hospitals (about 40,000 hospital beds) prospectively collected nonduplicate Enterobacteriaceae using 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 pneumoniae and Escherichia coli. A total of 702 Enterobacteriaceae isolates 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. pneumoniae and 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. coli is 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. C arbapenemase-producing Enterobacteriaceae (CPE), mainly Klebsiella pneumoniae, are an emerging threat to public and individual health worldwide. These microorganisms are often resistant to almost all available antibiotics (1, 2), so there are few alternative treatment options. The most common carbapenemases are KPC (class A); VIM, IMP, and NDM (class B); and the OXA-48 types (class D). However, the extent to which health care systems have been affected and the carbapenemase types that are predominant differ substantially from country to country (3).A multicenter study performed in Spain in 2009 revealed 43 (0.04%) cases of CPE, which were mostly VIM-1 and IMP-22 (4). After that, we reported a rapid increase in the number of cases of CPE, mainly OXA-48-producing K. pneumoniae, in this country from 2010 to 2012 (5-7).Because previous studies (5, 6) were based on voluntary reports without taking into account key important issues, in this paper, we present data on the impact of CPE as obtained from a prospective, multicenter, and population-based study. We show that carbapenemase production in this country is widely and irregularly distributed; however, the rates of susceptibility to meropenem and colistin were still high.(The preliminary results of this study were presented in part at the 24th European Congress of Clinical Microbiology and Infectious Diseases Annual Meeting, 10 to 13 May 2014 in Barcelona, Spain,).

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Objectives There is little information about carbapenemase-producing (CP) Citrobacter spp. We studied the molecular epidemiology and microbiological features of CP Citrobacter spp. isolates collected in Spain (2013–15). Methods In total, 119 isolates suspected of being CP by the EUCAST screening cut-off values were analysed. Carbapenemases and ESBLs were characterized using PCR and sequencing. The genetic relationship among Citrobacter freundii isolates was studied by PFGE. Results Of the 119 isolates, 63 (52.9%) produced carbapenemases, of which 37 (58.7%) produced VIM-1, 20 (31.7%) produced OXA-48, 12 (19%) produced KPC-2, 2 (3.2%) produced NDM-1 and 1 (1.6%) produced VIM-2; 9 C. freundii isolates co-produced VIM-1 plus OXA-48. Fourteen isolates (22.2%) also carried ESBLs: 8 CTX-M-9 plus SHV-12, 2 CTX-M-9, 2 SHV-12 and 2 CTX-M-15. Fifty-seven isolates (90.5%) were C. freundii, 4 (6.3%) were Citrobacter koseri, 1 (1.6%) was Citrobacter amalonaticus and 1 (1.6%) was Citrobacter braakii. By EUCAST breakpoints, eight (12.7%) of the CP isolates were susceptible to the four carbapenems tested. In the 53 CP C. freundii analysed by PFGE, a total of 44 different band patterns were observed. Four PFGE clusters were identified: cluster 1 included eight isolates co-producing VIM-1 and OXA-48; blaVIM-1 was carried in a class 1 integron (intI–blaVIM-1–aacA4–dfrB1–aadA1–catB2–qacEΔ1/sul1) and blaOXA-48 was carried in a Tn1999.2 transposon. Conclusions We observed the clonal and polyclonal spread of CP Citrobacter spp. across several Spanish geographical areas. Four species of Citrobacter spp. produced up to five carbapenemase types, including co-production of VIM-1 plus OXA-48. Some CP Citrobacter spp. isolates were susceptible to the four carbapenems tested, a finding with potential clinical implications.

KPC-producing Enterobacteriaceae are increasing in Spain. Most KPC-producing K. pneumoniae isolates belonged to only five clones: ST11 and ST512 caused interregional spread, ST101 caused regional spread and ST1961 and ST678 produced independent hospital outbreaks. ST101/KPC-2 had the highest number of resistance and virulence genes. ST101/KPC-2 and ST512/KPC-3 were recently implicated in the spread of KPC in Italy.

A progressive increase in the prevalence of CP E. coli was observed, mainly due to the dissemination of OXA-48 producers. The most sensitive method for detecting decreased susceptibility of CP E. coli to carbapenems was disc diffusion with ertapenem using the EUCAST screening cut-offs. The spread of CP E. coli was due to a polyclonal population. The Pasteur scheme showed the highest discrimination power. Surveillance is crucial for the early detection of CP E. coli.

Objectives NDM carbapenemases have spread worldwide. However, little information exists about the impact of NDM-producing Enterobacteriaceae in Spain. By WGS, we sought to elucidate the population structure of NDM-like-producing Klebsiella pneumoniae and Escherichia coli in Spain and to determine the plasmids harbouring blaNDM-like genes. Methods High-resolution SNP typing, core-genome MLST and plasmid reconstruction (PlasmidID) were performed on 59 NDM-like-producing K. pneumoniae and 8 NDM-like-producing E. coli isolated over an 8 year period in Spain. Results Five major epidemic clones of NDM-producing K. pneumoniae caused five important nationwide outbreaks: ST437/NDM-7, ST437/NDM-1, ST147/NDM-1, ST11/NDM-1 and ST101/NDM-1; in contrast, the spread of NDM-producing E. coli was polyclonal. Three blaNDM types were identified: blaNDM-1, 61.2%; blaNDM-7, 32.8%; and blaNDM-5, 6%. Five K. pneumoniae isolates co-produced other carbapenemases (three blaOXA-48 and two blaVIM-1). The average number of acquired resistance genes was higher in K. pneumoniae than in E. coli. The plasmids encoding blaNDM-like genes belonged to IncFII, IncFIB, IncX3, IncR, IncN and IncC types, of which IncF, IncR and IncC were associated with MDR. The genetic surroundings of blaNDM-like genes showed a highly variable region upstream of ISAba125. Conclusions In recent years NDM-producing K. pneumoniae and E. coli have emerged in Spain; the spread of a few high-risk K. pneumoniae clones such as ST437/NDM-7, ST437/NDM-1, ST147/NDM-1, ST11/NDM-1 and ST101/NDM-1 have caused several interregional outbreaks. In contrast, the spread of NDM-producing E. coli has been polyclonal. Plasmid types IncFII, IncFIB, IncX3, IncR, IncN and IncC carried blaNDM, and the same IncX3 plasmid was detected in K. pneumoniae and E. coli.

mWe conducted a prospective multicenter study in Spain to characterize the mechanisms of resistance to amoxicillin-clavulanate (AMC) in Escherichia coli. Up to 44 AMC-resistant E. coli isolates (MIC > 32/16 g/ml) were collected at each of the seven participant hospitals. Resistance mechanisms were characterized by PCR and sequencing. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and by multilocus sequence typing. Overall AMC resistance was 9.3%. The resistance mechanisms detected in the 257 AMC-resistant isolates were OXA-1 production (26.1%), hyperproduction of penicillinase (22.6%), production of plasmidic AmpC (19.5%), hyperproduction of chromosomic AmpC Escherichia coli is an important etiologic agent for both nosocomial-and community-acquired infections in humans (9, 13, 22). Amoxicillin-clavulanate (AMC) is one of the most widely used antibiotics in many countries (3,11,15). In Spain, a 34.7% increase in community use of AMC was recorded from 2000 to 2006 (20). Recently, blood isolates of E. coli nonsusceptible to AMC increased from 9.3% (2003) Enzymatic mechanisms of E. coli resistance to AMC include hyperproduction of plasmid-mediated class A ␤-lactamases such as TEM-1 and SHV-1 (18, 31), plasmid-mediated AmpC-type ␤-lactamase (p-AmpC) (21), chromosomal AmpC ␤-lactamase (c-AmpC) (21), production of inhibitor-resistant TEM (IRT) ␤-lactamases (17, 27), plasmid-mediated ␤-lactamase OXA-1 (32), and complex mutant TEM (CMT) enzymes than combine IRT-and extended-spectrum ␤-lactamase (ESBL)-type substitutions (26).In spite of the significant increase in AMC use in the last several years, there is little recent information available about the prevalence of AMC resistance mechanisms in E. coli; most previous studies analyzed strains isolated more than 10 years ago from single hospitals in the United States (12, 28), France (16), and Spain (19,24). Accordingly, the aim of this prospective Spanish national multicenter study was to investigate the epidemiology and mechanisms of AMC resistance in clinical isolates of E. coli causing both community and nosocomial infections. MATERIALS AND METHODS Study design and bacterial isolates.A prospective multicenter study was designed to obtain E. coli isolates resistant to AMC (MIC of Ն 32/16 g/ml and/or disk inhibition zone of Յ13 mm according to the Clinical and Laboratory Standards Institute [CLSI]) (7) from clinical samples col-

The rapid increase in CIPNS E. coli causing bacteraemia was closely related to the increase in resistance to amoxicillin/clavulanic acid, production of ESBLs and resistance to aminoglycosides. Community use of fluoroquinolones (mainly moxifloxacin and levofloxacin) and of amoxicillin/clavulanic acid represents a significant driver in the progression of fluoroquinolone resistance in bacteraemic E. coli.