalues for ampicillin, ceftazidime, and cefepime and, to a lesser extent, imipenem and meropenem, are clearly lower than those of IMP-1. The His306Gln mutation may induce important modifications of the L3 loop and thus of substrate accessibility and hydrolysis and be the main reason for this behavior.

ABSTRACTAbout 7% of 7,252 nonduplicated clinical Escherichia colistrains from a Spanish hospital showed reduced susceptibility to amoxicillin-clavulanate. Of these, 0.37% produced the IRTs TEM-30, TEM-31, TEM-33, TEM-34, TEM-37, TEM-40, TEM-51, and TEM-54; 5.3% were probable class C β-lactamase overproducers; 0.8% were probable TEM-1 hyperproducers; 0.18% produced OXA-30; 0.15% overexpressed SHV-1; and 0.03% produced a PSE-1 enzyme.

ABSTRACTFor the present report, a novel complex class 1 integron, In60, was characterized. Part of this integron includes the blaCTX-M-9gene and its downstream nucleotide sequence, which shares 81% and 78% nucleotide identity with those of kluA-1β-lactamase and orf3of K. ascorbata, respectively. Furthermore, a new insertion sequence, IS3000, has been found in In60. PCR analysis indicates that integron In60 is present in 33 of 34 nonclonal enterobacterial isolates carrying the putative β-lactamase CTX-M-9.

ABSTRACTA new CTX-M-type β-lactamase (CTX-M-9) has been cloned from a clinical cefotaxime-resistant Escherichia colistrain. Despite the close identity that exists between the CTX-M-9 and Toho-2 β-lactamases (88%), the 35 amino acids located between residues Ala-185 and Ala-219 are totally different in both enzymes. Outside of this region there are only six amino acids substitutions between both proteins.

In this study, the plasmid content of clinical and commensal strains was analyzed and compared. The replicon profile was similar in both populations, except for L, M, A/C, and N (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and F were the most frequent replicons, only IncI1, sequence type 12 (ST12) was associated with blaCMY-2in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid.

ABSTRACTTwo strains of Enterobacteriaceae, Escherichia coliand Klebsiella pneumoniae, producing VIM-1 were isolated for the first time in Spain. In both strains, blaVIM-1was found to be carried on a gene cassette inserted into a class 1 integron. The blaVIM-1-containing integron was located on a transferable plasmid.

ABSTRACTWe studied in parallel the population structure of 90 carbapenemase-producing and 88 carbapenemase-susceptible Klebsiella pneumoniaeisolates collected in 20 Spanish hospitals, in the context of the EuSCAPE project. Fourteen and 50 multilocus sequence types (MLSTs) were detected among the carbapenemase-producing and carbapenem-susceptible isolates, respectively. ST11 and ST15 clones were more frequent in the carbapenemase-producing group than in the carbapenemase-susceptible group (P< 0.0001). Among the members of the carbapenem-suceptible group, the cefotaxime-resistant population showed population parameters that differed between the populations of the wild-type strains and the carbapenemase producers.

ABSTRACTThe increase in the level of quinolone resistance ofHaemophilus influenzaeclinical isolates during ofloxacin therapy of a patient with recurrent respiratory infections was investigated. The first isolate (MIC of ciprofloxacin of 2 μg/ml) and the second isolate (MIC of 32 μg/ml) belonged to the same clone, as shown by pulsed-field gel electrophoresis, and the increase in the resistance level was associated with a substitution in Ser-84 to Arg in the ParC protein. These results emphasize the potential risk of development of quinolone-resistant H. influenzaeduring fluoroquinolone therapy in patients with recurrent respiratory infection.