Characterization of Carbapenemase-Producing Klebsiella oxytoca in Spain, 2016-2017.

There is little information about carbapenemase-producing (CP) Klebsiella oxytoca, an important nosocomial pathogen. We characterized CP K. oxytoca isolates collected from different Spanish hospitals between January 2016 and October 2017. During the study period, 139 nonduplicate CP K. oxytoca isolates were identified; of these, 80 were studied in detail. Carbapenemase and extended-spectrum ß-lactamase genes were identified by PCR and sequencing. Genetic relatedness was studied by pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS), carried out on 12 representative isolates, was used to identify the resistome, to elucidate the phylogeny, and to determine the plasmids harboring carbapenemase genes. Forty-eight (60%) isolates produced VIM-1, 30 (37.5%) produced OXA-48, 3 (3.7%) produced KPC-2, 2 (2.5%) produced KPC-3, and 1 (1.2%) produced NDM-1; 4 isolates coproduced two carbapenemases. By PFGE, 69 patterns were obtained from the 80 CP K. oxytoca isolates, and four well-defined clusters were detected: cluster 1 consisted of 11 OXA-48-producing isolates, and the other three clusters included VIM-1-producing isolates (5, 3, and 3 isolates, respectively). In the 12 sequenced isolates, the average number of acquired resistance genes was significantly higher in VIM-1-producing isolates (10.8) than in OXA-48-producing isolates (2.3). All 12 isolates had chromosomally encoded genes of the blaOXY-2 genotype, and by multilocus sequence typing, most belonged to sequence type 2 (ST2). Carbapenemase genes were carried by IncL, IncHI2, IncFII, IncN, IncC, and IncP6 plasmid types. The emergence of CP K. oxytoca was principally due to the spread of VIM-1- and OXA-48-producing isolates in which VIM-1- and OXA-48 were carried by IncL, IncHI2, IncFII, and IncN plasmids. ST2 and the genotype blaOXY-2 predominated among the 12 sequenced isolates.

Emergence of NDM-producing Klebsiella pneumoniae and Escherichia coli in Spain: phylogeny, resistome, virulence and plasmids encoding blaNDM-like genes as determined by WGS.

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.

The Carbapenemase-Producing Klebsiella pneumoniae Population Is Distinct and More Clonal than the Carbapenem-Susceptible Population.

We studied in parallel the population structure of 90 carbapenemase-producing and 88 carbapenemase-susceptible Klebsiella pneumoniae isolates 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.

Carbapenem-resistant Citrobacter spp. isolated in Spain from 2013 to 2015 produced a variety of carbapenemases including VIM-1, OXA-48, KPC-2, NDM-1 and VIM-2.

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.

Survey of Carbapenemase-Producing Enterobacteriaceae in Companion Dogs in Madrid, Spain.

We found a low prevalence (0.6%) of carbapenemase-producingEnterobacteriaceae(CPE) in fecal microbiota of companion dogs. A single VIM-1-producingKlebsiella pneumoniaeisolate belonging to sequence type (ST) 2090 was detected.blaVIM-1was carried on a class 1 integron and an untypeable ∼48-kb plasmid. Emergence and spread of CPE in this group of animals may be a threat to public health in human and veterinary medicine. This finding supports the need for active surveillance studies in companion animals that live close to humans, as interspecies transmission may occur within the same household.

Carbapenemase-producing Escherichia coli is becoming more prevalent in Spain mainly because of the polyclonal dissemination of OXA-48.

OBJECTIVES: The objective of this study was to analyse the microbiological traits and the population structure of carbapenemase-producing (CP) Escherichia coli isolates collected in Spain between 2012 and 2014. METHODS: Two-hundred-and-thirty-nine E. coli isolates non-susceptible to carbapenems were studied. The carbapenemase genes and the phylogenetic groups were characterized using PCR. MLST was carried out using the typing schemes of the University of Warwick and the Institut Pasteur. The diversity of the population structure was estimated by calculating a simple diversity index (SDI). RESULTS: One-hundred-and-twenty-one isolates (50.6%) produced carbapenemases, of which 87 (71.9%) were OXA-48, 27 (22.3%) were VIM-1, 4 (3.3%) were KPC-2, 2 (1.7%) were NDM and 1 (0.8%) was IMP-22; 4 isolates were collected in 2012, 40 in 2013 and 77 in 2014. Ertapenem was more sensitive than imipenem or meropenem for screening for OXA-48-producing E. coli. Using the Warwick typing scheme, 59 different STs were identified, the most prevalent being ST131 (16.5%). The population diversity was higher among VIM-1-producing isolates (SDI = 81.5%) than among OXA-48-producing isolates (SDI = 44.8%). The Pasteur scheme had a higher discrimination capability (SDI = 55.4%) than the Warwick scheme (SDI = 48.8%). CONCLUSIONS: 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.

The spread of KPC-producing Enterobacteriaceae in Spain: WGS analysis of the emerging high-risk clones of Klebsiella pneumoniae ST11/KPC-2, ST101/KPC-2 and ST512/KPC-3.

OBJECTIVES: We analysed the microbiological traits and population structure of KPC-producing Enterobacteriaceae isolates collected in Spain between 2012 and 2014. We also performed a comparative WGS analysis of the three major KPC-producing Klebsiella pneumoniae clones detected. METHODS: Carbapenemase and ESBL genes were sequenced. The Institut Pasteur MLST scheme was used. WGS data were used to construct phylogenetic trees, to identify the determinants of resistance and to de novo assemble the genome of one representative isolate of each of the three major K. pneumoniae clones. RESULTS: Of the 2443 carbapenemase-producing Enterobacteriaceae isolates identified during the study period, 111 (4.5%) produced KPC. Of these, 81 (73.0%) were K. pneumoniae and 13 (11.7%) were Enterobacter cloacae. Three major epidemic clones of K. pneumoniae were identified: ST11/KPC-2, ST101/KPC-2 and ST512/KPC-3. ST11/KPC-2 differed from ST101/KPC-2 and ST512/KPC-3 by 27 819 and 6924 SNPs, respectively. ST101/KPC-2 differed from ST512/KPC-3 by 28 345 SNPs. Nine acquired resistance genes were found in ST11/KPC-2, 11 in ST512/KPC-3 and 13 in ST101/KPC-2. ST101/KPC-2 had the highest number of virulence genes (20). An 11 bp deletion at the end of the mgrB sequence was the cause of colistin resistance in ST512/KPC-3. CONCLUSIONS: 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.

Suppression of cell migration is promoted by miR-944 through targeting of SIAH1 and PTP4A1 in breast cancer cells.

BACKGROUND: Aberrant expression of microRNAs has been associated with migration of tumor cells. In this study, we aimed to investigate the biological significance of miR-944 whose function is unknown in breast cancer. METHODS: MiR-944 expression in breast cancer cells and tumors was evaluated by Taqman qRT-PCR assays. Transcriptional profiling of MDA-MB-231 cells expressing miR-944 was performed using DNA microarrays. Cell viability, migration and invasion were assessed by MTT, scratch/wound-healing and transwell chamber assays, respectively. The luciferase reporter assay was used to evaluate targeting of SIAH1, PTP4A1 and PRKCA genes by miR-944. SIAH1 protein levels were measured by Western blot. Silencing of SIAH1 gene was performed by RNA interference using shRNAs. RESULTS: Our data showed that miR-944 expression was severely repressed in clinical specimens and breast cancer cell lines. Suppression of miR-944 levels was independent of hormonal status and metastatic potential of breast cancer cells. Gain-of-function analysis indicated that miR-944 altered the actin cytoskeleton dynamics and impaired cell migration and invasion. Genome-wide transcriptional profiling of MDA-MB-231 cells that ectopically express miR-944 showed that 15 genes involved in migration were significantly repressed. Notably, luciferase reporter assays confirmed the ability of miR-944 to bind the 3´UTR of SIAH1 and PTP4A1 genes, but not PRKCA gene. Congruently, an inverse correlation between miR-944 and SIAH1 protein expression was found in breast cancer cells. Moreover, SIAH1 was upregulated in 75 % of miR-944-deficient breast tumors. Finally, SIAH1 gene silencing by RNA interference significantly impaired cell migration of breast cancer cells. CONCLUSIONS: Our results pointed out that miR-944 is a novel upstream negative regulator of SIAH1 and PTP4A1 genes and provided for the first time evidence for its functional role in migration and invasion of breast cancer cells. They also suggest that miR-944 restoration may represent a potential strategy for breast cancer therapy.

Prospective multicenter study of carbapenemase-producing Enterobacteriaceae from 83 hospitals in Spain reveals high in vitro susceptibility to colistin and meropenem.

The 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 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.

Characterizing carbapenemase-producing Escherichia coli isolates from Spain: high genetic heterogeneity and wide geographical spread.

Introduction: Carbapenemase-Producing Escherichia coli (CP-Eco) isolates, though less prevalent than other CP-Enterobacterales, have the capacity to rapidly disseminate antibiotic resistance genes (ARGs) and cause serious difficult-to-treat infections. The aim of this study is phenotypically and genotypically characterizing CP-Eco isolates collected from Spain to better understand their resistance mechanisms and population structure. Methods: Ninety representative isolates received from 2015 to 2020 from 25 provinces and 59 hospitals Spanish hospitals were included. Antibiotic susceptibility was determined according to EUCAST guidelines and whole-genome sequencing was performed. Antibiotic resistance and virulence-associated genes, phylogeny and population structure, and carbapenemase genes-carrying plasmids were analyzed. Results and discussion: The 90 CP-Eco isolates were highly polyclonal, where the most prevalent was ST131, detected in 14 (15.6%) of the isolates. The carbapenemase genes detected were bla OXA-48 (45.6%), bla VIM-1 (23.3%), bla NDM-1 (7.8%), bla KPC-3 (6.7%), and bla NDM-5 (6.7%). Forty (44.4%) were resistant to 6 or more antibiotic groups and the most active antibiotics were colistin (98.9%), plazomicin (92.2%) and cefiderocol (92.2%). Four of the seven cefiderocol-resistant isolates belonged to ST167 and six harbored bla NDM. Five of the plazomicin-resistant isolates harbored rmt. IncL plasmids were the most frequent (45.7%) and eight of these harbored bla VIM-1. bla OXA-48 was found in IncF plasmids in eight isolates. Metallo-ß-lactamases were more frequent in isolates with resistance to six or more antibiotic groups, with their genes often present on the same plasmid/integron. ST131 isolates were associated with sat and pap virulence genes. This study highlights the genetic versatility of CP-Eco and its potential to disseminate ARGs and cause community and nosocomial infections.

Carbapenemase-producing enterobacteriaceae in Spain in 2012.

We report the epidemiological impact of carbapenemase-producing Enterobacteriaceae (CPE) in Spain in 2012. Of the 237 carbapenemases detected, 163 were from the OXA-48 group, 60 were from VIM-1, 8 were from KPC-2, 5 were from IMP, and 1 was from NDM-1. Interhospital spread of carbapenemase-producing Klebsiella pneumoniae was due to a limited number of multilocus sequence types (MLST) and carbapenemase types, including ST15-VIM-1, ST11-OXA-48, ST405-OXA-48, ST101-KPC-2, and ST11-VIM-1. The number of CPE cases in Spain has increased sharply in recent years, due mainly to the emergence of OXA-48.

Emergence of OXA-48-producing Klebsiella pneumoniae and the novel carbapenemases OXA-244 and OXA-245 in Spain.

OBJECTIVES: To describe the molecular and population-level characterization of a selected group of OXA-48-like-producing Klebsiella pneumoniae isolates collected in Spain between January 2011 and May 2012. METHODS: During the study period, 151 OXA-48-like-producing K. pneumoniae isolates were collected from 10 hospitals in six different Spanish regions. From these, a representative sample of 21 isolates that caused hospital outbreaks and single infections was selected for further in-depth analysis. Molecular epidemiology was investigated using PFGE and multilocus sequence typing (MLST). Resistance genes were characterized by PCR and sequencing. Plasmids carrying bla(OXA-48-like) were studied by PFGE with S1 nuclease digestion. RESULTS: All 21 isolates had ertapenem MICs ≥ 1 mg/L, but 47.6% remained susceptible to imipenem and meropenem; bla(OXA-48) was identified in 19 isolates (90.5%) and the novel bla(OXA-244) and bla(OXA-245) genes were detected in 1 isolate each. With one exception, all isolates that contained bla(OXA-48-like) also contained bla(CTX-M-15). PFGE typing revealed six clusters comprising isolates that belonged to MLST types ST11, ST16, ST392, ST405, ST437 and ST663, respectively. Two main clusters were identified: PFGE cluster 1 (12 isolates, belonging either to ST405 or ST663, from seven hospitals), and PFGE cluster 2 (4 ST16 isolates from two hospitals). Six of seven donor isolates conjugated successfully; bla(OXA-48-like) (but not bla(CTX-M-15)) was carried on ≈ 60 kb Inc L/M plasmids. CONCLUSIONS: Multidrug-resistant K. pneumoniae producing OXA-48-like carbapenemase are emerging as important pathogens in Spain due to intra- and inter-hospital, clonal and non-clonal dissemination.

Carbapenemase-Producing Klebsiella pneumoniae in COVID-19 Intensive Care Patients: Identification of IncL-VIM-1 Plasmid in Previously Non-Predominant Sequence Types.

During the COVID-19 pandemic, intensive care units (ICUs) operated at or above capacity, and the number of ICU patients coinfected by nosocomial microorganisms increased. Here, we characterize the population structure and resistance mechanisms of carbapenemase-producing Klebsiella pneumoniae (CP-Kpn) from COVID-19 ICU patients and compare them to pre-pandemic populations of CP-Kpn. We analyzed 84 CP-Kpn isolates obtained during the pandemic and 74 CP-Kpn isolates obtained during the pre-pandemic period (2019) by whole genome sequencing, core genome multilocus sequence typing, plasmid reconstruction, and antibiotic susceptibility tests. More CP-Kpn COVID-19 isolates produced OXA-48 (60/84, 71.4%) and VIM-1 (18/84, 21.4%) than KPC (8/84, 9.5%). Fewer pre-pandemic CP-Kpn isolates produced VIM-1 (7/74, 9.5%). Cefiderocol (97.3-100%) and plazomicin (97.5-100%) had the highest antibiotic activity against pandemic and pre-pandemic isolates. Sequence type 307 (ST307) was the most widely distributed ST in both groups. VIM-1-producing isolates belonging to ST307, ST17, ST321 and ST485, (STs infrequently associated to VIM-1) were detected during the COVID-19 period. Class 1 integron Int1-blaVIM-1-aac(6')-1b-dfrB1-aadAI-catB2-qacEΔ1/sul1, found on an IncL plasmid of approximately 70,000 bp, carried blaVIM-1 in ST307, ST17, ST485, and ST321 isolates. Thus, CP-Kpn populations from pandemic and pre-pandemic periods have similarities. However, VIM-1 isolates associated with atypical STs increased during the pandemic, which warrants additional monitoring and surveillance.

Corrigendum: Phenotypic and molecular characterization of IMP-producing Enterobacterales in Spain: predominance of IMP-8 in Klebsiella pneumoniae and IMP-22 in Enterobacter roggenkampii.

[This corrects the article DOI: 10.3389/fmicb.2022.1000787.].

Widespread Detection of Yersiniabactin Gene Cluster and Its Encoding Integrative Conjugative Elements (ICEKp) among Nonoutbreak OXA-48-Producing Klebsiella pneumoniae Clinical Isolates from Spain and the Netherlands.

In this study, we determined the presence of virulence factors in nonoutbreak, high-risk clones and other isolates belonging to less common sequence types associated with the spread of OXA-48-producing Klebsiella pneumoniae clinical isolates from The Netherlands (n = 61) and Spain (n = 53). Most isolates shared a chromosomally encoded core of virulence factors, including the enterobactin gene cluster, fimbrial fim and mrk gene clusters, and urea metabolism genes (ureAD). We observed a high diversity of K-Locus and K/O loci combinations, KL17 and KL24 (both 16%), and the O1/O2v1 locus (51%) being the most prevalent in our study. The most prevalent accessory virulence factor was the yersiniabactin gene cluster (66.7%). We found seven yersiniabactin lineages-ybt 9, ybt 10, ybt 13, ybt 14, ybt 16, ybt 17, and ybt 27-which were chromosomally embedded in seven integrative conjugative elements (ICEKp): ICEKp3, ICEKp4, ICEKp2, ICEKp5, ICEKp12, ICEKp10, and ICEKp22, respectively. Multidrug-resistant lineages-ST11, ST101, and ST405-were associated with ybt 10/ICEKp4, ybt 9/ICEKp3, and ybt 27/ICEKp22, respectively. The fimbrial adhesin kpi operon (kpiABCDEFG) was predominant among ST14, ST15, and ST405 isolates, as well as the ferric uptake system kfuABC, which was also predominant among ST101 isolates. No convergence of hypervirulence and resistance was observed in this collection of OXA-48-producing K. pneumoniae clinical isolates. Nevertheless, two isolates, ST133 and ST792, were positive for the genotoxin colibactin gene cluster (ICEKp10). In this study, the integrative conjugative element, ICEKp, was the major vehicle for yersiniabactin and colibactin gene clusters spreading. IMPORTANCE Convergence of multidrug resistance and hypervirulence in Klebsiella pneumoniae isolates has been reported mostly related to sporadic cases or small outbreaks. Nevertheless, little is known about the real prevalence of carbapenem-resistant hypervirulent K. pneumoniae since these two phenomena are often separately studied. In this study, we gathered information on the virulent content of nonoutbreak, high-risk clones (i.e., ST11, ST15, and ST405) and other less common STs associated with the spread of OXA-48-producing K. pneumoniae clinical isolates. The study of virulence content in nonoutbreak isolates can help us to expand information on the genomic landscape of virulence factors in K. pneumoniae population by identifying virulence markers and their mechanisms of spread. Surveillance should focus not only on antimicrobial resistance but also on virulence characteristics to avoid the spread of multidrug and (hyper)virulent K. pneumoniae that may cause untreatable and more severe infections.

Extended-spectrum-ß-lactamase-producing Escherichia coli as a cause of pediatric infections: report of a neonatal intensive care unit outbreak due to a CTX-M-14-producing strain.

Little information is available about pediatric infections caused by extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli. We characterized an outbreak caused by a CTX-M-14-producing E. coli isolate in a neonatal intensive care unit (NICU) and studied other infections caused by ESBL-producing E. coli in non-NICU pediatric units. All children ≤4 years old who were infected or colonized by ESBL-producing E. coli isolates between January 2009 and September 2010 were included. Molecular epidemiology was studied by phylogroup analysis, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. Antibiotic resistance genes were analyzed by PCR and sequencing. Plasmids were studied by PFGE with S1 nuclease digestion and by incompatibility group analysis using a PCR-based replicon-typing scheme. Of the ESBL-producing E. coli isolates colonizing or infecting the 30 newborns, identical PFGE results were observed for 21 (70%) isolates, which were classified as CTX-M-14-producing E. coli of ST23 phylogroup A. bla(CTX-M-14a) was linked to ISEcp1 and was carried on an ∼80-bp IncK plasmid. A smaller ongoing outbreak due to SHV-12-producing ST131 E. coli was also identified in the same NICU. Fifteen additional infections with ESBL-producing E. coli were identified in non-NICU pediatric units, but none was caused by the CTX-M-14-producing E. coli epidemic clone. Overall, CTX-M-14 (71.1%), CTX-M-15 (13.3%), and SHV-12 (13.3%) were the most important ESBLs causing pediatric infections in this study. Infections of newborns with CTX-M-14-producing E. coli were caused by both clonal and nonclonal isolates.

Phenotypic and molecular characterization of IMP-producing Enterobacterales in Spain: Predominance of IMP-8 in Klebsiella pneumoniae and IMP-22 in Enterobacter roggenkampii.

Objectives: Little is known about IMP-producing Enterobacterales (IMP-Ent) in Europe. We analyzed at genomic and phenotypic level IMP-Ent isolates circulating in Spain in a 9-year period. Materials and methods: IMP-Ent isolates submitted to our reference laboratory were included. Antibiotic susceptibility was performed using microdilution method (EUCAST), and IMP-carbapenemase activity was measured with carbapenemase inhibitors, the ß-CARBA method, the modified Hodge test (MHT), and the modified carbapenemase inhibition method (mCIM). All isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: Fifty IMP-Ent isolates, collected from 19 hospitals in 13 Spanish provinces, were detected: Klebsiella pneumoniae (IMP-Kpn) (24; 48%), Enterobacter roggenkampii (13; 26%), Enterobacter hormaechei (8, 16%), Klebsiella oxytoca (two; 4%), Enterobacter asburiae (one, 2%), Serratia marcescens (one; 2%) and Escherichia coli (one; 2%). All isolates were positive by the MHT and ß-CARBA tests; 48 (96%) were mCIM positive; 12 (24%) and 26 (52%) displayed positive inhibition with dipicolinic (meropenem) and EDTA (ertapenem), respectively. Five IMP-carbapenemase types were identified: IMP-8 (22; 44%), IMP-22 (17; 34%), IMP-13 (7; 14%), IMP-28 (two; 4%), and IMP-15 (two; 4%), predominating IMP-8 in K. pneumoniae and IMP-22 in E. roggenkampii. IMP-28 was exclusively identified in K. oxytoca and IMP-15 in E. hormaechei. Predominant STs were ST405 (29.2%), ST15 (25%) and ST464 (20.8%) in IMP-Kpn; ST96 (100%) in E. roggenkampii and ST182 (62.5%) in E. hormachei. Colistin and amikacin were the most active non-carbapenem antibiotics against IMP-Ent. Conclusion: IMP-Ent isolates remain infrequent in Spain, although in recent years have been circulating causing nosocomial outbreaks, being IMP-8-producing K. pneumoniae and IMP-22-producing E. roggenkampii the most frequently detected in this study. Inhibition with EDTA or dipicolinic acid presented false negative results in some IMP-producing strains. Active microbiological and molecular surveillance is essential for a better comprehension and control of IMP-Ent dissemination.

Prevalence, detection and characterisation of fosfomycin-resistant Escherichia coli strains carrying fosA genes in Community of Madrid, Spain.

OBJECTIVES: The aim of this this study was to describe the presence of different variants of the fosA gene in fosfomycin-resistant Escherichia coli strains in Madrid, Spain. METHODS: fos genes were searched for in 55 E. coli strains collected from seven representative hospitals located in Madrid. A phenotypic screening test was performed following the disk diffusion method with sodium phosphonoformate added as described by Nakamura et al. Additionally, a molecular study based on PCR was used to confirm the screening results. Positive strains for fos genes were further subjected to whole-genome sequencing (WGS). RESULTS: Phenotypic screening was positive in 9/55 strains (16.4%), although genotypic detection was positive in only 3 (fosA3, fosA4 and fosA6). Thus, the prevalence of fos genes in Madrid was 5.5% (3/55). WGS data were not available for the fosA6-positive strain. One isolate with fosA3 (ST69) carried a blaCTX-M-55 gene and seven virulence genes (air, eilA, iha, iss, lpfA, sat and senB). The fosA4-positive isolate (ST4038) carried the virulence genes iss, lpfA, iroN and mchF. Both fos genes were located between two IS26 mobile elements of a plasmid. CONCLUSION: We detected the presence of different variants of plasmid-mediated fosA genes in fosfomycin-resistant E. coli strains in Madrid, Spain. Despite the few reports in Europe, it would be of interest to monitor the spread of these acquired resistance genes.