Deciphering mechanisms affecting cefepime-taniborbactam in vitro activity in carbapenemase-producing Enterobacterales and carbapenem-resistant Pseudomonas spp. isolates recovered during a surveillance study in Spain.

PURPOSE: To characterize the resistance mechanisms affecting the cefepime-taniborbactam combination in a collection of carbapenemase-producing Enterobacterales (CPE) and carbapenem-resistant Pseudomonas spp. (predominantly P. aeruginosa; CRPA) clinical isolates. METHODS: CPE (n = 247) and CRPA (n = 170) isolates were prospectively collected from patients admitted to 8 Spanish hospitals. Susceptibility to cefepime-taniborbactam and comparators was determined by broth microdilution. Cefepime-taniborbactam was the most active agent, inhibiting 97.6% of CPE and 67.1% of CRPA (MICs ≤ 8/4 mg/L). All isolates with cefepime-taniborbactam MIC > 8/4 mg/L (5 CPE and 52 CRPA) and a subset with MIC ≤ 8/4 mg/L (23 CPE and 24 CRPA) were characterized by whole genome sequencing. RESULTS: A reduced cefepime-taniborbactam activity was found in two KPC-ST307-Klebsiella pneumoniae isolates with altered porins [KPC-62-K. pneumoniae (OmpA, OmpR/EnvZ), KPC-150-K. pneumoniae (OmpK35, OmpK36)] and one each ST133-VIM-1-Enterobacter hormaechei with altered OmpD, OmpR, and OmpC; IMP-8-ST24-Enterobacter asburiae; and NDM-5-Escherichia coli with an YRIN-inserted PBP3 and a mutated PBP2. Among the P. aeruginosa (68/76), elevated cefepime-taniborbactam MICs were mostly associated with GES-5-ST235, OXA-2+VIM-2-ST235, and OXA-2+VIM-20-ST175 isolates also carrying mutations in PBP3, efflux pump (mexR, mexZ) and AmpC (mpl) regulators, and non-carbapenemase-ST175 isolates with AmpD-T139M and PBP3-R504C mutations. Overall, accumulation of these mutations was frequently detected among non-carbapenemase producers. CONCLUSIONS: The reduced cefepime-taniborbactam activity among the minority of isolates with elevated cefepime-taniborbactam MICs is not only due to IMP carbapenemases but also to the accumulation of multiple resistance mechanisms, including PBP and porin mutations in CPE and chromosomal mutations leading to efflux pumps up-regulation, AmpC overexpression, and PBP modifications in P. aeruginosa.

Automatic Discrimination of Species within the Enterobacter cloacae Complex Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Supervised Algorithms.

The Enterobacter cloacae complex (ECC) encompasses heterogeneous clusters of species that have been associated with nosocomial outbreaks. These species may have different acquired antimicrobial resistance and virulence mechanisms, and their identification is challenging. This study aims to develop predictive models based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles and machine learning for species-level identification. A total of 219 ECC and 118 Klebsiella aerogenes clinical isolates from three hospitals were included. The capability of the proposed method to differentiate the most common ECC species (Enterobacter asburiae, Enterobacter kobei, Enterobacter hormaechei, Enterobacter roggenkampii, Enterobacter ludwigii, and Enterobacter bugandensis) and K. aerogenes was demonstrated by applying unsupervised hierarchical clustering with principal-component analysis (PCA) preprocessing. We observed a distinctive clustering of E. hormaechei and K. aerogenes and a clear trend for the rest of the ECC species to be differentiated over the development data set. Thus, we developed supervised, nonlinear predictive models (support vector machine with radial basis function and random forest). The external validation of these models with protein spectra from two participating hospitals yielded 100% correct species-level assignment for E. asburiae, E. kobei, and E. roggenkampii and between 91.2% and 98.0% for the remaining ECC species; with data analyzed in the three participating centers, the accuracy was close to 100%. Similar results were obtained with the Mass Spectrometric Identification (MSI) database developed recently (https://msi.happy-dev.fr) except in the case of E. hormaechei, which was more accurately identified with the random forest algorithm. In short, MALDI-TOF MS combined with machine learning was demonstrated to be a rapid and accurate method for the differentiation of ECC species.

Outbreak by KPC-62-producing ST307 Klebsiella pneumoniae isolates resistant to ceftazidime/avibactam and cefiderocol in a university hospital in Madrid, Spain.

OBJECTIVES: Ceftazidime/avibactam and cefiderocol are two of the latest antibiotics with activity against a wide variety of Gram-negatives, including carbapenem-resistant Enterobacterales. We sought to describe the phenotypic and genotypic characteristics of ceftazidime/avibactam- and cefiderocol-resistant KPC-Klebsiella pneumoniae (KPC-Kp) detected during an outbreak in 2020 in the medical ICU of our hospital. METHODS: We collected 11 KPC-Kp isolates (6 clinical; 5 surveillance samples) resistant to ceftazidime/avibactam and cefiderocol from four ICU patients (November 2020 to January 2021), without prior exposure to these agents. All patients had a decontamination regimen as part of the standard ICU infection prevention protocol. Additionally, one ceftazidime/avibactam- and cefiderocol-resistant KPC-Kp (June 2019) was retrospectively recovered. Antibiotic susceptibility was determined by broth microdilution. ß-Lactamases were characterized and confirmed. WGS was also performed. RESULTS: All KPC-Kp isolates (ceftazidime/avibactam MIC  ≥16/4 mg/L; cefiderocol MIC ≥4 mg/L) were KPC + CTX-M-15 producers and belonged to the ST307 high-risk-clone (ST307-HRC). KPC-62 (L168Q) was detected in all isolates involved in the 2020 outbreak, contained in January 2021. KPC-31 (D179Y) was identified in the KPC-Kp from 2019. Cloning experiments demonstrated that both blaKPC-62 and blaKPC-31 were responsible for ceftazidime/avibactam resistance (MIC >16 mg/L) and an increased cefiderocol MIC. Additionally, mutations in OmpA and EnvZ/OmpR porin proteins (in KPC-62-Kp) and in PBP2 (in KPC-31-Kp) were found and may be involved in cefiderocol resistance. CONCLUSIONS: The emergence of resistance to both ceftazidime/avibactam and cefiderocol in KPC-Kp-HRCs, together with the diversification of novel KPC enzymes displaying different antibiotic resistance phenotypes, is an epidemiological and clinical risk.

Significant increase of CTX-M-15-ST131 and emergence of CTX-M-27-ST131 Escherichia coli high-risk clones causing healthcare-associated bacteraemia of urinary origin in Spain (ITUBRAS-2 project).

OBJECTIVES: To assess the microbiological characteristics of Escherichia coli causing healthcare-associated bacteraemia of urinary origin (HCA-BUO) in Spain (ITUBRAS-2 project), with particular focus on ESBL producers and isolates belonging to ST131 high-risk clone (HiRC). Clinical characteristics and outcomes associated with ST131 infection were investigated. METHODS: A total of 222 E. coli blood isolates were prospectively collected from patients with HCA-BUO from 12 tertiary-care hospitals in Spain (2017-19). Antimicrobial susceptibility and ESBL/carbapenemase production were determined. ST131 subtyping was performed. A subset of 115 isolates were selected for WGS to determine population structure, resistome and virulome. Clinical charts were reviewed. RESULTS: ESBL-producing E. coli prevalence was 30.6% (68/222). ST131 represented 29.7% (66/222) of E. coli isolates and accounted for the majority of ESBL producers (46/68, 67.6%). The C2/H30-Rx subclone accounted for most ST131 isolates (44/66) and was associated with CTX-M-15 (37/44) and OXA-1 enzymes (27/44). Cluster C1-M27 was identified in 4/10 isolates belonging to subclade C1/H30-R1 and associated with CTX-M-27. Additionally, ST131 isolates showed a high content of other acquired resistance genes, and clade C/ST131 isolates carried characteristic QRDR mutations. They were categorized as uropathogenic E. coli and had higher aggregate virulence scores. ST131 infection was associated with more complex patients, prior use of cephalosporins and inadequate empirical treatment but was not associated with worse clinical outcomes. CONCLUSIONS: ST131 HiRC is the main driver of ESBL-producing E. coli causing HCA-BUO in Spain, mainly associated with the expansion of subclade CTX-M-15-C2/H30-Rx and the emergence of CTX-M-27-C1/H30-R1 (Cluster C1-M27).

Multicenter evaluation of BioFire JI panel related to improved microbiological diagnostics on acute osteoarticular infections.

Microbiological diagnosis of osteoarticular infections (OI) is crucial for a successful treatment. A prospective multicenter study including 262 synovial fluids with suspicion of acute OI was performed between July 2021 and October of 2022. BioFire Joint Infection Panel multiplex-PCR test was performed and results were compared with conventional cultures of synovial fluid specimens. In total, 136 microorganisms were detected, and fourteen samples were positive for more than one microorganism. In monomicrobial infections (n = 87) agreement with culture was 69%. In 26 samples, the multiplex PCR yield an additional positive result when culture result was negative. It helped in the detection of fastidious microorganisms as K. kingae and N. gonorrhoeae. This multiplex PCR has proven to be a useful technique that can be used for patients with high suspicion of acute OI in a rapid and automated manner.

In Vitro Activity of Cefepime-Taniborbactam against Carbapenemase-Producing Enterobacterales and Pseudomonas aeruginosa Isolates Recovered in Spain.

Novel ß-lactam-ß-lactamase inhibitor combinations currently approved for clinical use are poorly active against metallo-ß-lactamase (MBL)-producing strains. We evaluated the in vitro activity of cefepime-taniborbactam (FTB [formerly cefepime-VNRX-5133]) and comparator agents against carbapenemase-producing Enterobacterales (n = 247) and carbapenem-resistant Pseudomonas species (n = 170) clinical isolates prospectively collected from different clinical origins in patients admitted to 8 Spanish hospitals. FTB was the most active agent in both Enterobacterales (97.6% MICFTB, ≤8/4 mg/L) and Pseudomonas (67.1% MICFTB, ≤8/4 mg/L) populations. The MICFTB was >8 mg/L in 6/247 (2.4%) Enterobacterales isolates (3 KPC-producing Klebsiella pneumoniae isolates, 1 VIM-producing Enterobacter cloacae isolate, 1 IMP-producing E. cloacae isolate, and 1 NDM-producing Escherichia coli isolate) and in 56/170 (32.9%) Pseudomonas isolates, 19 of them carbapenemase producers (15 producers of VIM, 2 of GES, 1 of GES+VIM, and 1 of GES+KPC). Against the Enterobacterales isolates with meropenem MICs of >2 mg/L (138/247), FTB was the most active agent against both serine-ß-lactamases (107/138) and MBL producers (31/138) (97.2 and 93.5% MICFTB, ≤8/4 mg/L, respectively), whereas the activity of comparators was reduced, particularly against the MBL producers (ceftazidime-avibactam, 94.4 and 12.9%, meropenem-vaborbactam, 85.0 and 64.5%, imipenem-relebactam, 76.6 and 9.7%, ceftolozane-tazobactam, 1.9 and 0%, and piperacillin-tazobactam, 0 and 0%, respectively). Among the meropenem-resistant Pseudomonas isolates (163/170; MIC, >2 mg/L), the activities of FTB against serine-ß-lactamase (35/163) and MBL (43/163) producers were 88.6 and 65.1%, respectively, whereas the susceptibilities of comparators were as follows: ceftazidime-avibactam, 88.5 and 16.0%, meropenem-vaborbactam, 8.5 and 7.0%, imipenem-relebactam, 2.9 and 2.3%, ceftolozane-tazobactam, 0 and 2.3%, and piperacillin-tazobactam, 0 and 0%, respectively. Microbiological results suggest FTB as a potential therapeutic option in patients infected with carbapenemase-producing Enterobacterales and carbapenem-resistant Pseudomonas isolates, including MBL producers.

Impact of Ceftazidime-Avibactam Treatment in the Emergence of Novel KPC Variants in the ST307-Klebsiella pneumoniae High-Risk Clone and Consequences for Their Routine Detection.

The emergence of Klebsiella pneumoniae isolates carrying novel blaKPC variants conferring ceftazidime-avibactam (CAZ/AVI) resistance is being increasingly reported. We evaluated the accuracy of phenotypic methods commonly used in routine clinical laboratories in the detection of novel K. pneumoniae carbapenemase (KPC) enzymes. Additionally, we characterized by whole-genome sequencing (WGS) the KPC-ST307-K. pneumoniae isolates recovered in our hospital before and after CAZ/AVI therapy. Rectal colonization or infection by carbapenem-resistant KPC-3 K. pneumoniae isolates (imipenem MIC, 16 mg/L; meropenem MIC, 8 to >16 mg/L) and CAZ/AVI-susceptible isolates (CAZ/AVI MIC, 1 to 2 mg/L) were first detected in three intensive care unit (ICU) patients admitted between March 2020 and July 2020. KPC K. pneumoniae isolates with increased CAZ/AVI MICs (8 to 32 mg/L) and carbapenem susceptibility (imipenem and meropenem MIC, <1 mg/L) were recovered within 6 to 24 days after CAZ/AVI treatment. WGS confirmed that all KPC K. pneumoniae isolates belonged to the sequence type 307 (ST307) high-risk clone and carried identical antimicrobial resistance genes and virulence factors. The presence of the novel blaKPC-46, blaKPC-66, and blaKPC-92 genes was confirmed in the K. pneumoniae isolates with increased CAZ/AVI MICs and restored carbapenem activity. KPC production was confirmed by immunochromatography, the eazyplex Superbug CRE system, and the Xpert Carba-R assay in all KPC K. pneumoniae isolates, but not in any isolate using chromogenic agar plates for carbapenemase producers (ChromID-CARBA), the KPC/MBL/OXA-48 Confirm kit, and the ß-CARBA test. Nevertheless, all grew in chromogenic agar plates for extended-spectrum ß-lactamase (ESBL) producers (ChromID-ESBL). We report the failure of the most common phenotypic methods used for the detection of novel KPC carbapenemases but not of rapid molecular or immunochromatography assays, thus highlighting their relevance in microbiology laboratories.

Characterization of carbapenemase-producing Serratia marcescens and whole-genome sequencing for plasmid typing in a hospital in Madrid, Spain (2016-18).

OBJECTIVES: Carbapenemase-producing Enterobacterales (CPE) are increasingly recognized in nosocomial infections, also affecting ICU patients. We aimed to characterize the carbapenemase-producing Serratia marcescens (CPSm) isolates recovered in our hospital in Madrid (Spain) between March 2016 and December 2018. METHODS: Overall, 50 isolates from clinical and epidemiological surveillance samples were recovered from 24 patients admitted to the medical ICU and 10 non-ICU-related patients based on their phenotypic resistance. Carbapenemase characterization, antibiotic susceptibility, PFGE clonal relatedness, plasmid characterization, WGS (Illumina-NovaSeq 6000) and phylogenetic analysis were performed. RESULTS: A single isolate was finally considered for each patient, except for Patient 8 that was colonized by two different isolates (n = 35). Isolates were characterized as VIM-1 (n = 29) or OXA-48 producers (n = 6). Up to seven genetic lineages were found by PFGE, with dominance of two clones. Plasmid characterization confirmed that almost all CPSm carried the same ∼60 kb IncL OXA-48- or VIM-1-encoding plasmid, which was related to the globally disseminated IncL-pOXA-48a. WGS allowed plasmid reconstruction with two variants: IncL-pVIM-1 (∼65 kb) and IncL-pOXA-48 (∼62 kb). blaOXA-48-Tn1999 (∼5 kb) was the unique antibiotic resistance gene in pOXA-48, whereas pVIM-1 plasmids (∼8 kb) harboured a class 1 integron containing 5'-blaVIM-1+aacA4+dfrB1+aadA1+catB2+qacEDelta1+sul1-3'. CONCLUSIONS: Our results confirm the dissemination of CPSm within our institution in both ICU and non-ICU environments, representing two prevalent CPSm clones, and the same IncL-pOXA-48 plasmid previously described in other Enterobacterales, but containing the blaVIM-1 gene. This also reinforces the relevance of species different from Klebsiella pneumoniae or Escherichia coli in the CPE landscape and circulating lineages and plasmids in local CPE epidemiology.

Evaluation of CHROMagar™-Serratia agar, a new chromogenic medium for the detection and isolation of Serratia marcescens.

A comparative analysis of the performance of the new selective chromogenic CHROMagar™-Serratia culture medium for detection and isolation of Serratia marcescens was undertaken. A total of 134 clinical isolates (95 S. marcescens with and without carbapenemase production and 39 non-S. marcescens isolates) and 96 epidemiological samples (46 rectal swabs and 50 from environmental surfaces) were studied. Diagnostic values when compared with CHROMagar™-Orientation medium were 96.8% sensitivity, 100% specificity, 100% positive predictive value and 88.5% negative predictive value. In conclusion, CHROMagar™-Serratia shows an excellent ability for differentiation of S. marcescens among clinical isolates and in environmental samples.

Nosocomial infections associated to COVID-19 in the intensive care unit: clinical characteristics and outcome.

Bacterial and fungal co-infection has been reported in patients with COVID-19, but there is limited experience on these infections in critically ill patients. The objective of this study was to assess the characteristics and ouctome of ICU-acquired infections in COVID-19 patients. We conducted a retrospective single-centre, case-control study including 140 patients with severe COVID-19 admitted to the ICU between March and May 2020. We evaluated the epidemiological, clinical, and microbiological features, and outcome of ICU-acquired infections. Fifty-seven patients (40.7%) developed a bacterial or fungal nosocomial infection during ICU stay. Infection occurred after a median of 9 days (IQR 5-11) of admission and was significantly associated with the APACHE II score (p = 0.02). There were 91 episodes of infection: primary (31%) and catheter-related (25%) bloodstream infections were the most frequent, followed by pneumonia (23%), tracheobronchitis (10%), and urinary tract infection (8%) that were produced by a wide spectrum of Gram-positive (55%) and Gram-negative bacteria (30%) as well as fungi (15%). In 60% of cases, infection was associated with septic shock and a significant increase in SOFA score. Overall ICU mortality was 36% (51/140). Infection was significantly associated with death (OR 2.7, 95% CI 1.2-5.9, p = 0.015) and a longer ICU stay (p < 0.001). Bacterial and fungal nosocomial infection is a common complication of ICU admission in patients with COVID-19. It usually presents as a severe form of infection, and it is associated with a high mortality and longer course of ICU stay.

Antimicrobial resistance in ICUs: an update in the light of the COVID-19 pandemic.

PURPOSE OF REVIEW: To describe current antimicrobial resistance in ESKAPE Gram-negative microorganisms and their situation in the ICUs, the implication of the so-called high-risk clones (HiRCs) involved in the spread of antimicrobial resistance as well as relevance of the COVID-19 pandemic in the potential increase of resistance. RECENT FINDINGS: Extended-spectrum and carbapenemase producing Enterobacterales and multidrug and extensive drug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii have increased worldwide. Sequence type (ST)131 Escherichia coli, ST258, ST11, ST10, ST147 and ST307 Klebsiella pneumoniae, ST111, ST175, ST235 and ST244 P. aeruginosa HiRCs are responsible for this increase in the ICUs, and some of them are implicated in the emergence of resistance mechanisms affecting new antimicrobials. A similar situation can be found with European clonal complex 1 and clonal complex 2 of A. baumannii. The high use of antimicrobials during the COVID-19 pandemic, particularly in ICUs, might have a negative influence in future trends of antimicrobial resistance. SUMMARY: The increase of antimicrobial resistance in ICUs is mainly due to the spread of HiRCs and is exemplified with the ESKAPE Gram-negative microorganisms. The COVID-19 pandemic might have a negative impact in the increase of antimicrobial resistance and should be monitored through specific surveillance studies in ICUs.

First Report of an OXA-48- and CTX-M-213-Producing Kluyvera Species Clone Recovered from Patients Admitted in a University Hospital in Madrid, Spain.

Enterobacterales species other than Klebsiella pneumoniae also contribute to OXA-48 carbapenemase endemicity. We studied the emergence of an OXA-48-producing Kluyvera species clone, which expresses the novel CTX-M-213 enzyme, colonizing patients in our hospital. Rectal swabs from patients admitted in four wards (March 2014 to March 2016; R-GNOSIS project) were seeded onto Chromo ID-ESBL) and Chrom-CARB/OXA-48 chromogenic agar plates. Carbapenemases and extended-spectrum ß-lactamases (ESBLs) were characterized (PCR, sequencing, cloning, and site-directed mutagenesis), and antibiotic susceptibility was determined. Clonal relatedness was established (XbaI pulsed-field gel electrophoresis [XbaI-PFGE]), and plasmid content was studied (transformation, S1 nuclease digestion-PFGE, SB-hybridization, restriction fragment length polymorphism [RFLP] analysis [DraI and HpaI], and PCR [incompatibility group and repA, traU, and parA genes]). Whole-genome sequencing (WGS) (Illumina HiSeq-2500) and further bioinformatics analysis of plasmids (PLACNET and plasmidSPAdes) were performed. Patients' charts were reviewed. Six unrelated patients (median age, 75 years [range, 59 to 81 years]; 4/6 male patients) colonized with OXA-48-producing Kluyvera species isolates (>95% similarity of the PFGE pattern) were identified. Nosocomial acquisition was demonstrated. In two patients, OXA-48-producing Kluyvera species isolates coexisted with OXA-48-producing Raoultella ornithinolytica, K. pneumoniae, and Escherichia coli The blaOXA-48 gene was located on an ∼60-kb IncL plasmid related to IncL/M-pOXA-48a and the novel blaCTX-M-213 gene in a conserved chromosomal region of Kluyvera species isolates. CTX-M-213, different from CTX-M-13 (K56E) but conferring a similar ß-lactam resistance profile, was identified. Genomic analysis also revealed a 177-kb IncF plasmid (class I integron harboring sul1 and aadA2) and an 8-kb IncQ plasmid (IS4-blaFOX-8). We describe the first blaOXA-48 plasmid in Kluyvera spp. and the novel chromosomal CTX-M-213 enzyme and highlight further nosocomial dissemination of blaOXA-48 through clonal lineages or plasmids related to IncL/M-pOXA-48a.

The what, when and how in performing and interpreting microbiological diagnostic tests in skin and soft tissue infections.

PURPOSE OF REVIEW: To summarize and classify the most recent and relevant microbiological studies for each type of skin and soft tissue infection (SSTI). RECENT FINDINGS: Following Infectious Diseases Society of America and Food and Drug Administration classifications of SSTIs, we differentiate between two large groups, the superficial or uncomplicated infections and the complicated infections with deep involvement. It is not usually necessary to obtain microbiological samples in uncomplicated infections, except in cases of recurrences or for epidemiological control purposes. In the case of complicated infections, the samples are of two different types: those obtained from the affected area (surgical samples, punctures of abscesses or swabs) and systemic samples (i.e. blood cultures). The clinical condition also determines the type of samples to be obtained. In cases of systemic involvement, blood cultures are mandatory. For immunocompromised patients, who may present atypical infections, detection of antigens, serologies or molecular biology techniques may be helpful. The rapid diagnosis is currently the goal to be pursued by implementing techniques such as matrix assisted laser desorption ionization-time of flight, commercial real-time PCR or the promising metagenomics. SUMMARY: Microbiological diagnosis is one of the cornerstones of the management of SSTIs. Prompt obtaining and processing of the necessary samples, depending on the clinical situation of the patient, is of relevance in the decision-making process. Rapid and fluid reporting of the results (identification, mechanisms of resistance and antibiogram) will improve the management of these patients.

Characterization of carbapenemase-producing Enterobacteriaceae from colonized patients in a university hospital in Madrid, Spain, during the R-GNOSIS project depicts increased clonal diversity over time with maintenance of high-risk clones.

Objectives: To describe the incidence and microbiological features of carbapenemase-producing Enterobacteriaceae (CPE) from colonized patients in a Spanish university hospital during a cluster-randomized study [the Resistance of Gram-Negative Organisms: Studying Intervention Strategies (R-GNOSIS) project] on isolation strategies for faecal ESBL carriers. Methods: From March 2014 to March 2016, 15 556 rectal swabs from 8209 patients admitted in two surgical wards and two medical wards were collected and seeded on ESBL and CPE chromogenic agars. Carbapenemase characterization (PCR and sequencing) was performed, and antibiotic susceptibility (MIC), clonality (PFGE and MLST) and diversity (Simpson diversity index estimation) were determined. Results: One hundred and ninety-eight CPE isolates, mainly Klebsiella pneumoniae (53.5%) and Escherichia coli (19.2%), were identified in 162 patients (2%). Prevalence of CPE carriage remained unchanged over time. Overall, amikacin (9.6%), tigecycline (9.6%) and colistin (0.5%) showed low non-susceptibility. The most frequent carbapenemase was OXA-48 (64.1%), followed by VIM-1 (26.8%), NDM-1 (5.3%) and KPC-3 (3.5%), and these were co-produced with ESBLs in 43.9%. OXA-48 plus CTX-M-15 was the most frequent association. Two major K. pneumoniae clones were identified (OXA-48-CTX-M-15-ST11 and VIM-1-SHV-12-ST54) with considerable genetic diversity among the remaining isolates, including OXA-48-E. coli. Species diversity tended to decrease from 0.75 in the first 6 months of the study to 0.43 in the final months. The emergence of new clones (i.e. OXA-48-Kluyvera spp. and NDM-1-K. pneumoniae ST437 and ST101) and displacement of other particular clones were also demonstrated. Conclusions: We describe a polyclonal and changeable CPE population over time. Coexistence of worldwide disseminated clones, such as ST11-OXA-48- K. pneumoniae, with unrelated and emerging OXA-48-E. coli clones, depicts a disturbing CPE epidemiology in our institution.

Long-term clonal dynamics of Enterococcus faecium strains causing bloodstream infections (1995-2015) in Spain.

OBJECTIVES: To investigate the population structure of Enterococcus faecium causing bloodstream infections (BSIs) in a tertiary Spanish hospital with low glycopeptide resistance, and to enhance our knowledge of the dynamics of emergence and spread of high-risk clonal complexes. METHODS: All available E. faecium causing BSIs (n = 413) in our hospital (January 1995-May 2015) were analysed for antibiotic susceptibility (CLSI), putative virulence traits (PCR, esp, hylEfm) and clonal relationship (SmaI-PFGE, MLST evaluated by goeBURST and BAPS). RESULTS: The increased incidence of BSIs caused by enterococci [2.3‰ of attended patients (inpatients and outpatients) in 1996 to 3.0‰ in 2014] significantly correlated with the increase in BSIs caused by E. faecium (0.33‰ of attended patients in 1996 to 1.3‰ in 2014). The BSIs Enterococcus faecalis:E. faecium ratio changed from 5:1 in 1996 to 1:1 in 2014. During the last decade an increase in E. faecium BSIs episodes in cancer patients (10.9% in 1995-2005 and 37.1% in 2006-15) was detected. Ampicillin-susceptible E. faecium (ASEfm; different STs/BAPS) and ampicillin-resistant E. faecium (AREfm; ST18/ST17-BAPS 3.3a) isolates were recovered throughout the study. Successive waves of BAPS 2.1a-AREfm (ST117, ST203 and ST80) partially replaced ASEfm and ST18-AREfm since 2006. CONCLUSIONS: Different AREfm clones (belonging to BAPS 2.1a and BAPS 3.3a) consistently isolated during the last decade from BSIs might be explained by a continuous and dense colonization (favouring both invasion and cross-transmission) of hospitalized patients. High-density colonization by these clones is probably enhanced in elderly patients by heavy and prolonged antibiotic exposure, particularly in oncological patients.

Characterization and rapid control of a vancomycin-resistant Enterococcus faecium (VREF) outbreak in a renal transplant unit in Spain: The environment matters.

OBJECTIVE: To describe a clonal outbreak due to vancomycin-resistant Enterococcus faecium (VREF) in the nephrology and renal transplant unit of a tertiary teaching hospital in Barcelona, Spain, and to highlight how active patient and environment surveillance cultures, as well as prompt and directed intervention strategies, mainly environmental, helped to successfully bring it under control. PATIENTS AND METHODS: A study was conducted on patients admitted to the nephrology ward with any culture positive for VREF over a 6-month period (August 2012-January 2013). Based on the identification of a clonal link between the isolates, weekly rectal screening using swabs was implemented for all patients, as well as environmental cultures and cleaning of medical equipment and the ward. VREF isolates were identified by MicroScan and confirmed by Etest. Bacterial identification was confirmed by MALDI-TOF MS. The presence of van genes, and esp and hyl virulence genes was determined using PCR. The clonal relationship between the isolates was studied first with DiversiLab (bioMérieux), and then by PFGE-Smal and MLST. A two-tier sequence of infection control measures was implemented. RESULTS: During the study period, VREF was isolated from 13 patients. All cases were colonized with no criteria for infection. VREF isolates were also extensively recovered from the environment and medical equipment. Isolates carried the vanA gene, and were multidrug-resistant, including high-level resistance (MIC >16mg/L) to vancomycin and teicoplanin. Molecular analysis showed that all VREF isolates belonged to sequence type 17 (ST17) carrying hyl virulence genes. After implementing infection control measures in a two-tier sequence, and reinforcing particularly environmental and medical equipment cleaning, no further cases were detected in the follow-up year. CONCLUSION: A clonal outbreak of VREF-ST17 involving only colonization is reported. The prompt implementation of aggressive infection control measures in patients and the environment was effective in controlling the outbreak and avoided the potential emergence of infection among patients.

Detection of Carbapenemase Production in a Collection of Enterobacteriaceae with Characterized Resistance Mechanisms from Clinical and Environmental Origins by Use of Both Carba NP and Blue-Carba Tests.

Rapid-screening methods to confirm the presence of resistance mechanisms in multidrug-resistant bacteria are currently recommended. Carba NP and Blue-Carba tests were evaluated in carbapenemase-producing Enterobacteriaceae from hospital (n = 102) and environmental (n = 57) origins for detecting the different molecular classes among them. Both methods showed to be fast and cost-effective, with high sensitivity (98% to 100%) and specificity (100%), and may be easily introduced in the routine laboratory.

A single-day point-prevalence study of faecal carriers in long-term care hospitals in Madrid (Spain) depicts a complex clonal and polyclonal dissemination of carbapenemase-producing Enterobacteriaceae.

OBJECTIVES: The objective of this study was to describe the prevalence and microbiological characteristics of carbapenemase-producing Enterobacteriaceae (CPE) colonizing patients in long-term care hospitals (LTCHs) in Madrid, Spain. METHODS: Three LTCHs were included in a single-day point-prevalence survey (September 2013). Rectal swabs, collected from all hospitalized patients (137 in LTCH-A, 121 in LTCH-B and 83 in LTCH-C), were plated onto chromogenic media. Population structure (PFGE and MLST), genes encoding carbapenemases and ESBLs and plasmids carrying carbapenemase genes were characterized. RESULTS: The prevalence of CPE carriers was 4.1% (14/341) [2.9% (4/137), LTCH-A; 4.1% (5/121), LTCH-B; and 6.0% (5/83), LTCH-C]. OXA-48 was the most prevalent carbapenemase (nine Klebsiella pneumoniae, two Escherichia coli, one Enterobacter cloacae and one Citrobacter braakii) followed by VIM-1 (one K. pneumoniae and one Raoultella ornithinolytica). One patient (LTCH-C) was co-colonized with OXA-48-producing K. pneumoniae and E. coli. K. pneumoniae and E. coli isolates also coproduced CTX-M-15 (n = 11) or CTX-M-9 (n = 1) enzymes. K. pneumoniae clustered into six PFGE types corresponding to ST11 (n = 1), ST15 (n = 6), ST307 (n = 1) and ST405 (n = 2). E. coli from LTCH-A and LTCH-C exhibited two different PFGE types associated with ST68. OXA-48 and VIM-1 enzymes were found in different clones in LTCH-A and LTCH-C. However, OXA-48 was the only carbapenemase detected in LTCH-B, mainly associated with K. pneumoniae ST15. KPC, IMP and NDM enzymes were not detected. blaOXA-48 was located on an ∼ 60 kb plasmid with a pOXA-48a-IncL/M backbone. CONCLUSIONS: We describe the first point-prevalence study of CPE faecal carriers in LTCHs in Spain. OXA-48, the most prevalent carbapenemase, showed a complex dissemination pattern with clonal and polyclonal bacterial populations.

CTX-M-15-H30Rx-ST131 subclone is one of the main causes of healthcare-associated ESBL-producing Escherichia coli bacteraemia of urinary origin in Spain.

OBJECTIVES: The objective of this study was to assess the prevalence and molecular epidemiology of ESBL-producing Escherichia coli causing healthcare-associated (HCA) and community-associated (CA) bacteraemia of urinary origin (BUO) in Spain. METHODS: An observational cohort study was conducted at eight hospitals from different Spanish geographical areas (2010-11). BUO episodes (n = 425) were classified as HCA (n = 215) and CA (n = 210), and one blood isolate per episode was collected. Susceptibility testing was performed, ESBLs were screened by double-disc diffusion test and ESBL and OXA-1 genes were characterized (PCR and sequencing). Population structure (phylogenetic groups, XbaI-PFGE and MLST) and ST131 subtyping (PCR) were determined. Virulence genes were detected by PCR and virulence score, profiles and extraintestinal pathogenic E. coli (ExPEC) status calculated. RESULTS: ESBL-producing E. coli prevalence was 9.2% (39/425). ESBL-producing E. coli episodes were significantly associated with HCA-BUO episodes [14% (30/215) versus 4.3% (9/210); P = 0.001]. The highest non-susceptibility proportions corresponded to ciprofloxacin (97.4%), amoxicillin/clavulanate (74.4%), co-trimoxazole (69.2%) and tobramycin (61.5%). Of the 39 ESBL-producing E. coli isolates, 34 produced CTX-M enzymes (21 CTX-M-15, 11 CTX-M-14 and 2 CTX-M-1). Fifteen STs were identified, the B2-ST131 clone being the most prevalent (54%; 21/39). All ST131 isolates were ExPEC and had the highest virulence scores, but they showed less diversity in virulence profiles than other STs. The H30Rx subclone accounted for most ST131 isolates (20/21), co-produced CTX-M-15 (20/20) and OXA-1 (19/20) enzymes and was associated with HCA episodes (16/20). CONCLUSIONS: The CTX-M-15-ST131-H30Rx subclone is a relevant MDR pathogen causing BUO, mainly HCA episodes. The dominance of this subclone with comparatively less diversity of virulence profiles reflects the spread of a successful and MDR ESBL ST131 lineage in Spain.