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Objectives: Cefiderocol is a novel siderophore-conjugated cephalosporin developed for the treatment of multidrug-resistant Gram-negative bacterial (GNB) infections. However, the current gold standard for cefiderocol susceptibility testing, broth microdilution (BMD) using iron-depleted cation-adjusted Mueller-Hinton broth, presents challenges for many microbiology laboratories. In this study, we evaluate the real-world performance of disc diffusion (DD) and a commercial BMD method (ComASP ® ) to test cefiderocol susceptibility in a series of isolates collected prospectively from severely ill patients in a multicentre study., Methods: The susceptibilities of 1472 isolates (632 Enterobacterales, 532 Pseudomonas aeruginosa , 84 Acinetobacter spp. and 224 Stenotrophomonas maltophilia ) collected in 60 Spanish hospitals were analysed following the EUCAST 2023 and 2024 criteria. We assessed the performance of DD (cefiderocol 30 μg disc, Liofilchem) and a commercial BMD method (ComASP ® Cefiderocol, Liofilchem)., Results: A total of 1408 and 1450 isolates were susceptible by DD and ComASP ® BMD, respectively. Overall, the agreement between both methods was 96.9%. Forty-four isolates were resistant by DD but susceptible by ComASP ® BMD, and two were susceptible by DD but resistant by ComASP ® BMD ( Acinetobacter baumannii isolates). Adoption of the updated 2024 EUCAST DD breakpoints and areas of technical uncertainty (ATUs) led to a decrease in susceptibility among Enterobacterales (95.3% versus 92.6%)., Conclusions: DD is a straightforward, rapid and accessible method for routine determination of cefiderocol susceptibility in real-world practice. ComASP ® BMD shows a high agreement with DD in susceptible isolates and may help to resolve DD interpretability concerns in isolates with susceptibility results within the ATU, but caution is warranted when testing resistant isolates., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Objectives: To analyse the susceptibility profile to cefepime, carbapenems and new β-lactam/β-lactamase inhibitor combinations in Enterobacter cloacae complex and Klebsiella aerogenes isolated from intra-abdominal, urinary, respiratory and bloodstream infections in the SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study in Spain., Methods: The susceptibilities of 759 isolates (473 E. cloacae complex and 286 K. aerogenes ) collected in 11 Spanish hospitals from 2016 to 2022 were analysed following the EUCAST 2023 criteria. Molecular characterization looking for β-lactamase genes was performed through PCR and DNA sequencing analysis., Results: E. cloacae complex showed resistance to third-generation cephalosporins in 25% of the cases, whereas K. aerogenes was resistant in 35%. Regarding cefepime, resistance in E. cloacae was higher (10%) than in K. aerogenes (2%). Carbapenems showed >85% activity in both microorganisms. Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam had good activity against these microorganisms (>95%). In contrast, the activity of ceftolozane/tazobactam was lower (80%). A high proportion of the isolates resistant to new β-lactam/β-lactamase inhibitor combinations carried a carbapenemase, mainly OXA-48-like and VIM-1., Conclusions: Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam show high activity against both E. cloacae complex and K. aerogenes isolates recovered in the SMART-Spain study. In contrast, differences have been found in the case of cefepime, showing more activity against K. aerogenes than E. cloacae complex. These results are useful for antimicrobial stewardship programmes and for the implementation of local and national guidelines., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Objectives: To assess the microbiological characteristics of Escherichia coli causing healthcare-associated bacteraemia of urinary origin (HCA-BUO) in Spain (ITUBRAS-2 project), with particular focus on ESBL producers and isolates belonging to ST131 high-risk clone (HiRC). Clinical characteristics and outcomes associated with ST131 infection were investigated., Methods: A total of 222 E. coli blood isolates were prospectively collected from patients with HCA-BUO from 12 tertiary-care hospitals in Spain (2017-19). Antimicrobial susceptibility and ESBL/carbapenemase production were determined. ST131 subtyping was performed. A subset of 115 isolates were selected for WGS to determine population structure, resistome and virulome. Clinical charts were reviewed., Results: ESBL-producing E. coli prevalence was 30.6% (68/222). ST131 represented 29.7% (66/222) of E. coli isolates and accounted for the majority of ESBL producers (46/68, 67.6%). The C2/H30-Rx subclone accounted for most ST131 isolates (44/66) and was associated with CTX-M-15 (37/44) and OXA-1 enzymes (27/44). Cluster C1-M27 was identified in 4/10 isolates belonging to subclade C1/H30-R1 and associated with CTX-M-27. Additionally, ST131 isolates showed a high content of other acquired resistance genes, and clade C/ST131 isolates carried characteristic QRDR mutations. They were categorized as uropathogenic E. coli and had higher aggregate virulence scores. ST131 infection was associated with more complex patients, prior use of cephalosporins and inadequate empirical treatment but was not associated with worse clinical outcomes., Conclusions: ST131 HiRC is the main driver of ESBL-producing E. coli causing HCA-BUO in Spain, mainly associated with the expansion of subclade CTX-M-15-C2/H30-Rx and the emergence of CTX-M-27-C1/H30-R1 (Cluster C1-M27)., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To evaluate the impact of time to results (TTR) on the outcome of patients with carbapenemase-producing Enterobacterales bloodstream infections (CPE-BSI)., Methods: Times-series study conducted from January 2014 to December 2021, selecting patients with first CPE-BSI episodes. Periods of intervention were defined according to implementation of diagnostic bundle tests in the microbiology laboratory: pre-intervention (January 2014-December 2017) and post-intervention (January 2018-December 2021). TTR was defined as time elapsed from positivity time of the blood culture bottles to physicians' notification of CPE-BSI episodes, and was evaluated in patients who received inappropriate empirical and switched to appropriate targeted treatment (switch group). Analysis of a composite unfavourable outcome (mortality at Day 30 and/or persistent and/or recurrent bacteraemia) was performed for the total episodes and in the switch group., Results: One hundred and nine episodes were analysed: 66 pre-intervention and 43 post-intervention. Compared with pre-intervention, patients in the post-intervention period were younger (68 versus 63 years, P = 0.04), had INCREMENT score > 7 (31.8% versus 53.5%, P = 0.02) and unfavourable outcome (37.9% versus 20.9%, P = 0.04). Proportion of TTR > 30 h was more frequent pre-intervention than post-intervention (61.7% versus 35.5%, P = 0.02). In multivariate analysis of the 109 episodes, source other than urinary or biliary (OR 2.76, 95% CI 1.11-6.86) was associated with unfavourable outcome, while targeted appropriate treatment trended to being protective (OR 0.17, 95% CI 0.03-1.00). Considering the switch group (n = 78), source other than urinary or biliary (OR 14.9, 95% CI 3.25-69.05) and TTR > 30 h (OR 4.72, 95% CI 1.29-17.22) were associated with unfavourable outcome., Conclusions: Decreased TTR in the post-intervention period was associated with the outcome in patients with CPE-BSI episodes., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To study the in vitro activity of imipenem/relebactam and comparators and the imipenem/relebactam resistance mechanisms in a Pseudomonas aeruginosa collection from Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17) surveillance studies., Methods: P. aeruginosa isolates (n = 474) were prospectively recovered from complicated urinary tract (cUTI), complicated intra-abdominal (cIAI) and lower respiratory tract (LRTI) infections in 11 Portuguese and 8 Spanish ICUs. MICs were determined (ISO broth microdilution). All imipenem/relebactam-resistant P. aeruginosa isolates (n = 30) and a subset of imipenem/relebactam-susceptible strains (n = 32) were characterized by WGS., Results: Imipenem/relebactam (93.7% susceptible), ceftazidime/avibactam (93.5% susceptible) and ceftolozane/tazobactam (93.2% susceptible) displayed comparable activity. The imipenem/relebactam resistance rate was 6.3% (Portugal 5.8%; Spain 8.9%). Relebactam restored imipenem susceptibility to 76.9% (103/134) of imipenem-resistant isolates, including MDR (82.1%; 32/39), XDR (68.8%; 53/77) and difficult-to-treat (DTR) isolates (67.2%; 45/67). Among sequenced strains, differences in population structure were detected depending on the country: clonal complex (CC)175 and CC309 in Spain and CC235, CC244, CC348 and CC253 in Portugal. Different carbapenemase gene distributions were also found: VIM-20 (n = 3), VIM-1 (n = 2), VIM-2 (n = 1) and VIM-36 (n = 1) in Spain and GES-13 (n = 13), VIM-2 (n = 3) and KPC-3 (n = 2) in Portugal. GES-13-CC235 (n = 13) and VIM type-CC175 (n = 5) associations were predominant in Portugal and Spain, respectively. Imipenem/relebactam showed activity against KPC-3 strains (2/2), but was inactive against all GES-13 producers and most of the VIM producers (8/10). Mutations in genes affecting porin inactivation, efflux pump overexpression and LPS modification might also be involved in imipenem/relebactam resistance., Conclusions: Microbiological results reinforce imipenem/relebactam as a potential option to treat cUTI, cIAI and LRTI caused by MDR/XDR P. aeruginosa isolates, except for GES-13 and VIM producers., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Although pneumococcal conjugate vaccines (PCVs) effectively prevent invasive pneumococcal disease (IPD), serotype replacement has occurred., Objectives: We studied the pangenome, antibiotic resistance mechanisms and presence of mobile elements in predominant non-PCV13 serotypes causing adult IPD after PCV13 vaccine introduction in Spain., Methods: We conducted a multicentre study comparing three periods in six Spanish hospitals and analysed through whole genome sequencing representative strains collected in the pre-PCV13, early-PCV13 and late-PCV13 periods., Results: Among 2197 cases of adult IPD identified, 110 pneumococci expressing non-PCV13 capsules were sequenced. Seven predominant serotypes accounted for 42.6% of IPD episodes in the late-PCV13 period: serotypes 8 (14.4%), 12F (7.5%), 9N (5.2%), 11A (4.1%), 22F (3.9%), 24F (3.9%) and 16F (3.6%). All predominant non-PCV13 serotypes were highly clonal, comprising one or two clonal complexes (CC). In general, CC538, CC4048, CC3016F, CC43322F and CC669N, related to predominant non-PCV13 serotypes, were antibiotic susceptible. CC15611A was associated with resistance to co-trimoxazole, penicillin and amoxicillin. CC23024F was non-susceptible to penicillin and resistant to erythromycin, clindamycin, and tetracycline. Six composite transposon structures of the Tn5252-family were found in CC23024F, CC98912F and CC3016F carrying different combinations of erm(B), tet(M), and cat. Pangenome analysis revealed differences in accessory genomes among the different CC, with most variety in CC3016F (23.9%) and more conservation in CC15611A (8.5%)., Conclusions: We identified highly clonal predominant serotypes responsible for IPD in adults. The detection of not only conjugative elements carrying resistance determinants but also clones previously associated with vaccine serotypes (CC15611A and CC23024F) highlights the importance of the accessory genome., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To determine the prevalence of penicillin susceptibility among MSSA causing bloodstream infections (BSIs) in 16 Spanish hospitals and to characterize the penicillin-susceptible MSSA (MSSA-PENS) isolates., Methods: A total of 1011 Staphylococcus aureus isolates were collected from blood cultures in 16 Spanish hospitals during 2018-19 (6-12 months) and their susceptibility to 18 antimicrobials was determined. The MSSA-PENS isolates were selected and examined by PCR to determine the presence of the blaZ gene, other resistance genes and the genes lukF/lukS-PV, eta, etb and tst. The immune evasion cluster (IEC) type was also analysed. All the MSSA-PENS isolates were submitted to S. aureus protein A (spa) typing and the clonal complexes (CCs) were assigned according to their spa type., Results: The prevalence of MSSA was 74.6% (754/1011) and 14.9% (151/1011) were MSSA-PENS-blaZnegative. MSSA-PENS-blaZnegative isolates (n = 151) were ascribed to 88 spa types and 11 CCs. The most frequent CCs were CC5 (35/151) and CC398 (25/151), with t002-CC5 and t571-CC398 being the most common lineages. Pan-susceptibility was identified in 117 of the 151 MSSA-PENS-blaZnegative isolates (77.5%). In the remaining isolates, erythromycin and clindamycin resistance was the most frequent resistance found, although tobramycin, ciprofloxacin, fusidic acid, mupirocin and/or tetracycline resistance was also detected. Thirty-eight MSSA-PENS-blaZnegative isolates were IEC negative and four isolates were Panton-Valentine leucocidin ('PVL') positive., Conclusions: A high penicillin susceptibility rate was detected among MSSA, opening therapeutic opportunities for BSIs. The emergence of new successful MSSA-PENS clones could be responsible for these data. The detection among MSSA-PENS-blaZnegative isolates of the clonal lineage CC398 or the absence of an IEC raises questions about their possible animal origin, requiring further analysis., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To analyse the epidemiology, the resistome and the virulome of ceftolozane/tazobactam-susceptible or -resistant Pseudomonas aeruginosa clinical isolates recovered from surveillance studies in Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17)., Methods: P. aeruginosa isolates were recovered from intra-abdominal, urinary tract and lower respiratory tract infections in ICU patients admitted to 11 Portuguese and 8 Spanish hospitals. MICs were determined (ISO-standard broth microdilution, EUCAST 2020 breakpoints). A subset of 28 ceftolozane/tazobactam-resistant P. aeruginosa isolates were analysed and compared with 28 ceftolozane/tazobactam-susceptible P. aeruginosa strains by WGS., Results: Clonal complex (CC) 235 (27%) and CC175 (18%) were the most frequent, followed by CC244 (13%), CC348 (9%), CC253 (5%) and CC309 (5%). Inter-hospital clonal dissemination was observed, limited to a geographical region (CC235, CC244, CC348 and CC253 in Portugal and CC175 and CC309 in Spain). Carbapenemases were detected in 25 isolates (45%): GES-13 (13/25); VIM type (10/25) [VIM-2 (4/10), VIM-20 (3/10), VIM-1 (2/10) and VIM-36 (1/10)]; and KPC-3 (2/25). GES-13-CC235 (13/15) and VIM type-CC175 (5/10) associations were observed. Interestingly, KPC-3 and VIM-36 producers showed ceftolozane/tazobactam-susceptible phenotypes. However, ceftolozane/tazobactam resistance was significantly associated with GES-13 and VIM-type carbapenemase production. Six non-carbapenemase producers also displayed ceftolozane/tazobactam resistance, three of them showing known ceftolozane/tazobactam resistance-associated mutations in the PBP3 gene, ftsI (R504C and F533L). Overall, an extensive virulome was identified in all P. aeruginosa isolates, particularly in carbapenemase-producing strains., Conclusions: GES-13-CC235 and VIM type-CC175 were the most frequent MDR/XDR P. aeruginosa clones causing infections in Portuguese and Spanish ICU patients, respectively. Ceftolozane/tazobactam resistance was mainly due to carbapenemase production, although mutations in PBP-encoding genes may additionally be involved., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To analyse by WGS the ceftolozane/tazobactam (C/T) resistance mechanisms in Escherichia coli and Klebsiella spp. isolates recovered from complicated intra-abdominal and urinary tract infections in patients from Spanish ICUs (SUPERIOR surveillance study, 2016-17)., Methods: The clonal relatedness, the resistome and the virulome of 45 E. coli and 43 Klebsiella spp. isolates with different C/T susceptibility profiles were characterized., Results: In E. coli , two (C/T susceptible) carbapenemase producers (VIM-2-CC23, OXA-48-ST38) were detected. The most relevant clone was ST131-B2-O25:H4-H30 (17/45), particularly the CTX-M-15-ST131-H30-Rx sublineage (15/17). ST131 strains were mainly C/T susceptible (15/17) and showed an extensive virulome. In non-ST131 strains (28/45), CTX-M enzymes [CTX-M-14 (8/24); CTX-M-15 (6/24); CTX-M-1 (3/24); CTX-M-32 (2/24)] were found in different clones. C/T resistance was detected in non-clonal E. coli isolates (13%, 6/45) with ESBL (4/6) and non-ESBL (2/6) genotypes. Among Klebsiella spp., Klebsiella pneumoniae (42/43) and Klebsiella michiganensis (1/43) species were identified; 42% (18/43) were carbapenemase producers and 58% showed a C/T resistance phenotype (25/43). OXA-48-ST11 (12/18), OXA-48-ST392 (2/18), OXA-48-ST15 (2/18), NDM-1-ST101 (1/18) and OXA-48+VIM-2-ST15 (1/18) isolates were found, all C/T resistant. Correlation between carbapenemase detection and resistance to C/T was demonstrated ( P  < 0.001). In non-carbapenemase-producing K. pneumoniae (25/43), C/T resistance (28%, 7/25) was detected in ESBL (3/7) and AmpC (2/7) producers. Overall, an extensive virulome was found and was correlated with carbapenemase carriage ( P  < 0.001) and C/T resistance ( P  < 0.05), particularly in OXA-48-ST11 strains ( P  < 0.05)., Conclusions: Prediction of antimicrobial susceptibility profiles using WGS is challenging. Carbapenemase-encoding genes are associated with C/T resistance in K. pneumoniae , but other resistance mechanisms might be additionally involved., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

In 2013-2014, an outbreak involving 14 patients infected by an extensively drug-resistant strain of Pseudomonas aeruginosa was detected in a hospital in Madrid, Spain. Our objective was to evaluate an alternative strategy for investigating the outbreak in depth by means of molecular and genomic approaches. Pulsed-field gel electrophoresis (PFGE) was applied as a first-line approach, followed by a more refined whole genome sequencing analysis. Single nucleotide polymorphisms identified by whole genome sequencing were used to design a specific polymerase chain reaction (PCR) for screening unsuspected cases infected by the outbreak strain. Whole genome sequencing alerted us to the existence of greater genetic diversity than was initially assumed, splitting the PFGE-associated outbreak isolates into 4 groups, 2 of which represented coincidental transmission unrelated to the outbreak. A multiplex allele-specific PCR targeting outbreak-specific single nucleotide polymorphisms was applied to 290 isolates, which allowed us to identify 25 additional cases related to the outbreak during 2011-2017. Whole genome sequencing coupled with an outbreak-strain-specific PCR enabled us to markedly redefine the initial picture of the outbreak by 1) ruling out initially suspected cases, 2) defining likely independent coincidental transmission events, 3) predating the starting point of the outbreak, 4) capturing new unsuspected cases, and 5) revealing that the outbreak was still active., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Tetracycline resistance (TetR) is a marker of livestock-associated MRSA of lineage CC398., Objectives: To determine the MRSA CC398 prevalence among TetR-MRSA recovered in Spanish hospitals located in regions with different pig-farming densities, and the influence of pig density as a key risk factor for its acquisition., Methods: TetR-MRSA isolates (n = 232) recovered from clinical and epidemiological samples during January-June 2016 in 20 hospitals in 13 regions with different pig-farming densities were analysed. MRSA CC398 identification, detection of spa types, methicillin resistance genes and immune evasion cluster (IEC) genes were performed by PCR/sequencing. Statistical analyses were performed to establish the relationships between MRSA CC398 prevalence and pig density., Results: The global MRSA prevalence was 29.7% (6.9% TetR-MRSA/MRSA), with 137 CC398 isolates recovered, representing 4.1% of total MRSA and 59.1% of TetR-MRSA. Among MRSA CC398, 16 different spa types were recorded (t011: 72.3%), and all but two strains were IEC negative. Higher pig-density regions were associated with significant MRSA CC398 increases in hospitals located in adjacent regions (P < 0.001). Linear regression models explained the relationships between MRSA CC398 and pig density (P < 0.001), with an increase of 6.6 MRSA CC398 cases per 100 MRSA per increase of 100 pigs/km2 in a region., Conclusions: High pig density leads to a significant increase in MRSA CC398 in hospitals in Spain, and its combination with a high human population could help its dissemination. In Spain, the prevalence of the zoonotic CC398 lineage is closely related to pig-farming density; therefore, specific tools could be implemented in order to detect its dissemination., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: The objective of this study was to assess the prevalence and molecular epidemiology of ESBL-producing Escherichia coli causing healthcare-associated (HCA) and community-associated (CA) bacteraemia of urinary origin (BUO) in Spain., Methods: An observational cohort study was conducted at eight hospitals from different Spanish geographical areas (2010-11). BUO episodes (n = 425) were classified as HCA (n = 215) and CA (n = 210), and one blood isolate per episode was collected. Susceptibility testing was performed, ESBLs were screened by double-disc diffusion test and ESBL and OXA-1 genes were characterized (PCR and sequencing). Population structure (phylogenetic groups, XbaI-PFGE and MLST) and ST131 subtyping (PCR) were determined. Virulence genes were detected by PCR and virulence score, profiles and extraintestinal pathogenic E. coli (ExPEC) status calculated., Results: ESBL-producing E. coli prevalence was 9.2% (39/425). ESBL-producing E. coli episodes were significantly associated with HCA-BUO episodes [14% (30/215) versus 4.3% (9/210); P = 0.001]. The highest non-susceptibility proportions corresponded to ciprofloxacin (97.4%), amoxicillin/clavulanate (74.4%), co-trimoxazole (69.2%) and tobramycin (61.5%). Of the 39 ESBL-producing E. coli isolates, 34 produced CTX-M enzymes (21 CTX-M-15, 11 CTX-M-14 and 2 CTX-M-1). Fifteen STs were identified, the B2-ST131 clone being the most prevalent (54%; 21/39). All ST131 isolates were ExPEC and had the highest virulence scores, but they showed less diversity in virulence profiles than other STs. The H30Rx subclone accounted for most ST131 isolates (20/21), co-produced CTX-M-15 (20/20) and OXA-1 (19/20) enzymes and was associated with HCA episodes (16/20)., Conclusions: The CTX-M-15-ST131-H30Rx subclone is a relevant MDR pathogen causing BUO, mainly HCA episodes. The dominance of this subclone with comparatively less diversity of virulence profiles reflects the spread of a successful and MDR ESBL ST131 lineage in Spain., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: In Spain, despite the high rates of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA), the incidence of community-associated (CA) MRSA seems to be low on the basis of a small number of studies. We analysed the evolution of CA-MRSA in Spain from 2004 to 2012, and identified the clonal lineages and population structure., Methods: The study included 8326 MRSA strains. Susceptibility to 18 antimicrobials was determined. Isolates were tested for the presence of mecA, Panton-Valentine leucocidin (PVL) and the arginine catabolic mobile element (ACME) by PCR, and typed by staphylococcal cassette chromosome mec, PFGE, spa, multilocus sequence typing and agr., Results: Among the 8326 isolates, 246 (2.9%) were CA-MRSA. We identified genotypically 226 PVL-positive CA-MRSA isolates (88% agr type I, 10.2% agr type III and 1.8% agr type II) and 20 PVL-negative CA-MRSA isolates (all agr type I) from children and adults (82.1% from wounds) from 13 different geographical areas. A significant increase in the rates of CA-MRSA was observed when comparing 2004-07 (0.43%) with 2008-12 (5.44%). Resistance rates were as follows: only β-lactams, 84.5%; erythromycin, 12.8%; tetracycline, 8.8%; clindamycin, 4.9%; ciprofloxacin, 3.1%; fusidic acid, 2.0%; others, 0.4%; and multiresistant, 6.2% (six isolates USA300). The strains belonged to the PVL-positive clones ST8-IVc (69.9%), ST8-IVa-ACME-positive (USA300, 8.9%), ST8-IVa-ACME-negative (0.8%), ST30-IVc (4.5%), ST80-IVc (2.0%), ST5-IVc (1.2%) and others (ST59, ST72, ST88, ST642, ST1472 and ST1829; 4.5%) and to the PVL-negative ST398-V (8.1%)., Conclusions: We confirm an increase in CA-MRSA in Spain, the predominance of the ST8-IVc clone, the emergence of the USA300 clone, a high genetic diversity among PVL-positive CA-MRSA isolates and the recent emergence of the pig-associated ST398-V clone., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: The epidemiology of invasive Haemophilus influenzae has changed in recent years. β-Lactamase-negative ampicillin-resistant (BLNAR) invasive isolates have recently been described in Europe but their clinical significance is unclear. Our main goal was to determine whether invasive H. influenzae remains susceptible to β-lactam antibiotics indicated in the treatment of invasive infections., Methods: The antibiotic susceptibility of 307 invasive H. influenzae isolates to seven β-lactam antibiotics was determined by microdilution and interpreted by EUCAST and CLSI breakpoints. We also identified the bla genes, the amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3), the molecular epidemiology of invasive BLNAR isolates by PFGE and MLST, and the time-kill curves of two isolates with PBP3 mutations conferring reduced susceptibility to aminopenicillins and cephalosporins., Results: Of the invasive isolates, 86.6% were non-typeable and 62% were isolated from adults. Decreased susceptibility to β-lactams was due to the BLNAR genotype (gBLNAR; 19.2%) and to β-lactamase production (16.9%). Susceptibility rates to amoxicillin/clavulanic acid, cefotaxime, cefixime and imipenem were greater than 98%. Of 18 gBLNAR non-typeable isolates studied by MLST, 15 different STs were obtained. Amoxicillin and cefotaxime were bactericidal after 2 and 4 h of incubation, respectively., Conclusions: Invasive H. influenzae disease was mainly due to non-typeable isolates infecting adults, and the most common mechanism of β-lactam resistance was mutations in the transpeptidase domain of PBP3. The gBLNAR non-typeable isolates were genetically diverse. The majority of invasive H. influenzae remained susceptible to third-generation cephalosporins; amoxicillin and cefotaxime were bactericidal in two gBLNAR isolates.

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) strains carrying the mecC gene have been reported from humans and animals from several European countries, but never from Spain. We describe the first isolates of mecC-positive MRSA of human origin collected in Spain and report a fatal case of bacteraemia., Methods: Isolates were tested for phenotypic resistance using cefoxitin, tested for the mecA/mecC genes and toxin genes by PCR, and typed by staphylococcal cassette chromosome mec (SCCmec), PFGE, spa, multilocus sequence typing and agr., Results: During 2008-13 five MRSA isolates showing resistance to cefoxitin and carrying the mecC gene were recovered at one hospital in Spain. In a review of 5505 S. aureus strains received at the Spanish National Reference Centre for Staphylococci from the same period, we found two additional mecC-positive isolates. The isolates were recovered from blood (two), wounds (two), joint fluid (one), urine (one) and a nasal swab (one). All MRSA were mecA negative, presented SCCmecXI, belonged to agr group III and to clonal complex 130, and were negative for the production of the toxin genes tst1, eta, etb, etd and Panton-Valentine leucocidin. Six isolates belonged to spa type t843 (ST130 and ST1945, where ST stands for sequence type) and one to spa type t6220 (ST1945). One patient with mecC-positive MRSA sepsis died in the emergency department., Conclusions: We confirm the presence of MRSA carrying the mecC gene in Spain, the ability of this livestock-associated MRSA to cause severe infections in humans and the need to perform culture-based susceptibility testing methods in order to detect these emerging strains.

Objectives: To determine the mechanisms of resistance to β-lactam antibiotics in clinical isolates of Haemophilus parainfluenzae., Methods: Twenty clinical isolates of H. parainfluenzae with decreased susceptibility to aminopenicillins were examined and compared with a control group of 20 fully susceptible isolates. In this collection, the presence of amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3), β-lactamase production and the surrounding genetic regions of blaTEM genes in selected isolates were analysed., Results: Of the 20 non-susceptible isolates, 8 produced TEM β-lactamase (gBLPAR), 7 had mutations in the transpeptidase domain of the ftsI gene related to decreased susceptibility to β-lactams (gBLNAR) and 5 had both resistance mechanisms (gBLPACR). No resistance mechanisms were identified in the susceptible control group (gBLNAS). gBLNAR isolates had MIC90 values 4- to 16-fold higher than gBLNAS isolates for ampicillin, amoxicillin/clavulanic acid, cefuroxime, cefotaxime and cefixime, and the most common PBP3 mutation was Asn526Ser. The additional Ser385Thr substitution (III-like group) may confer decreased susceptibility to cefotaxime, cefixime and aztreonam, as in Haemophilus influenzae. In two β-lactamase-positive isolates without PBP3 mutations, the inhibitor-resistant TEM (IRT) β-lactamases TEM-34 and the novel TEM-182 were detected and carried by a TnA transposon of the Tn2 type; both isolates had an amoxicillin/clavulanic acid MIC of ≥8 mg/L. The TnA transposons of two β-lactamase-positive isolates (TEM-1 and TEM-182) were inserted between the tfc20 and tfc21 genes, typically associated with integrative and conjugative elements in Haemophilus spp.; the TEM-34 IRT β-lactamase was harboured in a ∼5.5 kb plasmid., Conclusions: Clinical isolates of H. parainfluenzae express a variety of aminopenicillin resistance mechanisms, either alone or in combination, including PBP3 modifications, blaTEM-1 and IRT β-lactamase production.

Background: Recent studies have reported a greater probability of vancomycin treatment failure in methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections caused by strains with a vancomycin MIC ≥ 1.5 mg/L. However, previous reports included patients treated without adjustments based on vancomycin serum levels and with different methods to evaluate MICs, which may render different results., Methods: Over a 5 year period (2005-09), vancomycin MICs were determined for 361 MRSA isolates recovered from 309 patients with bloodstream infection using microdilution and the Etest simultaneously. The relationship between the vancomycin MICs determined by each method was assessed. To assess the outcome of patients treated with vancomycin, 104 patients for whom serum vancomycin levels had been determined were selected., Results: The percentage of MRSA strains with MIC values ≥ 1.5 mg/L according to the Etest was 66.5% compared with only 3.6% according to microdilution. No correlation between mortality and any MIC value obtained with either method was observed, independently of the vancomycin serum levels measured., Conclusions: There is a poor correlation between vancomycin MIC values obtained by microdilution and by Etest. No association between mortality rate and any MIC value was observed, not even in patients with suboptimal vancomycin trough serum levels. These data do not support replacing or complementing the standard microdilution test with the Etest for determination of MICs of vancomycin in microbiology laboratories.

Background: Ventilator-associated pneumonia is the most frequently observed nosocomial infection in intensive care units, and it is associated with high morbidity and mortality. Early microbiological diagnosis and the initial administration of appropriate antimicrobial therapy are associated with decreased mortality and potentially reduced costs. Our study evaluates the clinical and financial impact of performing rapid antimicrobial susceptibility tests directly on samples obtained from the lower respiratory tract., Methods: A prospective, randomized study was performed over a 2-year period. Patients who had a lower respiratory tract infection that was acquired during mechanical ventilation and for whom samples obtained from the respiratory tract were sent for culture were randomized to 1 of 2 groups. Samples were cultured for the control group, and results were reported using standard procedures. Samples were also cultured for the test subject group using standard procedures, but in addition, a rapid antibiogram was immediately performed by placing E-test antibiotic strips (AB Biodisk) directly on respiratory tract samples. Patients in the E-test group received a preliminary laboratory report when it became available. The 2 patient groups were compared according to the following variables: type and severity of underlying conditions, total days of antimicrobial use, number of defined daily doses, cost of acquisition of the antimicrobial agent per episode, days of fever, days receiving mechanical ventilation, days in the intensive care unit, incidence of Clostridium difficile-associated diarrhea, and mortality., Results: Reporting a rapid E-test was associated with fewer days of fever, fewer days of antibiotic administration until resolution of the episode of ventilator-associated pneumonia, decreased antibiotic consumption, less C. difficile-associated diarrhea, lower costs of antimicrobial agents, and fewer days receiving mechanical ventilation., Conclusions: A rapid E-test of respiratory tract samples improves antimicrobial use in cases of ventilator-associated pneumonia.

Objectives: Little is known about the antibiotic susceptibility and clinical significance of non-type b capsulated Haemophilus influenzae. We studied the antibiotic resistance patterns, plasmid carriage and clinical features of H. influenzae type f infections in Spain during 1996-2002., Patients and Methods: Forty-nine H. influenzae type f recovered from Spanish hospitals were analysed at a central laboratory where full microbiological and molecular epidemiological studies were carried out. Antimicrobial susceptibility testing was performed in accordance with NCCLS guidelines., Results: Twelve strains (24.5%) were resistant to ampicillin and 22 (44.9%) to co-trimoxazole. Decreased susceptibility to clarithromycin, tetracycline, chloramphenicol and rifampicin was found in 16.3%, 12.2%, 14.3% and 2% of strains, respectively. Multidrug resistance was present in nine (18.4%) of the 49 isolates. The most prevalent resistance phenotype was ampicillin/tetracycline/co-trimoxazole/chloramphenicol, which was detected in five isolates. All six strains that were simultaneously resistant to ampicillin, tetracycline and chloramphenicol had conjugative plasmids. The main clinical diagnoses were pneumonia (32.6%), sepsis (18.4%) and meningitis (16.3%). Thirty-two patients (65.3%) had previous underlying predisposing conditions, principally respiratory diseases (20.4%). Twenty-one patients (42.8%) had impaired immunity. Thirty-seven (75.5%) patients were >14 years old, 12 (24.5%) were < or =14 years, and seven were < or =5 years. Most isolates were clonally related., Conclusions: A high prevalence of antibiotic resistance, including multiresistance, was detected in Spanish H. influenzae type f isolates. Carriage of large conjugative plasmids was strongly associated with antibiotic resistance. H. influenzae type f is mainly an opportunistic pathogen, although it may cause primary severe infections, such as meningitis in children.

Surveillance after introduction of Haemophilus influenzae serotype b vaccination in Spain identified 26 H. influenzae serotype e (HiE) isolates. Of these, 16 (61.5%) were recovered from patients aged >16 years and 10 (38.5%) from children <16 years of age. HiE caused respiratory infections in 14 patients (9 with pneumonia), conjunctivitis in 4, vaginitis in 2, abscess in 2, and cellulitis, peritoneal infection, sepsis and meningitis in 1 patient each. HiE was strongly clonal and highly resistant to ampicillin and cotrimoxazole, and the incidence of HiE infection did not increase over time.

The in vitro activity of gemifloxacin (SB-265805) was compared with that of other fluoroquinolones against 302 clinical isolates of Streptococcus pneumoniae, 300 clinical isolates of Haemophilus influenzae and 28 clinical isolates of Moraxella catarrhalis, including multiply resistant strains. Gemifloxacin at 0.12 mg/L inhibited all microorganisms tested. MIC(90) values of gemifloxacin, trovafloxacin, grepafloxacin and levofloxacin against all (630) isolates tested were 0.03, 0.12, 0.12 and 1 mg/L, respectively. MIC(90) values of the same fluoroquinolones against S. pneumoniae were 0.06, 0.25, 0.12 and 1 mg/L, respectively.

The in vitro activity of the oxazolidinone linezolid was compared with the activities of vancomycin and teicoplanin against 450 Gram-positive clinical isolates, including a variety of multiply resistant strains. Linezolid inhibited all microorganisms tested at < or = 4 mg/L, including methicillin- and teicoplanin-resistant staphylococci, glycopeptide-resistant enterococci, penicillin- and multiply resistant pneumococci and viridans streptococci, and erythromycin-resistant beta-haemolytic streptococci. The MIC(90) of linezolid for all isolates was 2 mg/L.

We compare a rapid dilution method for the determination of antiviral susceptibility of herpes simplex virus (HSV) with the plaque reduction assay. A total of 84 HSV clinical isolates were studied by both methods to detect in-vitro resistance to acyclovir and foscarnet. The rapid method showed for the detection of HSV isolates resistant to acyclovir and foscarnet, a sensitivity of 96. 8% and 100% and specificity of 100% and 100%, respectively. This method provides an easy and accurate screening procedure for the susceptibility testing of HSV to antiviral agents.

The in-vitro activity of sparfloxacin and eight other antimicrobial agents against 64 non-tuberculous mycobacteria (non-MAI) (40 rapidly growing and 24 slowly growing) was compared with those of ciprofloxacin, ofloxacin, amikacin, tobramycin, cefoxitin, imipenem, clarithromycin and doxycycline. Sparfloxacin presented the same activity as ciprofloxacin, being highly active against rapidly growing mycobacteria and showing good in-vitro activity against slowly growing mycobacteria. Amikacin was very active against rapidly growing mycobacteria and clarithromycin presented good activity against all mycobacteria tested. These results suggest that sparfloxacin is a valid agent to be considered in the treatment of Mycobacterium spp. infections.

During investigation of an outbreak of vancomycin resistant Enterococcus faecium in a paediatric hospital, an isolate of Enterococcus durans resistant to vancomycin, teicoplanin, ampicillin and highly resistant to gentamicin and streptomycin was found in the stools of a patient also colonized with a strain of E. faecium with the same resistance pattern. Minimal inhibitory concentrations of vancomycin and teicoplanin were 512 and 64 mg/mL, respectively. Resistance to vancomycin as well as high-level resistance to gentamicin was transferable to an E. faecium recipient strain. Both multiresistant E. faecium and E. durans isolates as well as the transconjugant presented only one plasmid. The vanA gene was detected and localized to the high molecular weight plasmid by DNA hybridization with a vanA gene probe. Growth in vancomycin resulted in induction of an approximately 40 kDa protein visible in membrane preparations from these cells. Genetic linkage between vancomycin and gentamicin resistance genes in the same plasmid is suggested.

Leishmania (Leishmania) infantum is the causative agent of both the cutaneous and visceral forms of leishmaniasis in southwest Europe; the dog is the main reservoir. In order to identify the L. (L.) infantum zymodemes present in Spain, a total number of 85 Leishmania stocks isolated from dogs (31), HIV-positive patients (46) with visceral or cutaneous leishmaniasis, a patient with visceral leishmaniasis complicating renal transplantation (1) and immunocompetent patients (7) with visceral or cutaneous leishmaniasis, have been characterized by isoenzyme typing. All canine stocks were MON-1, which is the most widespread zymodeme in the Mediterranean area. In immunocompetent patients three zymodemes were found: MON-1 (2), MON-24 (2) and MON-34 (3). Nine different zymodemes were obtained in stocks from HIV co-infected patients, indicating a higher variability of L. (L.) infantum amongst them: MON-1 (in 21 stocks), MON-24 (7), MON-28 (1), MON-29 (3), MON-33 (7), MON-34 (1) and MON-183 (4). Two new zymodemes, MON-198 (1) and MON-199 (1), were described among HIV patients from Spain. The stock from the renal transplanted patient was MON-1. The exclusive presence of certain zymodemes in immunocompromised patients and their absence in typical cases of cutaneous and visceral leishmaniasis and in infected dogs suggests two possibilities: (i) an anthroponotic pattern of leishmaniasis where intravenous drug user-infected patients act as potential reservoir for these new zymodemes. In the latter, syringes could act as the vehicles for infected monocytes; (ii) the cellular immune system could select virulent from non-virulent zymodemes in immunocompetent visceral leishmaniasis patients.

To evaluate the incidence and the significance of resistance to erythromycin among clinical isolates of Streptococcus pneumoniae, we identified and prospectively followed all hospitalized patients in a 27-month period who had the organism isolated from any clinical sample. Patients who had an infection caused by pneumococci resistant to erythromycin (minimum inhibitory concentration, > 1 microgram/mL) were compared to those with infections caused by erythromycin-susceptible organisms. The incidence of erythromycin resistance among pneumococci doubled over the study period (from 7.6% in 1988 to 15.2% in 1992). Most strains (94%) showed resistance to multiple antibiotics, including other macrolides. By multivariate analysis, an age of < 5 years and nosocomial acquisition of the infection were independent risk factors for erythromycin resistance. Among patients with pneumococcal pneumonia caused by erythromycin-resistant organisms, 9 patients treated with third-generation cephalosporins were cured, while therapy with erythromycin failed for 2 of the 6 patients to whom it was administered. The rapid and significant increase of erythromycin resistance among clinical isolates of S. pneumoniae points to the need for routine surveillance of pneumococcal resistance.

The authors compared a three-minute fluorescence method (MUCAP test; Biolife Italiana, Milan, Italy) with standard microbiologic procedures for the presumptive identification of Salmonella from stool cultures on three solid culture media. From 976 suspect colonies, 176 proved to be Salmonella by the traditional methods. They all were detected by the MUCAP test (sensitivity, 100%). There were 65 false-positive results, corresponding mostly to Pseudomonas (specificity, 91.8%). Neither the culture medium used nor the production of H2S affected the accuracy of the test. The results from this study showed that the MUCAP test is a rapid, simple, and sensitive method for the presumptive identification of Salmonella species.

We performed a prospective study of all infections with Streptococcus pneumoniae documented during a 22-month period at our hospital. A total of 163 clinically significant strains of S. pneumoniae were isolated from 139 patients whose ages ranged from 8 days to 91 years (mean +/- SD, 42.6 years +/- 26.8 years). Twenty percent of the patients had cancer, and 18% were infected with the human immunodeficiency virus. Pneumococcal infection was nosocomially acquired in one-fourth of cases. One-third of patients had nonpneumonic disease. A wide range of serotypes were isolated, and 42.5% of all strains were nonsusceptible--i.e., showed either intermediate or high-level resistance--to penicillin. The rates of resistance to chloramphenicol, erythromycin, and tetracycline were 23%, 10.8%, and 48.2%, respectively. Twenty-two percent of the infected patients died, with a 15.8% mortality directly attributable to pneumococcal infection. Factors associated with infection by strains of S. pneumoniae not susceptible to penicillin included an age of less than or equal to 10 years, immunosuppression, the presence of a rapidly fatal underlying disease, previous antimicrobial therapy, and infection by serotypes 14 and 23. All clinically significant isolates of S. pneumoniae should be submitted for antimicrobial susceptibility studies, and, whenever a high prevalence of resistance to penicillin and macrolides is detected, the use of these well-established empirical therapeutic regimens should be reconsidered.

The distribution of plasmids as a function of aerobactin production, antibiotic resistance and antimicrobial agents production was studied in 139 Escherichia coli strains obtained from clinical sources. Ninety eight per cent of the strains analyzed presented plasmids with a median value of 2.97 plasmids per cell. Differences in the number of plasmids were observed for aerobactin production (3.52 for aerobactin producing strains, 2.56 (for non-producing ones) and antibiotic resistance (3.19 for antibiotic resistant strains and 2.58 for the sensitive ones). But this was not the case for antibacterial agent production (2.96 for the producing strains, 2.98 for the non-producing ones. Ecological implications of these results are discussed.