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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. β-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.

Treatment of infections caused by OXA-48 carbapenemase producing multidrug-resistant isolates often necessitates combination therapy. In vitro effect of different antibiotic combinations against multidrug-resistant (MDR) Klebsiella pneumoniae isolates were evaluated in this study. Meropenem-tobramycin (MER+TOB), meropenem-ciprofloxacin (MER+CIP), colistin-meropenem (COL+MER), colistin-ciprofloxacin (COL+CIP) and colistin-tobramycin (COL+TOB) combinations were tested by time kill-assays. Each antibiotic alone and in combination at their Cmax values were tested against 4 clinical K. pneumoniae isolates at 1, 2, 4, 6, 8, 12 and 24 h. Effect of colistin and its associations were also assessed at 30 min. Bactericidal activity was defined as ≥3log10 CFU mL−1 decrease compared with initial inoculum. Synergy was defined as ≥2log10CFU mL−1 decrease by the combination compared with the most active single agent. Presence of bla OXA-48, bla NDM, bla VIM, bla IMP, bla KPC and bla CTX-M-1 genes was screened by PCR using specific primers. The bla OXA-48 gene was identified together with bla CTXM-1 group gene in all isolates. COL+MER demonstrated to be synergistic and bactericidal. MER+TOB showed synergistic and bactericidal effect on two strains although, regrowth was seen on other two strains at 24 h. MER+CIP exhibited indifferent effect on the strains. Combination therapy could be a potential alternative to treat MDR K. pneumoniae infections. This combination might prevent resistance development and secondary effects of colistin monotherapy. MER+TOB and MER+CIP might have an isolate-dependent effect, that may not always result in synergism.

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.

Colistin resistance is caused by different lipopolysaccharide (LPS) modifications, and we propose to evaluate the effect on the innate immune response ofin vivoandin vitrocolistin resistance acquisition. We used 2 pairs of isogenic strains: (1)Escherichia coliATCC25922, susceptible to colistin and its isogenic transconjugant-carryingmcr-1 gene; and (2) OXA-48, CTX-M-15K. pneumoniaesusceptible to colistin (CS-Kp) isolated from a urinary infection and its colistin-resistant variant (CR-Kp) from the same patient after prolonged treatment with colistin. No mutation of described genes for colistin resistance (pmrA, pmrB, mgrB. phoP/QandcrrAB) were found in the CR-Kp genome; however, LPS modifications were characterized by negative-ion MALDI-TOF. The strains were co-cultured with human monocytes to determine their survival after phagocytosis and induction to apoptosis. Also, monocytes were stimulated with bacterial LPS to study cytokine and immunecheckpoint production. The addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A of CR-Kp accounted for the colistin resistance. CR-Kp survived significantly longer inside human monocytes after being phagocytosed compared with the CS-Kp strain, whereas no significant differences were observed for theE. coliisogenic strains. In addition, LPS from CR-Kp induced both higher apoptosis in monocytes and higher levels of cytokine and immune checkpoint production than LPS from CS-Kp. This effect was strictly the opposite forE. coli. Our data reveal a variable impact of colistin resistance on the innate immune system, depending on the responsible mechanism. Adding Ara4N to LPS increases bacterial survival after phagocytosis and elicits a higher inflammatory response than its colistin-susceptible counterpart.

Carbapenemase production is constantly increasing among different Enterobacterales species. We analyzed the microbiological characteristics and population structure of all carbapenemase-producing E...

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.

TheEnterobacter cloacaecomplex (ECC) encompasses heterogeneous clusters of species that have been associated with nosocomial outbreaks. These species may host different acquired antimicrobial resistance and virulence mechanisms and their identification are challenging. This study aims to develop predictive models based on MALDI-TOF MS spectral profiles and machine learning for species-level identification.A total of 198 ECC and 116K. aerogenesclinical isolates from the University Hospital Ramón y Cajal (Spain) and the University Hospital Basel (Switzerland) were included. The capability of the proposed method to differentiate the most common ECC species (E. asburiae, E. kobei, E. hormaechei, E. roggenkampii, E. ludwigii, E. bugandensis) andK. aerogeneswas demonstrated by applying unsupervised hierarchical clustering with PCA pre-processing. We observed a distinctive clustering ofE. hormaecheiandK. aerogenesand a clear trend for the rest of the ECC species to be differentiated over the development dataset. Thus, we developed supervised, non-linear predictive models (Support Vector Machine with Radial Basis Function and Random Forest). The external validation of these models with protein spectra from the two participating hospitals yielded 100% correct species-level assignment forE. asburiae, E. kobei, andE. roggenkampiiand between 91.2% and 98.0% for the remaining ECC species. Similar results were obtained with the MSI database developed recently (https://msi.happy-dev.fr/) except in the case ofE. hormaechei, which was more accurately identified by Random Forest.In short, MALDI-TOF MS combined with machine learning demonstrated to be a rapid and accurate method for the differentiation of ECC species.

Background Carbapenemase-producing Enterobacterales (CPE) infections have been occasionally described in patients with coronavirus disease-19 (COVID-19). We assess the clinical features and outcome of these infections. Methods In this retrospective single-centre, case-control study, we included 54 patients with CPE infection: 30 case-patients (COVID-19) and 24 controls (non-COVID-19), collected between March and May 2020. We compared the epidemiological, clinical features, and outcome between cases and controls. Results CPE infection was more frequent in COVID-19 patients than in controls (1.1 vs. 0.5%, p = .005). COVID-19 patients were younger, had a lower frequency of underlying diseases (p = .01), and a lower median Charlson score (p = .002). Predisposing factors such as antimicrobial use, mechanical ventilation, or ICU admission, were more frequent in COVID-19 patients (p

Background: The current spread of carbapenemase-producing Enterobacterales (CPE) is a great concern. Methods: We recovered 198 CPE from 162 patients admitted in our Hospital (March 2014-March 2016) during the R-GNOSIS European Project. Microbiological features and plasmid characteristics of CPE recovered from patients co-colonized with multiple CPE were studied. Findings: Thirty patients (18.5%; CI 95%= 12.5%–24.5%) presented co-colonization with multiple CPE producing the same (CPE-SC) (15.4%) or a different carbapenemase (CPE-DC) (4.3%). OXA-48 (83.3%) was the most frequent carbapenemase, followed by VIM-1 (26.7%), NDM-1 (10%) and KPC-3 (3.3%). CPE-DC-patients had longer admissions [63 days (20–107)] than the other patients. Moreover, hospital stay until CPE detection was lower [9 days (5–14)] (p = 0.0052) in CPE-SC-patients than in those with a single colonization; 56% showed co-colonization in the first positive sample, although most of them had previous admissions and had received multiple antibiotic treatments. CPE were more frequently recovered in clinical samples from co-colonized [CPE-DC (28.6%), CPE-SC (24%)] patients than from patients with a single CPE (15.2%). Among CPE-SCOXA-48 [80% (p = 0.11)], K. pneumoniae [88% (p = 0.006)] and E. coli [84% (p

Here we describe an outbreak due to NDM-1+CTX-M-15+DHA-1-producing Klebsiella pneumoniae (NDM-1-Kp) in Spain related to a patient previously admitted to a healthcare centre in an endemic area (Pakistan). Nine colonised patients were detected in the Neurosurgery ward between September 2015 and February 2016 during the R-GNOSIS European Project. NDM-1-Kp isolates from clinical samples were also recovered in three of these patients. Surveillance culture at admission was negative in the index case, but NDM-1-Kp colonisation was detected 27 days later after receiving antibiotic treatment. Co-colonisation with a second NDM-1-Kp isolate was identified in this patient 61 days post-admission. Overall length of stay (LOS = 75 days) (P0.01) and LOS until carbapenemase detection (LOS-1 = 36 days) was longer in NDM-1-Kp carriers than in patients with other carbapenemase-producing Enterobacterales. Intervention strategies were implemented after the outbreak declaration and NDM-1-Kp transmission was contained. Among the NDM-1-Kp isolates, two clones [ST437 (index case and Patient 2) and ST101 (index case and Patients 3-9)] with different IncFIB NDM-1-containing plasmids were identified. Whole-genome sequencing revealed a high content of antimicrobial resistance genes in both isolates in addition to a large number of virulence factors. Colonisation with other epidemic (OXA-48-ST11-K. pneumoniae and VIM-1-ST54-K. pneumoniae) and non-epidemic (VIM-1-ST908-K. pneumoniae and VIM-ST431-Escherichia coli) clones was also detected in two NDM-1 carriers. Implementation of adequate infection control measures and uninterrupted active surveillance programmes for detecting patients with a low colonisation status are crucial to prevent the introduction and dissemination of NDM-type enzymes in our region.

Dissemination of multidrug-resistant Klebsiella pneumoniae in the hospital environment represents a primary target of resistance containment and stewardship programs. At present, polymyxins, mostly in combination, exemplify a last-resort alternative. Colistin-resistant K. pneumoniae isolates harboring OXA-48 plus CTX-M-15 (n = 21) with the simultaneous colistin-susceptible counterparts (n = 9) were recovered from 14 hospitalized patients (January 2014–January 2015) admitted in different wards. In most cases, patients had not previously received colistin. Genetic relatedness experiments demonstrated that 93% (28/30) of isolates belonged to the ST11 high-risk clone. Heteroresistance and the fitness cost of colistin resistance were addressed in susceptible and resistant isolates as well as in in vitro–obtained stable mutants, and results appeared to be strain dependent. Whole genome sequencing demonstrated molecular changes in pmrA, pmrB, and mgrB genes. Plasmid-mediated colistin resistance genes were not found. Colistin resistance in multidrug-resistant K. pneumoniae isolates should be continuously monitored to detect its potential emergence, even in patients not previously exposed to colistin.

Objectives To determine the activity of murepavadin in comparison with tobramycin, colistin and aztreonam, against cystic fibrosis (CF) Pseudomonas aeruginosa isolates growing in biofilms. The biofilm-epidemiological cut-off (ECOFF) values that include intrinsic resistance mechanisms present in biofilms were estimated. Methods Fifty-three CF P. aeruginosa isolates from respiratory samples were tested using the Calgary (closed system) device, while 4 [2 clinical (one smooth, one mucoid) and 2 reference strains] were tested using the BioFlux, a microfluidic open model of biofilm testing. Biofilm was stained with SYTO9® and propidium iodide. The minimal biofilm inhibitory concentration (MBIC) and the minimal biofilm eradication concentration (MBEC) were determined. The MBIC-ECOFF and the MBEC-ECOFF were calculated. Results Colistin, tobramycin and murepavadin presented similar MBIC50/MBIC90 values (4/32, 8/64 and 2/32, respectively). Murepavadin exhibited the lowest MBEC90 (64 mg/L). Aztreonam MBIC and MBEC values were higher than those of the other antibiotics tested. Tobramycin and murepavadin had the lowest MBEC-ECOFF (64 and 128 mg/L, respectively), while those of aztreonam and colistin exceeded 512 mg/L. Using the BioFlux, for the PAO1, PAO mutS and the smooth clinical strain, a significant difference (P Conclusions Murepavadin demonstrated good activity against P. aeruginosa biofilms both in open and closed systems. The MBIC-ECOFF and the MBEC-ECOFF are proposed as new parameters to estimate the activity of antibiotics on biofilms.

Background Pseudomonas aeruginosa is of great concern among MDR bacteria and rapid and reliable in vitro antibiotic susceptibility testing methods are extremely necessary. Colistin is, in many cases, among the limited useful alternatives for these isolates. Unfortunately, only a few reliable in vitro methods are validated for testing susceptibility to colistin. Although EUCAST and CLSI recommend broth microdilution (BMD) as the standard method for antibiotic susceptibility testing, this method is not routinely performed in microbiology laboratories. However, some commercial products based upon BMD have tested well and offer consistent results. Objectives To evaluate the performance of the colorimetric Rapid Polymyxin Pseudomonas Test (RPPT) (ELITech Microbiology, France). Methods Eighty-seven clinical P. aeruginosa strains, prospectively collected in two microbiology laboratories exhibiting either susceptibility or various degrees of multidrug resistance, including to colistin, were used. Different susceptibility testing methods were simultaneously performed and compared with reference BMD and interpreted using 2020 EUCAST criteria. Results Results indicate an essential agreement (EA) of 97.7% for RPPT while the other tests did not reach 90% of EA [66.7% MicroScan, 63.2% Etest (bioMérieux, France) and 60.9% other MIC Test Strips (MTS, Liofilchem, Italy)]. The categorical agreement was 98.9% for RPPT, 87.4% for MTS, 85.1% for Etest and 64.4% for MicroScan. Conclusions The RPPT was able to accurately detect both colistin-susceptible and -resistant isolates within 4 h, offering a rapid alternative for a prompt decision about the inclusion of this antibiotic in a patient’s treatment.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) infections have been occasionally described in patients with coronavirus disease-19 (COVID-19). We assess the clinical features and outcome of these infections. METHODS: In this retrospective single-centre, case-control study, we included 54 patients with CPE infection: 30 case-patients (COVID-19) and 24 controls (non-COVID-19), collected between March and May 2020. We compared the epidemiological, clinical features, and outcome between cases and controls. RESULTS: CPE infection was more frequent in COVID-19 patients than in controls (1.1 vs. 0.5%, p = .005). COVID-19 patients were younger, had a lower frequency of underlying diseases (p = .01), and a lower median Charlson score (p = .002). Predisposing factors such as antimicrobial use, mechanical ventilation, or ICU admission, were more frequent in COVID-19 patients (p < .05). There were 73 episodes of infection (42 cases and 31 controls) that were more frequently hospital-acquired and diagnosed at the ICU in COVID-19 patients (p < .001). Urinary tract was the most common source of infection (47.9%), followed by pneumonia (23.3%). The frequency of severe sepsis or shock (p = .01) as well as the median SOFA score (p = .04) was higher in cases than in controls. Klebsiella pneumoniae (80.8%), Serratia marcescens (11%) and Enterobacter cloacae (4.1%) were the most common bacteria in both groups (KPC 56.2%, OXA-48 26% and VIM 17.8%). Overall 30-d mortality rate of COVID-19 patients and controls was 30 and 16.7%, respectively (p = .25). CONCLUSIONS: COVID-19 patients have an increased risk of CPE infections, which usually present as severe, nosocomial infections, appearing in critically-ill patients and associated with a high mortality.

The aim was to develop and validate a Pseudomonas aeruginosa genotypic resistance score, based on analysis of the whole genome sequence resistome, to predict antimicrobial susceptibility phenotypes.A scoring system based on the analysis of mutation-driven resistance in 40 chromosomal genes and horizontally acquired resistance (Resfinder) was developed for ceftazidime, ceftolozane/tazobactam, meropenem, ciprofloxacin and tobramycin. Resistance genes/mutations were scored from 0 (no effect) to 1 (EUCAST clinical resistance). One hundred wild-type strains obtained from 51 different hospitals during a 2017 multicentre study were fully sequenced and analysed in order to define a catalogue of natural polymorphisms in the 40 chromosomal resistance genes. The capacity of genotypic score to predict the susceptibility phenotype was tested in 204 isolates randomly selected from the 51 hospitals (four from each hospital).The analysis of the 100 wild-type isolates yielded a catalogue of 455 natural polymorphisms in the 40 genes involved in mutational resistance. However, resistance mutations and high-risk clones (such as ST235) were also documented among a few wild-type isolates. Overall, the capacity of the genotypic score (0.5) for predicting phenotypic susceptibility (S + I in the case of meropenem) was very high (95-100%). In contrast, the capacity of the genotypic score to predict resistance (≥1) was far more variable depending on the agent. Prediction of meropenem clinical resistance was particularly low (18/39, 46.1%), whereas it classified clinical ceftolozane/tazobactam resistance in 100% (7/7) of cases.Although a margin for improvement was evidenced in this proof of concept study, an overall good correlation between the genotypic resistance score and the susceptibility profile was documented. Further refining of the scoring system, automatization and testing of large international cohorts should follow.

Carbapenemase production is constantly increasing among different

Methicillin-resistant Staphylococcus aureus (MRSA) detection in cystic fibrosis (CF) is challenging. We compared different phenotypic methods among 157 S. aureus from 136 CF-patients: cefoxitin (FOX) and oxacillin (OXA) broth-microdilution; MicroScan-WalkAway®; FOX and OXA disk-diffusion (DD), and PBP2a-latex agglutination. PCR detection of mecA/mecC was the gold standard. Growth on ChromIDTM-MRSA agar was evaluated and compared with that of 157 blood culture (BC) isolates. ChromIDTM-MRSA was also tested on sputa from 111 CF-patients. 32 isolates (20%) were mecA-positive. Both FOX DD and MicroScan-WalkAway® (FOX/OXA) showed the highest sensitivity and specificity (100% and 100%, 96.9% and 99.2%, 96.9% and 100%). ChromIDTM-MRSA showed an excellent sensitivity for BC and CF-isolates (100% and 96.9%) but a poorer specificity for CF ones (95.5% vs. 73.7%), which was also observed when samples were seeded on this medium. FOX DD and MicroScan-WalkAway® are suitable for MRSA detection among CF-isolates and should be used to confirm ChromIDTM-MRSA positive CF-cultures.

Carbapenemase-producing Enterobacterales (CPE) have become an important public health concern. In our hospital, VIM enzymes were first detected in 2005, Klebsiella pneumoniae carbapenemase (KPC) enzymes in 2009, and OXA-48 enzymes in 2012. We assess the population biology of the first OXA-48-producing Enterobacterales isolates recovered in our hospital (2012 to 2013) where infections by other carbapenemases had been endemic for several years. Over a 21-month period, 71 isolates (61 Klebsiella pneumoniae, 5 Escherichia coli, 2 Klebsiella aerogenes, and 1 each of Enterobacter cloacae, Klebsiella oxytoca, and Citrobacter amalonaticus) recovered from clinical and surveillance specimens from 57 patients (22.8% nonhospitalized) were investigated for OXA-48-like-producing enzymes. Analyses for characterization and determination of the location of the blaOXA-48 gene, plasmid transferability, sequence, and clonal relatedness were performed. Most of the isolates also coproduced CTX-M-15 (57/71, 80.3%) and/or VIM-1 (7/71, 9.8%). K. pneumoniae was predominantly identified as sequence type 11 (ST11) (63.4%) and ST405 (9.8%) and E. coli as ST540, ST1406, ST3163, and ST4301. The blaOXA-48 gene was part of Tn1999.2 located at the tir gene of plasmids (ca. ≥50 kb) of the IncL/M group, also carrying blaVIM-1 and blaCTX-M-15 genes. We selected one ST11 K. pneumoniae isolate for whole-genome sequencing in which we studied the plasmid containing the blaOXA-48 gene. This plasmid was compared with indexed plasmids existing in NCBI database by the use of BRIG and MAUVE. Our data suggest a rapid spread of blaOXA-48 genes between commonly isolated high-risk clones of Enterobacterales species, frequently associated with antibiotic resistance. Moreover, the emergence of the multiresistant ST11 K. pneumoniae clone among nonhospitalized patients emphasizes the difficulty of preventing its dissemination into the community. IMPORTANCE We present results of microbiological analysis of the first Enterobacterales isolates that were isolated in 2012 in our institution expressing OXA-48 carbapenemase. This enzyme confers resistance to carbapenems, an important group of antibiotics widely used in the hospitals. OXA-48 carbapenemase is currently present in many parts of the world, but it is found particularly frequently in the Mediterranean area. It was disseminated at the Ramon y Cajal Hospital and found to be associated with a particular Klebsiella pneumoniae strain, so-called high-risk clone ST11, which was previously found in our institution in association with other enzymes such as CTX-M-15, VIM-1, and KPC-3. This clone might have acquired a plasmid harboring the blaOXA-48 gene. Our results point out the importance of local epidemiology in the dissemination and maintenance of multidrug-resistant bacteria.

We present results of microbiological analysis of the first Enterobacterales isolates that were isolated in 2012 in our institution expressing OXA-48 carbapenemase. This enzyme confers resistance to carbapenems, an important group of antibiotics widely used in the hospitals. OXA-48 carbapenemase is currently present in many parts of the world, but it is found particularly frequently in the Mediterranean area. It was disseminated at the Ramón y Cajal Hospital and found to be associated with a particular Klebsiella pneumoniae strain, so-called high-risk clone ST11, which was previously found in our institution in association with other enzymes such as CTX-M-15, VIM-1, and KPC-3. This clone might have acquired a plasmid harboring the blaOXA-48 gene. Our results point out the importance of local epidemiology in the dissemination and maintenance of multidrug-resistant bacteria., Carbapenemase-producing Enterobacterales (CPE) have become an important public health concern. In our hospital, VIM enzymes were first detected in 2005, Klebsiella pneumoniae carbapenemase (KPC) enzymes in 2009, and OXA-48 enzymes in 2012. We assess the population biology of the first OXA-48-producing Enterobacterales isolates recovered in our hospital (2012 to 2013) where infections by other carbapenemases had been endemic for several years. Over a 21-month period, 71 isolates (61 Klebsiella pneumoniae, 5 Escherichia coli, 2 Klebsiella aerogenes, and 1 each of Enterobacter cloacae, Klebsiella oxytoca, and Citrobacter amalonaticus) recovered from clinical and surveillance specimens from 57 patients (22.8% nonhospitalized) were investigated for OXA-48-like-producing enzymes. Analyses for characterization and determination of the location of the blaOXA-48 gene, plasmid transferability, sequence, and clonal relatedness were performed. Most of the isolates also coproduced CTX-M-15 (57/71, 80.3%) and/or VIM-1 (7/71, 9.8%). K. pneumoniae was predominantly identified as sequence type 11 (ST11) (63.4%) and ST405 (9.8%) and E. coli as ST540, ST1406, ST3163, and ST4301. The blaOXA-48 gene was part of Tn1999.2 located at the tir gene of plasmids (ca. ≥50 kb) of the IncL/M group, also carrying blaVIM-1 and blaCTX-M-15 genes. We selected one ST11 K. pneumoniae isolate for whole-genome sequencing in which we studied the plasmid containing the blaOXA-48 gene. This plasmid was compared with indexed plasmids existing in NCBI database by the use of BRIG and MAUVE. Our data suggest a rapid spread of blaOXA-48 genes between commonly isolated high-risk clones of Enterobacterales species, frequently associated with antibiotic resistance. Moreover, the emergence of the multiresistant ST11 K. pneumoniae clone among nonhospitalized patients emphasizes the difficulty of preventing its dissemination into the community. IMPORTANCE We present results of microbiological analysis of the first Enterobacterales isolates that were isolated in 2012 in our institution expressing OXA-48 carbapenemase. This enzyme confers resistance to carbapenems, an important group of antibiotics widely used in the hospitals. OXA-48 carbapenemase is currently present in many parts of the world, but it is found particularly frequently in the Mediterranean area. It was disseminated at the Ramón y Cajal Hospital and found to be associated with a particular Klebsiella pneumoniae strain, so-called high-risk clone ST11, which was previously found in our institution in association with other enzymes such as CTX-M-15, VIM-1, and KPC-3. This clone might have acquired a plasmid harboring the blaOXA-48 gene. Our results point out the importance of local epidemiology in the dissemination and maintenance of multidrug-resistant bacteria.

Introduction The lack of consensus of control measures to prevent extended-spectrum β-lactamase producing Enterobacterales (ESBL-E) transmission in the hospital setting is of great concern. We describe the prevalence and species distribution of ESBL-E and carbapenemase producing Enterobacterales (CPE) in patients admitted in a tertiary Hospital during an active surveillance screening program for detecting ESBL-E carriers and reducing the ESBL-E transmission (R-GNOSIS Project). Methods From March-2014 to March-2016, 15,556 rectal swabs were collected from 8209 patients admitted in two medical (Gastroenterology, Pneumology) and two surgical (Neurosurgery, Urology) wards. Swabs were seeded onto ChromoID-ESBL and -CARB/OXA-48 agar plates. Growing colonies were identified by MALDI-TOF MS. ESBL and carbapenemases were phenotypically detected. Changes in species diversity (SDI) and distribution over time were analyzed. Results ESBL-E incidence (8.4%) tended to decrease over time (p = 0.003) and CPE carrier prevalence remained unchanged during the study (2%). The contact isolation strategy targeted to reduce ESBL-E transmission was ineffective in reducing ESBL-E carriers but significant differences were observed with CPE (p = 0.017). SDI did not change among ESBL-E and E. coli was predominant (78.5%) during the study. K. pneumoniae (54%) was the most frequent CPE species, followed by E. coli (19%). SDI decreased among the CPE population over time mainly due to K. pneumoniae dominance and increased E. coli prevalence in the last part of the study. Conclusions During the R-GNOSIS project, contact precautions were not effective in reducing the ESBL-E transmission but may have had a positive collateral effect on the CPE containment.

Background The anaerobic infection management is usually based on empirical treatment because anaerobic culture techniques take a long time due to their fastidious nature. The aim of this study was to analyze the etiological profile of severe anaerobic infections and AST data from clinical anaerobic bacteria isolated in a tertiary hospital in Madrid (Spain). Material and methods A consecutive study was carried out over 19 months in Ramon y Cajal Universitary Hospital, Madrid. Clinical samples were processed in appropriate anaerobic media and incubated using Anoxomat system. Identification was performed by MALDI-TOF. AST were determined with gradient diffusion method using EUCAST (penicillin, co-amoxiclav, imipenem, clindamycine and metronidazole) or CLSI (cefoxitin) breakpoints. Results During the period of study, 503 anaerobic microorganisms isolated from 424 clinical samples were included. Twenty-six percent of the cultures were monomicrobial, while 70.0% also contained aerobic bacteria. The most common source of infection was abscesses (26%), while blood infections represented the 11%. Anaerobic gram-negative bacilli were predominant (41%), being Bacteroides fragilis (13%) the most prevalent overall; anaerobic gram-positive bacilli represented 35%, anaerobic gram-positive cocci 19% and anaerobic gram-negative cocci 5%. Metronidazole and imipenem were the most effective agents tested against anaerobic bacteria, while clindamycin presented higher resistance rates. Conclusion Antimicrobial susceptibility surveillance of anaerobic bacteria should be performed to monitor changes in resistance patterns and to be able to optimize empiric antimicrobial treatment. Reliable species identification and quick reporting of results would guide clinicians to select the optimal antimicrobial therapy.

Dalbavancin is a semisynthetic lipoglycopeptide approved for the treatment of acute skin and soft tissue infections due to Gram-positive microorganisms susceptible to this antimicrobial agent. The FDA (Food and Drug Administration) and the EUCAST (European Committee on Antimicrobial Susceptibility Testing) have established clinical breakpoints to interpret the results of the antibiogram (expressed as MIC [minimum inhibitory concentration]) with approved doses (1g intravenously [IV] followed by 0.5g IV at day 8 or 1.5g IV in a single dose). The EUCAST has also determined PK/PD (pharmacokinetic/pharmacodynamic) breakpoints -susceptible, ≤ 0.25mg/L; resistant, > 0.25mg/L-, established recommendations for in vitro susceptibility testing (addition of polysorbate-80 to the growth media) and subrogate values based on the vancomycin-susceptible category to interpret dalbavancin susceptibility in the absence of in vitro study of this antimicrobial.

Non-fermenting Gram negative bacteria are increasingly being cultured in respiratory samples from people with cystic fibrosis (CF). This study aimed to determine the susceptibility of clinical CF respiratory isolates from distinct geographical regions to a range of antimicrobials. A total of 286 isolates (106 Stenotrophomonas maltophilia, 100 Burkholderia spp., 59 Achromobacter spp., 12 Pandoraea spp. and 9 Ralstonia spp.) from The Netherlands, Northern Ireland, Spain, USA and Australia were tested. The MIC50, MIC90 and susceptibility categorization were determined. Cotrimoxazole was the most active compound for all the microorganisms (MIC50=0.12-4 mg/L, MIC90=1-16 mg/L). For S. maltophilia, 47% and 62% of isolates were susceptible to cotrimoxazole according to CLSI and EUCAST breakpoints, respectively. Ceftazidime presented a lower level of susceptibility (35%, MIC50=32 mg/L, MIC90=256 mg/L). Tobramycin and colistin MIC90 were >128 mg/L and >16 mg/L, respectively. Regarding Burkholderia isolates, 72%, 56% and 44% were susceptible to cotrimoxazole, ceftazidime and meropenem, respectively. For both ceftazidime and meropenem, the MIC50 and MIC90 values were within the intermediate or resistant category. The most active antibiotics for A. xylosoxidans were cotrimoxazole (MIC50=2, MIC90=8 mg/L) and imipenem (MIC50=2, MIC90=8 mg/L). Cotrimoxazole, imipenem, and ciprofloxacin were active against the 12 Pandoraea species (MIC50= 0.12-4 mg/L, MIC90= 1-8 mg/L). Ciprofloxacin (MIC50=4 mg/L) and cotrimoxazole (MIC50=1 mg/L) were the only active antibiotics for Ralstonia spp. There were no statistically significant differences in susceptibility rates between countries. Non-fermenting Gram-negative bacteria other than P. aeruginosa are potential pathogens in CF and cotrimoxazole has demonstrated to be the most active compound against them.

The genus Raoultella was excised from Klebsiella in 2001, but difficulties in its identification may have led to an underestimation of its incidence and uncertainty on its pathogenic role. Recently, clinical reports involving Raoultella have increased, probably through the introduction of mass-spectrometry in clinical microbiology laboratories and the development of accurate molecular techniques. We performed a retrospective analysis using our blood culture collection (2011-14) to identify Raoultella isolates that could have been erroneously reported as Klebsiella. PCR and gene sequencing of highly specific chromosomal class A β-lactamase genes was established as the reference method, and compared with 16S rRNA and rpoβ sequencing, as well as matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS), MicroScan Walkaway system and API20E biochemical identification. MALDI-TOF and rpoβ correctly identified all Raoultella isolates, whereas 16S rRNA provided inconclusive results, and MicroScan and API20E failed to detect this genus. The analysis of the clinical characteristics of all Raoultella bacteraemia cases reported in the literature supports the role of Raoultella as an opportunistic pathogen that causes biliary tract infections in elderly patients who suffer from some kind of malignancy or have undergone an invasive procedure. Two salient conclusions are that Raoultella shows tropism for the biliary tract and so its identification could help clinicians to suspect underlying biliary tract disease when bacteraemia occurs. Concomitantly, as most phenotypic identification systems are not optimized for the identification of Raoultella, the use of MALDI-TOF or additional phenotypic tests is recommended for the reliable identification of this genus.

Performance of the CHROMagar mSuperCARBA media was assessed in both well-characterized carbapenem-resistant Enterobacteriaceae ( n =52) and routine surveillance rectal swab specimens ( n =211). Limit of detection ranged between 10 1 and 10 2 CFU/mL except for OXA-48 producers with low-carbapenem MICs (10 6 CFU/mL). High sensitivity (100%) and specificity (100%) were obtained with rectal swabs.

To standardize the methodology for conducting direct antimicrobial susceptibility testing (AST) of Enterobacterales and Pseudomonas aeruginosa causing bacteremia from positive blood culture pellets. Two methods for processing positive blood cultures with Enterobacterales and P. aeruginosa were compared: a conventional method for identification and AST versus a direct method obtaining a pellet for both matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) identification and direct AST. A total of 157 (145 Enterobacterales, 12 P. aeruginosa) positive blood cultures were included. Microorganism identification showed 100% concordance between both methods at species and genus level. Definitive AST results were obtained 24 h earlier with the rapid method than the conventional one (p

Background More data are needed about the safety of antibiotic de-escalation in specific clinical situations as a strategy to reduce exposure to broad-spectrum antibiotics. The aims of this study were to investigate predictors of de-escalation and its impact on the outcome of patients with bloodstream infection due to Enterobacteriaceae (BSI-E). Methods A post hoc analysis was performed on a prospective, multicenter cohort of patients with BSI-E initially treated with ertapenem or antipseudomonal β-lactams. Logistic regression was used to analyze factors associated with early de-escalation (EDE) and Cox regression for the impact of EDE and late de-escalation (LDE) on 30-day all-cause mortality. A propensity score (PS) for EDE vs no de-escalation (NDE) was calculated. Failure at end of treatment and length of hospital stay were also analyzed. Results Overall, 516 patients were included. EDE was performed in 241 patients (46%), LDE in 95 (18%), and NDE in 180 (35%). Variables independently associated with a lower probability of EDE were multidrug-resistant isolates (odds ratio [OR], 0.50 [95% confidence interval {CI}, .30–.83]) and nosocomial infection empirically treated with imipenem or meropenem (OR, 0.35 [95% CI, .14–.87]). After controlling for confounders, EDE was not associated with increased risk of mortality; hazard ratios (HR) (95% CIs) were as follows: general model, 0.58 (.25–1.31); model with PS, 0.69 (.29–1.65); and PS-based matched pairs, 0.98 (.76–1.26). LDE was not associated with mortality. De-escalation was not associated with clinical failure or length of hospital stay. Conclusions De-escalation in patients with monomicrobial bacteremia due to Enterobacteriaceae was not associated with a detrimental impact on clinical outcome.

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 β-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 bla(OXA-48) gene was located on an ∼60-kb IncL plasmid related to IncL/M-pOXA-48a and the novel bla(CTX-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 β-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-bla(FOX-8)). We describe the first bla(OXA-48) plasmid in Kluyvera spp. and the novel chromosomal CTX-M-213 enzyme and highlight further nosocomial dissemination of bla(OXA-48) through clonal lineages or plasmids related to IncL/M-pOXA-48a.

Recent emergence of transferable plasmid-borne colistin resistance (mcr genes) raised fear for pan-resistance. We evaluated the performance of a new chromogenic medium [CHROMID® Colistin R agar (COLR)] for the screening of colistin-resistant Enterobacterales. Specificity was evaluated using 89 rectal swabs and 89 stools prospectively collected. COLR sensitivity was evaluated by seeding 59 negative clinical samples artificially contaminated (105 CFU/mL) with 59 colistin-resistant Enterobacterales, including 20 mcr-1–positive strains. Twelve samples with an Enterobacterales with nonintrinsic resistance to colistin were recovered during the specificity study, including one mcr-1–positive Escherichia coli, representing a 6.7% prevalence of colistin resistance in fecal carriage. Overall, specificity was 100.0% [95% CI: 97.8–100.0] and sensitivity yielded 88.1% [95% CI: 77.5–94.1]. False negatives corresponded to 3 Enterobacter spp. (MIC>64 mg/L), 2 Salmonella spp. (MIC = 16 mg/L), 1 E. coli (MIC = 4 mg/L), and 1 K. pneumoniae (MIC = 8 mg/L). COLR appears to be a sensitive and specific chromogenic agar for screening colistin-resistant Enterobacterales, including those carrying mcr-1 gene.

The antibacterial activity of fosfomycin-tobramycin combination was studied by time-kill assay in eight Pseudomonas aeruginosa clinical isolates belonging to the fosfomycin wild-type population (MIC = 64 μg/ml) but with different tobramycin susceptibilities (MIC range, 1 to 64 μg/ml). The mutant prevention concentration (MPC) and mutant selection window (MSW) were determined in five of these strains (tobramycin MIC range, 1 to 64 μg/ml) in aerobic and anaerobic conditions simulating environments that are present in biofilm-mediated infections. Fosfomycin-tobramycin was synergistic and bactericidal for the isolates with mutations in the mexZ repressor gene, with a tobramycin MIC of 4 μg/ml. This effect was not observed in strains displaying tobramycin MICs of 1 to 2 μg/ml due to the strong bactericidal effect of tobramycin alone. Fosfomycin presented higher MPC values (range, 2,048 to >2,048 μg/ml) in aerobic and anaerobic conditions than did tobramycin (range, 16 to 256 μg/ml). Interestingly, the association rendered narrow or even null MSWs in the two conditions. However, for isolates with high-level tobramycin resistance that harbored aminoglycoside nucleotidyltransferases, time-kill assays showed no synergy, with wide MSWs in the two environments. glpT gene mutations responsible for fosfomycin resistance in P. aeruginosa were determined in fosfomycin-susceptible wild-type strains and mutant derivatives recovered from MPC studies. All mutant derivatives had changes in the GlpT amino acid sequence, which resulted in a truncated permease responsible for fosfomycin resistance. These results suggest that fosfomycin-tobramycin can be an alternative for infections due to P. aeruginosa since it has demonstrated synergistic and bactericidal activity in susceptible isolates and those with low-level tobramycin resistance. It also prevents the emergence of resistant mutants in either aerobic or anaerobic environments.

A nationwide study was developed to evaluate the ability of 60 Spanish clinical microbiology laboratories to predict the underlying β-lactam resistance mechanisms of 12 Enterobacteriaceae strains (CCS01-CCS12). Results obtained by two reference laboratories were compared with those reported by the participant laboratories that used their own routine susceptibility testing methodology. Clinical and Laboratory Standards Institute (CLSI) interpretive criteria were used in 53.3% of centres, whilst 46.7% used European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Overall categorical agreement (CA) was 85.5%. Rates of very major errors (VME), minor errors (MinE) and major errors (ME) were 5%, 9.7% and 5.5%, respectively. The lowest CA values were obtained for carbapenems (56.7-78.1%). β-Lactam/β-lactamase inhibitors were also problematic compounds: VME for amoxicillin/clavulanic acid were 8% (EUCAST criteria) and MinE for piperacillin/tazobactam were 45.7% (CLSI criteria). OXA-48 (CCS02, CCS10, CCS11), VIM-1 (CCS09) and KPC-3 (CCS05) were correctly identified by 75-87%, 65% and 71% of centres, respectively. Strains with an extended-spectrum β-lactamase (ESBL) plus a carbapenemase (CCS03, CCS04, CCS06) were correctly detected in 32-68% of centres. Seven percent correct identifications were recorded for CCS08 (chromosomal K1 β-lactamase hyperexpression plus IMP-8). Strains with permeability deficiencies [CCS07 (ACT-1 plus porin deficit) and CCS12 (TEM-24 plus porin deficit)] were correctly detected in 17% and 10% of centres, respectively. The TEM-1-producer (CCS01) was detected in 40% of centres. Microbiologists should be aware of new antibiotic resistance mechanisms, and these surveillance studies are particularly useful for this purpose and for the communication of such traits.

Dalbavancin is a semisynthetic lipoglycopeptide approved for the treatment of acute skin and soft tissue infections due to Gram-positive microorganisms susceptible to this antimicrobial agent. The FDA (Food and Drug Administration) and the EUCAST (European Committee on Antimicrobial Susceptibility Testing) have established clinical breakpoints to interpret the results of the antibiogram (expressed as MIC [minimum inhibitory concentration]) with approved doses (1g intravenously [IV] followed by 0.5g IV at day 8 or 1.5g IV in a single dose). The EUCAST has also determined PK/PD (pharmacokinetic/pharmacodynamic) breakpoints -susceptible, ≤ 0.25mg/L; resistant,0.25mg/L-, established recommendations for in vitro susceptibility testing (addition of polysorbate-80 to the growth media) and subrogate values based on the vancomycin-susceptible category to interpret dalbavancin susceptibility in the absence of in vitro study of this antimicrobial.

Because of the increase in bacterial resistance, there is a need for new antimicrobial agents. In particular, Staphylococcus aureus is a frequent cause of severe infections and has an extraordinary capacity to develop antibiotic multiresistance, including resistance to glycopeptides, linezolid, and daptomycin. Although the incidence of methicillin-resistant S. aureus (MRSA) seems to have stabilized in the last few years, its wide dissemination in healthcare settings and in the community is a cause of concern. Ceftaroline is a new broad-spectrum cephalosporin with bactericidal activity against Gram-positive bacteria, including MRSA and multidrug-resistant Streptococcus pneumoniae. In addition, this drug is active against staphylococci showing resistance to glycopeptides, linezolid, and daptomycin. The ceftaroline MIC90 against MRSA ranges from 0.5-2mg/L and that against methicillin-resistant coagulase-negative staphylococci is 0.5mg/L. Ceftaroline has also good activity against respiratory pathogens including Haemophilus influenzae and Moraxella catarrhalis. Although this drug is active against Enterobacteriaceae, it does not retain activity when these isolates produce extended-spectrum beta-lactamases, carbapenemases or hyperproduce AmpC. Ceftaroline is not active against nonfermentative Gram-negative bacilli. Ceftaroline is an interesting addition to the therapeutic armamentarium against MRSA and constitutes an important option for the treatment of polymicrobial infections caused by multidrug-resistant Gram-positive microorganisms.

In 1986, a new syndrome was described in Taiwan secondary to hypervirulent K. pneumoniae (hvKP), and its main feature was the ability to cause severe infection in young and immunocompetent hosts. Their virulence is explained by the efficient acquisition of iron and an increase in capsule production, which confer the characteristic hypermucoviscous phenotype. Most of these cases have been described in Asia and subsequently spread to America and Europe, where their prevalence is much lower. We present four cases of bacteremia and liver abscesses secondary to hypervirulent K. pneumoniae, two of them associated with endophthalmitis. K. pneumoniae isolates recovered from two of the patients belonged to capsular serotype K1 (genes wzx_K1 and magA), while the other two were K2 (gene wzy_K2). Both of the K1 isolates were classified into a ST23, and isolates of serotype K2 belonged to the ST375 and ST881 clones.In Europe, hvKP isolates are less frequently recovered, mostly associated with Asian citizens or travelers, which was not the case in our patients. K1 capsular serotype is a major cause of primary liver abscess and secondary septic embolus, and K2 is associated with secondary liver abscess. Although these hypervirulent variants usually affect immunocompetent patients as in our cases, diabetes mellitus is a major risk factor for the most invasive cases, with concomitant poor prognosis. Identification of hypervirulent K. pneumoniae serotypes K1 and K2 should be considered as part of the microbiological diagnosis of community-acquired liver abscess due to their clinical implications. Keywords: Hypermucoviscous Klebsiella pneumoniae, Liver abscess, Community acquired infection, MLST

Objectives To analyse the ongoing epidemiology of KPC-producing Enterobacteriaceae after a non-ST258 KPC-3-producing Klebsiella pneumoniae outbreak in a university hospital in Madrid, Spain. Methods Enterobacterial isolates (one per patient based on bacterial identification and typing patterns) with carbapenem MICs higher than the EUCAST epidemiological cut-off values, a positive modified Hodge test and carbapenem/boronic acid combination disc test results were studied (16 March 2010 to 31 January 2012) and compared with KPC-producing isolates previously described in our institution (September 2009 to February 2010). The bacterial population structure (PFGE and multilocus sequence typing), carbapenemase genes and KPC plasmids were studied. Patients' clinical records were reviewed. Results Twenty-four KPC-producing Enterobacteriaceae (20 K. pneumoniae, 2 Escherichia coli and 2 Enterobacter cloacae) from 23 patients (13 males, median age 72 years) were studied. Most KPC-producer strains were considered as colonizers. Six K. pneumoniae clones (ST11, ST20, ST384, ST454, ST659 and ST971), two E. coli clones (ST224 and ST357) and one E. cloacae clone were identified. blaKPC-3 was located on IncFIIK plasmids of ∼100 kb (E. coli ST357 and K. pneumoniae ST384, ST454, ST659 and ST971 clones) and on IncN plasmids of ∼40 kb (K. pneumoniae ST11 clone). Non-typeable plasmids of ∼20 kb containing blaKPC-2 were detected in scarcely represented clones (K. pneumoniae ST20, E. coli ST224 and E. cloacae). Conclusions During the 2 year period following the emergence of non-ST258 KPC-3-producing K. pneumoniae isolates in our institution, the blaKPC-3 and blaKPC-2 genes efficiently penetrated other Enterobacteriaceae lineages. Non-ST258 K. pneumoniae isolates were mostly responsible for the dissemination of KPC enzymes, producing a complex epidemiological picture.

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.

Antimicrobial resistance mechanisms among clinically relevant gram-positive microorganisms can be demonstrated using phenotypic tests that enable the interpretation of underlying mechanisms responsible for the in vitro resistance. The reporting of these mechanisms, either inferred or demonstrated, helps in the adjustment of clinical treatments and the epidemiological follow up of resistance traits. In the present work, phenotypic tests for detection of antimicrobial resistance mechanisms involving the most frequent antimicrobial families used against Staphylococcus spp., Enterococcus spp. and Streptococcus pneumoniae are analysed. In the case of Staphylococcus, phenotypic tests to reveal the mechanisms of resistance against beta-lactams, macrolides, lincosamides and streptogramin B (MLS(B)), as well as intermediate susceptibility to glycopeptides, resistance to aminoglycosides, mupirocin and linezolid are reviewed. Tests to detect glycopeptide resistance and high-level aminoglycoside resistance among enterococci are analysed. Detection of penicillin resistance, as well as diminished susceptibility to third generation cephalosporins, together with diminished susceptibility or even resistance to fluoroquinolones is also detailed.

All Streptococcus bovis blood culture isolates recovered from January 2003 to January 2010 ( n = 52) at the Hospital Universitario Ramón y Cajal were reidentified on the basis of their genetic traits using new taxonomic criteria. Initial identification was performed by the semiautomatic Wider system (Fco. Soria-Melguizo, Spain) and the API 20 Strep system (bioMérieux, France). All isolates were reidentified by PCR amplification and sequencing of both the 16S rRNA and sodA genes and by mass spectrometry using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker, Germany). Results of 16S rRNA/ sod A gene sequencing were as follows: Streptococcus gallolyticus subsp. gallolyticus , 14/14 (number of isolates identified by 16S rRNA/number of isolates identified by sodA gene sequencing); Streptococcus gallolyticus subsp. pasteurianus , 24/24; Streptococcus spp., 7/0; Streptococcus infantarius subsp. infantarius , 0/2; Streptococcus lutetiensis , 0/5; Leuconostoc mesenteroides , 4/0; and Lactococcus lactis , 3/3. MALDI-TOF MS identified 27 S. gallolyticus isolates but not at the subspecies level, 4 L. mesenteroides isolates, 3 L. lactis isolates, and 6 S. lutetiensis isolates, whereas 12 isolates rendered a nonreliable identification result. Pulsed-field gel electrophoresis grouped all S. gallolyticus subsp. gallolyticus isolates into 3 major clusters clearly different from those of the S. gallolyticus subsp. pasteurianus isolates, which, in turn, exhibited no clonal relationship. The percentages of resistance to the tested antimicrobials were 38% for erythromycin, 23% for fosfomycin, 10% for levofloxacin, 6% for tetracycline, and 4% for co-trimoxazole. The most frequent underlying diseases were hepatobiliary disorders (53%), endocarditis (17%), and malignancies (12%). We conclude that sequencing of the sod A gene was the most discriminatory method and that S. gallolyticus subsp. pasteurianus appears to have a higher genetic diversity than S. gallolyticus subsp. gallolyticus .

Bacterial biofilms play an important role in the persistent colonization of the respiratory tract in cystic fibrosis (CF) patients. The trade‐offs among planktonic or sessile modes of growth, mutation frequency, antibiotic susceptibility and mutant prevention concentrations (MPCs) were studied in a well‐defined collection of 42 CF Pseudomonas aeruginosa isolates . MICs of ciprofloxacin, tobramycin, imipenem and ceftazidime increased in the biofilm mode of growth, but not the MPCs of the same drugs. The mutation frequency median was significantly higher in planktonic conditions (1.1 × 10 −8 ) than in biofilm (9.9 × 10 −9 ) (p 0.015). Isolates categorized as hypomutable increased their mutation frequency from 3.6 × 10 −9 in the planktonic mode to 6 × 10 −8 in biofilm, whereas normomutators (from 9.4 × 10 −8 to 5.3 × 10 −8 ) and hypermutators (from 1.6 × 10 −6 to 7.7 × 10 −7 ) decreased their mutation frequencies in biofilm. High and low mutation frequencies in planktonic growth converge into the normomutable category in the biofilm mode of growth of CF P. aeruginosa , leading to stabilization of MPCs. This result suggests that once the biofilm mode of growth has been established, the propensity of CF P. aeruginosa populations to evolve towards resistance is not necessarily increased.

During the last few years, insufficient efficacy of currently recommended antimicrobial agents has been observed, mainly in the case of glycopeptides, during the treatment of infections due to methicillin-resistant Staphylococcus aureus, even if these isolates show MIC values within the susceptible range. The phenomena associated with this observation are tolerance (a genetic event in which a bactericidal antibiotic fails to kill a bacterial population), persistence (a non-inherited and transient phenotypic phenomenon in which a bacterial subpopulation -0.1%-10%- survive lethal antimicrobial concentrations irrespective of the mechanisms of action) and heteroresistance (an epigenetic event in which less susceptible isogenic subpopulations are recovered when the entire population is challenged with concentrations exceeding MIC values). New antimicrobials, including daptomycin, are less affected by these phenomena and should be considered as the treatment of choice when these events are demonstrated or suspected.

Susceptibility results with low reproducibility by the same or different methods have been observed for metallo-beta-lactamase (MBL)-producing Enterobacteriaceae . Eighteen VIM-1-producing Klebsiella pneumoniae isolates (one per patient) belonging to a single epidemic clone in our hospital (2005 to 2008) but with different susceptibilities to carbapenems were studied. Imipenem MICs ranged from 8 to >128 mg/liter by standard CLSI microdilution, from ≤1 to >8 mg/liter by the semiautomatic Wider system, and from 0.75 to >32 mg/liter by Etest. Meropenem MICs ranged from 0.5 to 128, ≤1 to >8, and 0.38 to >32 mg/liter, respectively. Ertapenem MICs by CLSI microdilution and Etest ranged from 1 to 64 and 0.75 to >32 mg/liter, respectively. The rates of essential agreement (±1 log 2 dilution) for imipenem and meropenem MICs between the Wider system and the reference microdilution method were 45% and 49%, respectively. Those between Etest and the reference microdilution method for imipenem, meropenem, and ertapenem MICs were 33%, 67%, and 84%. The rates of very major errors for the Wider system and Etest were 33% and 28% for imipenem and 25% and 75% for meropenem, respectively. Low MIC reproducibility was observed even when the same inoculum was used (differences up to 4-fold dilutions). Heteroresistance was suspected due to the presence of colonies in the Etest inhibition zone. It was confirmed by population analysis profiles of 4 isolates displaying different imipenem MICs, with the exception of an OmpK36-porin-deficient isolate that homogeneously expressed carbapenem resistance (MIC, >128 mg/liter). Low carbapenem MIC reproducibility could be due to the presence of resistant subpopulations and variable expression of the resistance mechanisms. Since carbapenem MICs are not good markers of MBL production, reliable and reproducible phenotypic methods are needed to detect the presence of this mechanism.

INTRODUCTION AND OBJECTIVE Daptomycin is a bactericidal lipopeptide antibiotic, active against gram-positive bacteria. In this study, the comparative in vitro activity of daptomycin and other antimicrobial agents against isolates recovered in 3 Spanish hospitals from 2002 to 2006 was determined as part of the international SENTRY antimicrobial resistance surveillance program. The possible therapeutic role of daptomycin is addressed in the light of Spanish susceptibility data. METHODS Antimicrobial susceptibility testing was performed by the microdilution method with 1398 consecutively recovered gram-positive isolates. RESULTS All the staphylococci (n = 1024), enterococci (n = 228), and streptococci (n = 146) studied were susceptible to daptomycin. The highest MIC values were 1, 4, and 0.5 microg/mL, respectively, regardless of methicillin, vancomycin, or penicillin resistance status. All Staphylococcus aureus isolates were also susceptible to vancomycin, teicoplanin, linezolid, and quinupristin-dalfopristin. Coagulase-negative staphylococci were susceptible to vancomycin, linezolid, and quinupristin-dalfopristin. Only daptomycin and linezolid were active against all enterococcal isolates. In addition, vancomycin, teicoplanin, and ampicillin were fully active against Enterococcus faecalis. Daptomycin, vancomycin, teicoplanin, linezolid, and quinupristin-dalfopristin were active against all Streptococcus pyogenes, S. agalactiae, and S. viridans group isolates. The distribution of daptomycin MIC values in S. aureus and enterococci was homogeneously sustained along the 5-year study period. CONCLUSION The sustained antimicrobial activity of daptomycin against staphylococci, enterococci, and streptococci in Spain makes this antibiotic an excellent therapeutic option in severe infection caused by gram-positive microorganisms, including those with multiresistance.

Introduccion y objetivo La daptomicina es un antibiotico lipopeptidico bactericida frente a microorganismos grampositivos. En este trabajo estudiamos la actividad in vitro de daptomicina comparativamente con otros antibioticos frente a aislados de tres hospitales espanoles recogidos durante el periodo 2002-2006, en el marco del programa internacional de vigilancia de la resistencia, SENTRY, para establecer sus posibilidades terapeuticas con datos especificos de Espana. Metodos Los antibioticos se estudiaron por la tecnica de microdilucion frente a 1.398 microorganismos grampositivos aislados consecutivamente. Resultados Todos los estafilococos (n = 1.024), enterococos (n = 228) y estreptococos (n = 146) fueron sensibles a daptomicina y presentaban valores maximos de concentraciones inhibitorias minimas (CIM) de 1, 4 y 0,5 μg/ml, respectivamente, con independencia de la resistencia a meticilina, vancomicina o penicilina. Staphylococcus aureus fue tambien sensible a vancomicina, teicoplanina, linezolid y quinupristinadalfopristina y los estafilococos coagulasa negativa (SCN) a vancomicina, linezolid y quinupristina-dalfopristina. Frente a enterococos, solo daptomicina y linezolid fueron activos frente a la totalidad de estos. Ademas, vancomicina, teicoplanina y ampicilina fueron totalmente efectivos frente a Enterococcus faecalis . Frente a Streptococcus pyogenes , Streptococcus agalactiae y estreptococos del grupo viridans , daptomicina, vancomicina, teicoplanina, linezolid y quinupristina dalfopristina fueron plenamente activos. La distribucion de las CIM de daptomicina a traves de los 5 anos fue homogenea en S. aureus y enterococos. Conclusion La actividad de daptomicina frente a estafilococos, enterococos y estreptococos, mantenida a lo largo de los anos en Espana, la convierte en una excelente opcion terapeutica en infecciones graves por microorganismos grampositivos, incluyendo los multirresistentes.