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Objectives: Bacterial viability and enrichment of resistance resulting from three different amikacin administration schedules with respect to haemodialysis (HD) were assessed against three OXA-48-producing Klebsiella pneumoniae isolated during an outbreak in a Spanish hospital. Methods: A previously described two-compartment dynamic system was used. Three possible amikacin administration schedules were simulated: (i) haemodialysis immediately after amikacin infusion (pre-HD); (ii) infusion immediately after haemodialysis (post-HD); and (iii) infusion at 50% interdialytic period. Amikacin standard dose (SD) and double dose (DD) were simulated for each schedule. Values of Cmax/MIC, Cmax/MPC (mutant prevention concentration), AUC0-48h/MIC, AUC0-48h/MPC and %TMSW (percentage of time that the concentration was inside the mutant selection window) were determined with experimental data and were correlated with the area under the bacterial killing curve of the total population and the resistant subpopulation. Results: Both with SD and DD, the pre-HD schedule resulted in increases at 48h in bacterial counts of the total population at the expense of enrichment of pre-existing resistant subpopulations from 12h onwards for all strains. The estimated %TMSW that prevented enrichment of resistant mutants was

Acinetobacter baumannii is a highly versatile nosocomial pathogen. Multidrug resistance among A. baumannii isolates led to the use of colistin, subsequently giving rise to colistin-resistant strains. In this study, the genetic and phenotypic profiles of two colistin-resistant A. baumannii isolates were investigated. Two A. baumannii isolates were obtained from Patient 1 (C071 and C440) and three isolates were obtained from Patient 2 (C080, C314 and C428). Susceptibility profiles were determined by VITEK®2 and Etest. Clonality was determined by RAPD analysis and trilocus multiplex PCR. The pmrCAB operon was sequenced and common carbapenemase genes were screened for by PCR. Doubling times, haemolysis, surface motility, biofilm formation, siderophore production and proteolytic activity were phenotypically determined. Finally, whole-genome sequencing was performed for all five isolates. Isolates C440 and C428 were resistant to colistin and were clonally identical to their sensitive counterparts. The cause of colistin resistance was traced to the previously described P233S mutation in pmrB of C440 and to a novel ΔI19 mutation in pmrB of C428. blaOXA-58-like and blaGES-5 from the strains of Patients 1 and 2, respectively, were also detected. C440 had attenuated proteolytic activity and was positive for siderophore production compared with C071. No difference in in vitro virulence was detected between isolates C080, C314 and C428. In conclusion, one common and one novel mutation were encountered in pmrB from two distinct colistin-resistant A. baumannii isolates. These mutations caused colistin resistance during therapy in two distinct clones, and only one of them had altered in vitro virulence.

Introduction In 2011, a hospital-wide outbreak of OXA-48 producing Klebsiella pneumoniae occurred in our hospital, an epidemiological setting of high ESBL-producing K. pneumoniae rates. This study identifies risk factors for colonization with carbapenemase-producing enterobacteria (CPE) at Surgical Intensive Care Unit (SICU) admission. Methods A 2-year retrospective study was performed in all patients admitted to the SICU that following routine had a rectal swab collected upon admission. Results Of 254 patients admitted, 41 (16.1%) harbored CPE (five showing two carbapenemase-producing isolates). Most frequent carbapenemase-producing isolates and carbapenemases were K. pneumoniae (39/46, 84.8%) and OXA-48 (31/46; 76.1%), respectively. Carriers significantly had higher rates of chronic renal disease, previous digestive/biliary endoscopy, hospitalization, ICU/SICU admission, intraabdominal surgery, and antibiotic intake, as well as higher median values of clinical scores (SOFA, SAPS II and APACHE II). In the multivariate analysis (R2 = 0.309, p Conclusions A strong association between production of ESBLs and carriage of CPE (mainly OXA-48 producing K. pneumoniae) was found. According to the model, the co-selection of β-lactamases by previous exposure to broad-spectrum cephalosporins and β-lactam/β-lactamase inhibitors (with lower relative risk), abdominal surgery and prior digestive/biliary endoscopy were factors associated with CPE carriage.

The increasing rates of carbapenemase-producing Enterobacteriaceae (CPE) represent an important threat to health care systems and treatment of CPE infections is a challenge. The aim of the infection-carbapenem resistance evaluation surveillance trial (iCREST) was to determinate the prevalence of CPE in urine specimens in Spain and to evaluate the in vitro activity of ceftazidime-avibactam. Urine specimens (n = 11 826) were included and activity of ceftazidime-avibactam and comparators were investigated by broth microdilution in CPE. Carbapenemases were characterised by polymerase chain reaction (PCR) and sequencing as well as by whole genome sequencing (WGS). Overall prevalence of CPE was 1.6%. OXA-48 was the most prevalent (86.8%), followed by KPC (6.9%), VIM (4.8%), NDM (1.1%) and IMP (0.6%) carbapenemases. Klebsiella pneumoniae was the most common carbapenemase producer (87.8%). An uncommon carbapenemase type (IMP-8) in Spain was identify by WGS in an Enterobacter cloacae isolate, reinforcing the utility of surveillance programmes as effectives tools to detect unexpected genes that encode antimicrobial resistance. Ceftazidime-avibactam showed 100% susceptibility in KPC and OXA-48 producers and the rates of susceptibility in CPE non-susceptible to ceftazidime or meropenem were 92.1% and 96.9%, respectively. Ceftazidime-avibactam could be considered an adequate treatment option for urinary tract infections caused by KPC and OXA-48 producers.

Acinetobacter baumannii is a ubiquitous organism that has been involved in a wide range of nosocomial infections. Its ability to produce biofilms, among other characteristics, allows it to persist in hospitals for prolonged periods. In this study, in order to check the possible relationship between its resistance to different antibiotics and its ability to form biofilms on inert surfaces, the rate of biofilm formation as well as siderophore production and detection of OmpA and CsuE by PCR were investigated for 12 A. baumannii clinical isolates. The biofilms were cultured at 37 °C on steel coupons immersed in BHI broth and the attached viable cells were counted after 5, 24 and 48 h. Confocal Laser Scanning Microscopy (CLSM) images were obtained for some of the strains that were noted to produce a brown pigment. The biofilm volume and substratum coverage were estimated with an image analysis software program. Our data, though preliminary, show that the quicker biofilm formers were strains susceptible to aminoglycosides, whereas the biofilms providing thicker and more uniform surface coverage were produced by carbapenem-resistant strains, producing a brown pigment with a plausible siderophore role. Further investigation into a wider set of isolates could help better understand the relationship between biofilm formation and various clinical findings.

Objectives: The aim of this study was to investigate the structure of a broad and sustained hospital outbreak of OXA-48-producing Klebsiella pneumoniae (KpO48) belonging to sequence type 405 (ST405). Methods: Whole-genome sequencing and comparison of ten ST405 KpO48 isolates obtained from clinical samples in our hospital was performed. Using stringent criteria, 36 single nucleotide polymorphisms (SNPs) were detected (range 0–21 in pairwise comparisons), and allele-specific PCR was used to call the SNPs among a larger set of isolates. Results: Several haplotypes were identified within the population. The haplotypes did not show a spatial structure, but a temporal evolution of sequential haplotype replacements was observed. Conclusions: The dispersed spatial distribution suggests a reservoir formed by a large pool of colonised patients, and the temporal replacement pattern suggests that the sustained outbreak was composed of several small outbreaks that appeared and rapidly dispersed to several units., Ministerio de Economía y Competitividad [grant INNPACTO IPT-2011-0964- 900000 to PG-P and JM]; IdiPAZ [internal grant to JM]; the Instituto de Salud Carlos III, Ministerio de Economía y Compet-itividad [grant PI13/01218 to JM]; and REIPI RD12/0015 from Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, co-financed by the European Development Regional Fund

Carbapenem-resistant Klebsiella pneumoniae have spread globally throughout tertiary hospitals. Many Carbapenem-resistant K. pneumoniae clinical isolates are multidrug-resistant (MDR) and may become eventually pandrug-resistant (PDR). Here we present the closed genome of a PDR VIM-1-producing K. pneumoniae strain (KP1050) obtained in a tertiary hospital. The isolate belonged to sequence type 54 (ST54) and had five extrachromosomal elements (four plasmids and a circular phage genome). Most of the antimicrobial resistance genes (ARGs) were located in two clusters borne by two of the plasmids, comprising a class 1 integron that contained up to 14 ARGs including a VIM-1 metallo-β-lactamase gene, and an IS26 transposon that contained a mobile element from Acinetobacter baumannii encoding the amikacin resistance gene aac(6ʹ)-Ian. A MDR isolate obtained 6 years previously was identified (KP1048) retrospectively and was sequenced. Comparison of the two genomes showed that chromosomal mutations in outer membrane porins as well as mutations in the ramR and phoQ genes contributed to increase the resistance spectrum.

The main objective of our study was to describe the epidemiological and microbiological features of an oligoclonal hospital-wide outbreak caused by OXA-48-producing Enterobacteriaceae (OXA-48-PE). OXA-48 is a carbapenemase belonging to Ambler class D beta-lactamases, identified frequently in the Mediterranean and Southern European countries, and associated with several Enterobacteriaceae species. An outbreak of OXA-48-PE with a complex epidemic pattern was detected in January 2011. Initial control measures included contact precautions and the reinforcement of infection control practices, but despite all efforts made, the epidemiological situation hardly changed and new measures were implemented during 2013. An observational retrospective study was performed to describe the main features of the outbreak and to analyse the cumulative incidence (CI) trends. Eight hundred and 16 patients colonised or infected by OXA-48-PE were identified during the 2-year period (January 2013–December 2014), female 46%, mean age (s.d.), 71.6 (15.2). The samples isolated in the incident cases were rectal swabs (80%), urine samples (10.7%), blood samples (2.8%) and other clinical samples (6.6%). The most frequent OXA-48-PE was Klebsiella pneumoniae. Eleven different clones were identified, but K. pneumoniae sequence types 11 and 405 were predominant: ST11 (64.2%) and ST405 (29.3%). OXA-48-PE CI trend suffered a statistically significant change in August 2013, which continued the following months. Though we could not eradicate the outbreak, we observed a statistically significant drop in CI after an intervention for OXA-48-PE control, based on patient cohort, active surveillance, electronic alerts and reinforcement of infection control measures in a tertiary hospital.

INTRODUCTION Acinetobacter baumannii is a nosocomial pathogen that is showing increasing rates of carbapenem resistance. Multi-Drug Resistant (MDR) International Clones (ICs), associated with certain oxacillinases, are being reported globally. This organism also harbors numerous virulence determinants. In this study, we aim at characterizing A. baumannii isolated from a Spanish hospital in terms of antimicrobial susceptibility, clonality, carbapenemase genes harbored, and virulence determinants expressed. MATERIALS AND METHODS Fifty nine clinical bloodstream isolates were obtained from 2009 until 2013. Antimicrobial Susceptibility Testing was performed according to the CLSI guidelines. PFGE and tri-locus PCR typing were then performed in order to determine local and international clonality. PCRs for the detection of common carbapenemases were also performed. Production of hemolysis, biofilms, siderophores, surface motility, and proteolysis were determined phenotypically. Doubling times for selected strains were also calculated. Finally, statistical analysis for detecting associations between these factors was conducted. RESULTS AND DISCUSSION Carbapenem non-susceptibility was 84.75%, suggesting the immediate need for intervention. PFGE showed the distribution of the majority of the isolates among 7 clusters. Although all three ICs were detected, IC II was predominant at 71.19%. blaOXA-24-like was the most prevalent carbapenemase (62.71%), followed by blaOXA-58-like (13.56%), and blaOXA-23-like (11.86%). Strains pertaining to IC II, and those harboring blaOXA-24-like, were positively associated with α-hemolysis, production of strong biofilms, and siderophore production. Harboring blaOXA-23-like and blaOXA-58-like was associated with attenuated virulence. These associations suggest that an interplay exists between these factors that could be locally exploited. CONCLUSIONS An alarmingly high rate of carbapenem non-susceptibility has been detected in this study. There was a predominance of IC II and blaOXA-24-like, and those factors were associated with heightened expression of virulence determinants. This association could be exploited for modifying treatment regimens and for improving on infection control protocols in this hospital.

Bacteraemia due to carbapenemase-producing Enterobacteriaceae is an emerging medical problem. Management of this entity is complicated by the difficulty in Identifying resistance patterns and the limited therapeutic options. A cohort study was performed including all episodes of bloodstream infection due to OXA-48-producing Enterobacteriaceae (O48PE), occurring between July 2010 and April 2012. Data on predisposing factors, clinical presentation, therapy and outcome were collected from medical records. There were 40 cases of bacteraemia caused by O48PE, 35 Klebsiella pneumoniae and five Escherichia coli. Patients were elderly with significant comorbidities (57.5% underlying malignancy). Thirty-five cases (87.5%) were nosocomial, and five (12.5%) were healthcare-associated. Patients had frequently been exposed to antibiotics and to invasive procedures during hospitalization. The most common source of bacteraemia was the urinary tract followed by deep intra-abdominal surgical site infection. Clinical presentation was severe sepsis or shock in 18 cases (45%). Extended-spectrum β-lactamase production was detected in 92.5% of isolates. MIC90 for ertapenem, imipenem and meropenem were 32, 16 and 16 mg/L, respectively. Most frequently preserved antibiotics were amikacin, colistin, tigecycline and fosfomycin. These antibiotics combined are the basis of targeted therapies, including carbapenem in selected cases. Median delay in starting clinically adequate and microbiologically appropriate treatment was 3 days. Crude mortality during admission and within 30 days from bacteraemia was 65% and 50%, respectively. Bloodstream infections caused by O48PE have a poor prognosis. Delay in diagnosis and in initiation of optimal antimicrobial therapy is frequent. Suspicion and rapid identification could contribute to improving outcomes.

Background. Because most infections caused by carbapenemase-producing Enterobacteriaceae (CPE) begin during hospitalization, there are limited data about community-onset (CO) infections caused by CPE. Our aim is to describe the frequency of CO infections caused by CPE as well as the clinical features of CO bloodstream infections (CO-BSIs). Methods. This study includes retrospective case series of CO infections caused by CPE in a tertiary hospital from January 2010 to July 2014. Any clinical sample with a positive culture for CPE that had been ordered by primary care doctors or by doctors at the emergency room (ER) were classified as CO. Epidemiological and microbiological features of CO cases were assessed as were clinical features of CO-BSIs. Results. Of 780 clinical samples with CPE, 180 were requested at the ER or by primary care doctors (22.9%), 150 of which were produced by Klebsiella pneumoniae (83.3%). The blaOXA-48 gene was detected in 149 isolates (82.8%) followed by the blaVIM gene, 29 (16.1%). Sixty-one patients (33.9%) had a prior history of CPE infection/colonization. Thirty-four of the 119 (28.6%) patients without prior history of CPE infection/colonization did not fulfill Friedman criteria for healthcare-associated infections (HAIs). Considering previous hospitalization of up to 12 months as a criterion for defining HAI, only 16 (13.4%) cases were identified as community-acquired infections. The most frequent positive sample was urine (133 of 180; 73.9%). Twenty-one (11.7%) patients had a BSI, 9 of them secondary to urinary tract infections (42.9%). Thirty-day crude mortality among patients with BSI was 23.8% (5 of 21). Conclusions. Community-onset infections caused by CPE are an important subgroup of all CPE infections. The urinary tract is the main source. Bloodstream infections accounted for more than 10% of the cases.

We describe 12 cases of linezolid-resistant Enterococcus faecalis. The present study was done in 2 wards of Hospital Universitario La Paz in Madrid, Spain. The 2 wards involved were the intensive care unit (ICU) and reanimation unit. Twelve clinical strains of E. faecalis reported by the clinical laboratory as linezolid resistant based on MICs determined by E-test (AB Biodisk, Solna, Sweden) were collected between September 2005 and October 2006. The MIC of linezolid for all the resistant isolates was >128 microg/mL. The isolates were analyzed for the presence of the G2576T mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and pyrosequencing. Pyrosequencing showed that the first isolate had G and T at position 2576 in a 1:1 ratio, whereas the remaining ones had a wild type to mutant ratio of 1:3. PCR-RFLP showed that the mutations were in alleles 1, 3, and 4. The 12 isolates under investigation came from different patients but were indistinguishable by pulsed-field gel electrophoresis (n = 7) and repetitive extragenic palindromic sequence (REP)-PCR (n = 12). This is the first report of a clonal outbreak of linezolid-resistant E. faecalis in Spain. To prevent or minimize the emergence of resistance, we should use linezolid strictly after the therapeutic indications, courses of treatment should be kept as short as possible, and risk factors for resistance development should be considered before starting. In addition, we suggest that susceptibility testing of clinically significant Gram-positive pathogens should be done in all cases of treatment failure, and, depending on the local epidemiology of each ICU, it might be advisable to do it before starting treatment with linezolid.

Carbapenem-resistant or intermediate (MIC ≥1 mg/L) clinical isolates ( n = 12) of three species of Enterobacteriaceae ( Klebsiella pneumoniae, Klebsiella oxytoca and Escherichia coli ) were characterized. The isolates harboured integrons containing the VIM-1 metallo- β -lactamase gene together with other resistance gene cassettes. In particular, the CTX-M-2 gene was detected in four of the K. pneumoniae isolates. The patient population was mostly paediatric and characterized by severe underlying illnesses that involved long-term hospitalization, major surgery and/or immunosuppressive and broad-spectrum antibiotic therapy.

A study is presented on the presence of quinolone resistance qnrB1 genes in clinical isolates belonging to the largest series of infections caused by OXA-48-producing Klebsiella pneumoniae in a single-centre outbreak in Spain. Evidence is also provided, according to in vitro results, that there is a possibility of co-transfer of plasmid harbouring blaOXA-48 with an other plasmid harbouring qnrB1 in presence of low antibiotic concentrations of fluoroquinolones, showing the risk of multi-resistance screening.

The mechanisms of linezolid resistance among 86 staphylococcal isolates from two intensive care units were investigated. The most frequent was the G2576T mutation in the 23S rRNA (82%). The cfr gene was found in 17% of the isolates, seven S. aureus and eight S. epidermidis isolates. Four of the S. epidermidis isolates had the G2576T mutation and the cfr gene. In four S. haemolyticus isolates, the mechanism could not be identified.

Klebsiella pneumoniae KpO3210 is a OXA-48 carbapenemase-producing isolate obtained from a blood culture in the context of an outbreak in Hospital Universitario La Paz (Madrid, Spain) in 2010. It belongs to the major clone detected during the outbreak and is resistant to all beta-lactams and to several other antibiotics. © 2012, American Society for Microbiology., Ministerio de Economía y Competitividad; IdiPAZ

Klebsiella pneumoniae KpQ3 is a multidrug-resistant isolate obtained from a blood culture of a patient in a burn unit in the Hospital Universitario La Paz (Madrid, Spain) in 2008. The genome contains multiple antibiotic resistance genes, including a plasmid-mediated DHA-1 cephalosporinase gene.

We describe clinical and microbiological features of infections caused by OXA-48-producing Klebsiella pneumoniae (O48KP) in the setting of a prolonged, hospital-wide outbreak detected in January 2011.Clinical, demographic and microbiological data of patients with growth of O48KP in clinical specimens were collected until December 2011. PCR was used to detect carbapenemase and β-lactamase genes. The genetic relationships were determined by automated repetitive-sequence-based PCR.Seventy-one patients with clinically guided cultures showing growth of O48KP were identified. Nine were considered to be colonizing rather than causing infection. The most frequent source of infection was the urinary tract (22/62), followed by surgical site infections (17/62). Blood cultures were positive in 23/62 patients. Many patients had significant comorbidity and prolonged hospital stays. In-hospital mortality among patients with O48KP infections was 43.5%. The MIC(90)s of ertapenem, imipenem and meropenem were32, 16 and 16 mg/L, respectively. No single antimicrobial was active against all the isolates. The antibiotics most active against O48KP were amikacin (97.2% susceptible), colistin (90.1%), tigecycline (73%) and fosfomycin (66.2%). Although eight clones were identified, a predominant clone caused 73.2% of the infections. Multilocus sequence typing (MLST) of the predominant clone gave sequence type (ST) 405 and bla(TEM-1), bla(SHV-76), bla(CTX-M-15) and bla(OXA-1) genes and the insertion sequence IS1999 of the Tn1999 transposon were associated with bla(OXA-48) in this clone.To our knowledge, this is the largest reported series of infections caused by O48KP in the setting of a single-centre outbreak and provides further input on the clinical relevance of infections caused by O48KP and the difficulties associated with its detection and control.

Surveillance studies have shown the emergence of infections with linezolid-resistant bacteria. The relationship between appropriate linezolid use and the spread of linezolid resistance among Gram-positive microorganisms in a single tertiary referral centre was evaluated. In an initial observational study, a prospective prescription-indication study was conducted on intensive care areas and haematology, neurosurgery, vascular surgery and nephrology wards during 2009. An intervention through follow-up feedback on audit results from May-June 2010 was then conducted. From July-December 2010, a second drug-use study of linezolid was conducted, with the same objectives and methodology. To assess the antimicrobial pressure of linezolid, an ecological study was conducted from 2006-2010 in the same hospital wards. Indications for linezolid in the initial study were considered suitable in 38.5% of cases, whilst in the second study the rate was 51.2% (33% increase). Linezolid consumption fell by 57% in the second half of 2010. A significant correlation was found between its inadequate use (DDD/1000 patient-days) and the incidence of linezolid-resistant strains/1000 patient-days (r=0.93; P=6.9e-024); 85% of the variability in the incidence of linezolid resistance was predicted by its inadequate use. Its partial correlations were significant for Enterococcus faecium (r=0.407; P=0.049), Staphylococcus epidermidis (r=0.874; P=2.3e-008) and Staphylococcus haemolyticus (r=0.406; P=0.049) but not Staphylococcus aureus (r=0.051; P=0.704). A relationship was found between appropriate linezolid use and the incidence of linezolid-resistant strains of E. faecium, S. epidermidis and S. haemolyticus.

Summary Objectives The objective of this study was to evaluate the reliability and accuracy of the combined use of MALDI-TOF MS bacterial identification and the Vitek-2 Compact antimicrobial susceptibility testing (AST) directly from positive blood cultures. Methods Direct identification by MALDI-TOF MS and AST were performed in parallel to the standard methods in all positively flagged blood cultures bottles during the study period. Results Three hundred and twenty four monomicrobial positive blood cultures were included in the present study, with 257 Gram-negative and 67 Gram-positive isolates. MALDI-TOF MS identification directly from blood bottles reported the correct identification for Enterobacteriaceae in 97.7%, non-fermentative Gram-negative bacilli 75.0%, Staphylococcus aureus 7 5.8%, coagulase negative staphylococci 63.3% and enterococci 63.3%. A total 6156 isolate/antimicrobial agent combinations were tested. Enterobacteriaceae group and non-fermentative Gram-negative Bacilli showed an agreement of 96.67% and 92.30%, respectively, for the Gram-positive cocci the overall agreement found was 97.84%. Conclusions We conclude that direct identification by MALDI-TOF and inoculation of Vitek-2 Compact AST with positive blood culture bottles yielded very good results and decreased time between initial inoculation of blood culture media and determination of the antibiotic susceptibility for Gram-negative rods and Gram-positive cocci causing bacteremia.