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Journal of Antimicrobial Chemotherapy | 2010

Parallel increase in community use of fosfomycin and resistance to fosfomycin in extended-spectrum β-lactamase (ESBL)-producing Escherichia coli

Jesús Oteo; Verónica Bautista; Noelia Lara; Oscar Cuevas; Margarita Arroyo; Sara Fernández; Edurne Lázaro; Francisco J. de Abajo; José Campos

OBJECTIVES To document fosfomycin susceptibility of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC), analyse trends in fosfomycin use and investigate fosfomycin resistance in ESBL-EC isolated from urinary tract infections (UTIs). METHODS Twenty-seven Spanish hospitals participating in the European Antimicrobial Resistance Surveillance Network were requested to collect up to 10 sequential ESBL-EC for centralized susceptibility testing and typing. EUCAST guidelines were followed for antibiotic susceptibility testing, and bla(ESBL) type, phylogroups and O25b serotype were determined by PCR and sequencing. In addition, the trend in fosfomycin resistance among ESBL-EC causing UTIs was determined in 9 of the 27 hospitals. Total fosfomycin use for ambulatory care was established by WHO-recommended methods. RESULTS A total of 231 ESBL-EC (42.4% CTX-M-15, 34.2% SHV-12 and 23.4% CTX-M-14) were collected. The overall rate of fosfomycin resistance was 9.1%, but varied according to ESBL type (5.6% of CTX-M-14 isolates, 5.1% of SHV-12 and 15.3% of CTX-M-15). Of 67 O25b/B2 isolates, 11 (16.4%) were fosfomycin resistant. Predictors of infection with fosfomycin-resistant ESBL-EC were O25b/phylogroup B2 isolates, female gender and nursing home residence. Among 114 197 UTIs caused by E. coli 4740 (4.2%) were due to ESBL-EC. Fosfomycin resistance increased in these isolates from 4.4% (2005) to 11.4% (2009). The use of fosfomycin grew from 0.05 defined daily doses per 1000 inhabitants per day (1997) to 0.22 (2008), a 340% increase. CONCLUSIONS Key factors related to increased fosfomycin resistance in ESBL-EC causing UTIs could be the rapid growth in community use of fosfomycin, the widespread distribution of the 025b/B2 E. coli clone and the existence of a susceptible population comprising women residing in nursing home facilities.


Journal of Antimicrobial Chemotherapy | 2014

Frequent carriage of resistance mechanisms to β-lactams and biofilm formation in Haemophilus influenzae causing treatment failure and recurrent otitis media in young children

Silvia García-Cobos; Miriam Moscoso; Félix Pumarola; Margarita Arroyo; Noelia Lara; María Pérez-Vázquez; Belén Aracil; Jesús Oteo; Ernesto García; José Campos

OBJECTIVES Non-typeable Haemophilus influenzae are a major cause of acute otitis media (AOM), including chronic and recurrent otitis in young children. The objective of this study was to determine whether non-typeable H. influenzae isolates causing these infections produce biofilms and carry resistance mechanisms to β-lactams. METHODS A collection of 48 H. influenzae isolates was obtained by tympanocentesis or from otorrhoea samples from individual patients <3 years of age and diagnosed with recurrent or treatment failure AOM. Each isolate was surveyed for the presence of blaTEM genes, amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3) and biofilm formation in microtitre plates. RESULTS In 43 of the 48 isolates (89.6%), at least one of the three tested conditions was identified: biofilm formation (83.3%) and resistance mechanisms to β-lactams (33.3%), modifications in the transpeptidase domain of PBP3 being the most prevalent (22.9%), followed by β-lactamase production (10.4%). Additionally, 13 (27.1%) isolates had two or more of these three traits. In relation to biofilm formation, those isolates with an amoxicillin MIC ≤ 0.5 mg/L had higher optical density values than isolates with an amoxicillin MIC ≥ 1 mg/L (Mann-Whitney U-test, P=0.048). CONCLUSIONS These findings suggest that the successful treatment of non-typeable H. influenzae causing chronic and recurrent AOM in young children may be compromised by the high biofilm-forming capacity of the isolates and the presence of β-lactam resistance mechanisms, particularly PBP3 mutations.


Journal of Antimicrobial Chemotherapy | 2013

Novel mechanisms of resistance to β-lactam antibiotics in Haemophilus parainfluenzae: β-lactamase-negative ampicillin resistance and inhibitor-resistant TEM β-lactamases

Silvia García-Cobos; Margarita Arroyo; José Campos; María Pérez-Vázquez; Belén Aracil; Emilia Cercenado; Beatriz Orden; Noelia Lara; Jesús Oteo

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.


Journal of Antimicrobial Chemotherapy | 2016

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

Adriana Ortega; David Sáez; Verónica Bautista; Sara Fernández-Romero; Noelia Lara; Belén Aracil; María Pérez-Vázquez; José Campos; Jesús Oteo; José Esteban Aznar; Carolina Campelo; Isabel Sánchez-Romero; Rocío Martínez; Beatriz Orden; Alejandro González; Sonia Solís; Luisa García-Picazo; Emilia Cercenado; Almudena Alhambra; Santiago Salso; Carmen Elena Gómez; Juan Ignacio Alós; Mª Dolores Miguel-Martínez; Teresa Alarcón; Laura Llorca; Mª Teresa Ledo; Firdaous El Knaichi; Gloria Trujillo; Montserrat Morta; Belén Hernández

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


Journal of Antimicrobial Chemotherapy | 2014

Isolates of β-lactamase-negative ampicillin-resistant Haemophilus influenzae causing invasive infections in Spain remain susceptible to cefotaxime and imipenem

Silvia García-Cobos; Margarita Arroyo; María Pérez-Vázquez; Belén Aracil; Noelia Lara; Jesús Oteo; Emilia Cercenado; José Campos

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.


Antimicrobial Agents and Chemotherapy | 2017

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

Andrés Esteban-Cantos; Belén Aracil; Verónica Bautista; Adriana Ortega; Noelia Lara; David Sáez; Sara Fernández-Romero; María Pérez-Vázquez; Ferran Navarro; Hajo Grundmann; José Campos; Jesús Oteo

ABSTRACT We studied in parallel the population structure of 90 carbapenemase-producing and 88 carbapenemase-susceptible Klebsiella pneumoniae isolates collected in 20 Spanish hospitals, in the context of the EuSCAPE project. Fourteen and 50 multilocus sequence types (MLSTs) were detected among the carbapenemase-producing and carbapenem-susceptible isolates, respectively. ST11 and ST15 clones were more frequent in the carbapenemase-producing group than in the carbapenemase-susceptible group (P < 0.0001). Among the members of the carbapenem-suceptible group, the cefotaxime-resistant population showed population parameters that differed between the populations of the wild-type strains and the carbapenemase producers.


Journal of Antimicrobial Chemotherapy | 2017

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

David M Arana; Adriana Ortega; Eva González-Barberá; Noelia Lara; Verónica Bautista; Dolores Gómez-Ruíz; David Sáez; Sara Fernández-Romero; Belén Aracil; María Pérez-Vázquez; José Campos; Jesús Oteo

Objectives There is little information about carbapenemase-producing (CP) Citrobacter spp. We studied the molecular epidemiology and microbiological features of CP Citrobacter spp. isolates collected in Spain (2013-15). Methods In total, 119 isolates suspected of being CP by the EUCAST screening cut-off values were analysed. Carbapenemases and ESBLs were characterized using PCR and sequencing. The genetic relationship among Citrobacter freundii isolates was studied by PFGE. Results Of the 119 isolates, 63 (52.9%) produced carbapenemases, of which 37 (58.7%) produced VIM-1, 20 (31.7%) produced OXA-48, 12 (19%) produced KPC-2, 2 (3.2%) produced NDM-1 and 1 (1.6%) produced VIM-2; 9 C. freundii isolates co-produced VIM-1 plus OXA-48. Fourteen isolates (22.2%) also carried ESBLs: 8 CTX-M-9 plus SHV-12, 2 CTX-M-9, 2 SHV-12 and 2 CTX-M-15. Fifty-seven isolates (90.5%) were C. freundii, 4 (6.3%) were Citrobacter koseri, 1 (1.6%) was Citrobacter amalonaticus and 1 (1.6%) was Citrobacter braakii. By EUCAST breakpoints, eight (12.7%) of the CP isolates were susceptible to the four carbapenems tested. In the 53 CP C. freundii analysed by PFGE, a total of 44 different band patterns were observed. Four PFGE clusters were identified: cluster 1 included eight isolates co-producing VIM-1 and OXA-48; blaVIM-1 was carried in a class 1 integron (intI-blaVIM-1-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1) and blaOXA-48 was carried in a Tn1999.2 transposon. Conclusions We observed the clonal and polyclonal spread of CP Citrobacter spp. across several Spanish geographical areas. Four species of Citrobacter spp. produced up to five carbapenemase types, including co-production of VIM-1 plus OXA-48. Some CP Citrobacter spp. isolates were susceptible to the four carbapenems tested, a finding with potential clinical implications.


Antimicrobial Agents and Chemotherapy | 2005

Specific antibodies, levofloxacin, and modulation of capsule-associated virulence in Streptococcus pneumoniae.

David Tarragó; Noelia Lara; Asunción Fenoll; Julio Casal; María-José Giménez; Lorenzo Aguilar; David Sevillano

Amoxicillin subinhibitory concentrations produced 100% survival in passively immunized mice infected with non-amoxicillin-susceptible, poorly or highly encapsulated Streptococcus pneumoniae strains (2, 6), with negligible values of time that serum levels exceeded the MIC. We explored this phenomenon and its modulation by capsular production with levofloxacin against the same serotype 6B Streptococcus pneumoniae strain (6) (MIC of levofloxacin = 32 μg/ml) with two types of infecting inocula. (i) For the poorly encapsulated (PE) phenotype, the microorganism was grown in Todd-Hewitt broth supplemented with 0.5% yeast extract (Difco, Detroit, Mich.) until an absorbance of 0.3 at 580 nm (UV-visible spectrophotometer, Shimadzu UV-1203, Japan) was reached. (ii) For the highly encapsulated (HE) phenotype (4), after serial passages in mice, the microorganism was grown three times in Todd-Hewitt broth supplemented with 0.5% yeast extract (Difco, Detroit, Mich.) and enriched with 5% fetal bovine serum until an absorbance of 0.3 at 580 nm (UV-visible spectrophotometer, Shimadzu UV-1203, Japan) was reached. Eight-to 12-week old female BALB/c mice weighing 19 to 22 g were used. The challenge dose with the PE and the HE inocula (2, 6) was 4 × 108 CFU/ml. Previously described methods (6) were followed for hyperimmune serum production and determination of protection with and without levofloxacin doses decreasing on a twofold basis from 25 mg/kg of body weight. Groups of 10 animals per dose were used. Experiments were carried out in duplicate. Treatment was initiated 1 h after the intraperitoneal challenge, and a second dose was administered 24 h later. Levofloxacin concentrations were determined by bioassay using Escherichia coli ATCC 25922 in pooled sera from five animals per sampling time (predosing, 15 min, 30 min, 1 h, 2 h, and 4 h). Drug concentrations were analyzed by a noncompartmental approach using the WinNonlin Professional program (Pharsight, Mountain View, Calif.). Survival curves were obtained by the Kaplan-Meier method. An ordinal log-rank test was used to compare different study groups. Due to multiple comparisons, a P value of ≤0.001 was considered significant. Concentrations (μg/ml) of levofloxacin in serum obtained after a single 25-mg/kg dose were 144.54 at 15 min, 120.22 at 30 min, 4.67 at 1 h, 0.23 at 2 h, and undetectable at 4 h. Maximum concentration and area under the curve (AUC) were 144.54 μg/ml and 84.84 μg · h/ml. Table ​Table11 shows survival rates. No differences (P = 0.85) between the PE and the HE models were found with nonimmune serum or placebo controls. In the PE model, differences in survival rates between immunized and nonimmunized animals were nonsignificant (P = 0.03) with 6.25 mg/kg levofloxacin but significant (P < 0.0001) with the 12.5-mg/kg dose. Significant differences (P < 0.0001) were found, with higher survival rates in the PE than the HE model (0% from day 2 onwards), for each treatment regimen. TABLE 1. Survival rates produced by three levofloxacin doses over a 7-day follow-up period with both types of infecting inocula (PE and HE) in normal mice and previously immunized mice An AUC/MIC ratio of 25 to 30 has been classically related to favorable outcomes in humans infected with S. pneumoniae (1) despite data supporting lower values needed (5). Lower values are needed in rodents (3). In the present study, ratios of maximum concentration to MIC and AUC to MIC of 4.5 and 2.7, respectively, produced efficacy (80% survival) in the PE model. These values were not enough to produce efficacy when the strain was highly encapsulated (HE model), where an increase in capsule-associated virulence was noted. Human natural infections by S. pneumoniae occur with highly capsulated strains, suggesting that much higher AUC/MIC ratios are needed in natural infections. We express our gratitude to L. Alou (IPM, Madrid, Spain) for the statistical analysis.


Infectious Diseases in Clinical Practice | 2015

Surgical Site Infection Caused by Enterobacter cancerogenus: A Case Report and Review of Literature

Daniel Tena; María R. Lago; Juan Antonio Sáez-Nieto; María José Medina; Noelia Lara; Jesús Oteo

AbstractWe report the first case of surgical site infection caused by Enterobacter cancerogenus. The infection occurred in a patient who underwent surgery for a broken ankle. The source of infection was unclear and clinical outcome was good after treatment with amoxicillin/clavulanic acid for 10 days. The review of the literature shows that E. cancerogenus has been rarely implicated in skin and soft tissue infections. These infections occur usually after injuries or trauma related to environmental contamination. In conclusion, E. cancerogenus should be considered as a potential pathogen in patients with surgical site infections. Further cases should be reported in the future to know better the transmission routes of E. cancerogenus in patients with nosocomial infections.


Journal of Antimicrobial Chemotherapy | 2016

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

Jesús Oteo; María Pérez-Vázquez; Verónica Bautista; Adriana Ortega; Pilar Zamarrón; David Sáez; Sara Fernández-Romero; Noelia Lara; Raquel Ramiro; Belén Aracil; José Campos

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Jesús Oteo

Instituto de Salud Carlos III

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José Campos

Instituto de Salud Carlos III

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Belén Aracil

Instituto de Salud Carlos III

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Verónica Bautista

Instituto de Salud Carlos III

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Adriana Ortega

Instituto de Salud Carlos III

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David Sáez

Instituto de Salud Carlos III

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Margarita Arroyo

Instituto de Salud Carlos III

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Emilia Cercenado

Complutense University of Madrid

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