Nuestros investigadores

David González Fernández

Publicaciones científicas más recientes (desde 2010)

Autores: González Fernández, David (Autor de correspondencia); Gallagher, E.; Zúñiga, T.; et al.
Revista: INTERNATIONAL MICROBIOLOGY
ISSN 1139-6709  Vol. 23  Nº 2  2020  págs. 171 - 177
Presence of extended-spectrum beta-lactamase (ESBL-E), AmpC-producing and carbapenemase-producing (CPE) Enterobacteriaceae has been observed not only in the clinical environment, but also in the out-of-hospital environment. The objective of this study was to isolate and characterize strains of ESBL, AmpC, and CPE present in feces of healthy carriers in Navarra (n = 125). Despite the fact that no CPE strains were isolated, 16% and 11.2% of the studied population were ESBL-E and AmpC carriers, respectively. No significant differences were found by gender or age; young people (5-18 years old) showed the highest ESBL-E prevalence (31.8%). The isolates corresponded to E. coli (57.1%), Enterobacter spp. (28.6%), and Citrobacter freundii (14.3%), and all strains showed multidrug-resistant profiles. High resistance against cephalosporins, penicillins, and monobactams, and sensitivity to carbapenems, quinolones, and aminoglycosides were observed. With respect to ESBL producers, 52.4% were CTX-M-type (19.0% CTX-M-14, 9.5% CTX-M-1, and 28.6% CTX-M-15) and 47.6% were TEM-type (38.1% TEM-171). These results confirm the extensive dissemination of these resistances among a healthy population and pose the need to implement control measures and strategies according to the One Health approach in order to prevent the increase of severe and untreatable infections in a not far future.
Autores: Pérez Etayo, Lara (Autor de correspondencia); González Fernández, David; Vitas Pemán, Ana Isabel
Revista: MICROORGANISMS
ISSN 2076-2607  Vol. 8  Nº 4  2020  págs. 568
One of the main public health problems nowadays is the increase of antimicrobial resistance, both in the hospital environment and outside it (animal environment, food and aquatic ecosystems, among others). It is necessary to investigate the virulence-associated factors and the ability of horizontal gene transfer among bacteria for a better understanding of the pathogenicity and the mechanisms of dissemination of resistant bacteria. Therefore, the objective of this work was to detect several virulence factors genes (fimA, papC, papG III, cnf1, hlyA and aer) and to determine the conjugative capacity in a wide collection of extended-spectrum beta-lactamases-producing E. coli isolated from different sources (human, food, farms, rivers, and wastewater treatment plants). Regarding virulence genes, fimA, papC, and aer were distributed throughout all the studied environments, papG III was mostly related to clinical strains and wastewater is a route of dissemination for cnf1 and hlyA. Strains isolated from aquatic environments showed an average conjugation frequencies of 1.15 x 10(-1) +/- 5 x 10(-1), being significantly higher than those observed in strains isolated from farms and food (p < 0.05), with frequencies of 1.53 x 10(-4) +/- 2.85 x 10(-4) and 9.61 x 10(-4) +/- 1.96 x 10(-3), respectively. The reported data suggest the importance that the aquatic environment (especially WWTPs) acquires for the exchange of genes and the dispersion of resistance. Therefore, specific surveillance programs of AMR indicators in wastewaters from animal or human origin are needed, in order to apply sanitation measures to reduce the burden of resistant bacteria arriving to risky environments as WWTPs.
Autores: González Fernández, David; Gallagher, E.; Zúñiga, T.; et al.
Revista: INTERNATIONAL MICROBIOLOGY
ISSN 1139-6709  Vol. 23  Nº 2  2019  págs. 171 - 177
Presence of extended-spectrum ß-lactamase (ESBL-E), AmpC-producing and carbapenemase-producing (CPE) Enterobacteriaceae has been observed not only in the clinical environment, but also in the out-of-hospital environment. The objective of this study was to isolate and characterize strains of ESBL, AmpC, and CPE present in feces of healthy carriers in Navarra (n¿=¿125). Despite the fact that no CPE strains were isolated, 16% and 11.2% of the studied population were ESBL-E and AmpC carriers, respectively. No significant differences were found by gender or age; young people (5-18 years old) showed the highest ESBL-E prevalence (31.8%). The isolates corresponded to E. coli (57.1%), Enterobacter spp. (28.6%), and Citrobacter freundii (14.3%), and all strains showed multidrug-resistant profiles. High resistance against cephalosporins, penicillins, and monobactams, and sensitivity to carbapenems, quinolones, and aminoglycosides were observed. With respect to ESBL producers, 52.4% were CTX-M-type (19.0% CTX-M-14, 9.5% CTX-M-1, and 28.6% CTX-M-15) and 47.6% were TEM-type (38.1% TEM-171). These results confirm the extensive dissemination of these resistances among a healthy population and pose the need to implement control measures and strategies according to the One Health approach in order to prevent the increase of severe and untreatable infections in a not far future.
Autores: Pérez Etayo, Lara; González Fernández, David; Leiva León, José; et al.
Revista: MICROORGANISMS
ISSN 2076-2607  Vol. 8  Nº 9  2019  págs. 1425
Due to the global progress of antimicrobial resistance, the World Health Organization (WHO) published the list of the antibiotic-resistant "priority pathogens" in order to promote research and development of new antibiotics to the families of bacteria that cause severe and often deadly infections. In the framework of the One Health approach, the surveillance of these pathogens in different environments should be implemented in order to analyze their spread and the potential risk of transmission of antibiotic resistances by food and water. Therefore, the objective of this work was to determine the presence of high and critical priority pathogens included in the aforementioned list in different aquatic environments in the POCTEFA area (North Spain-South France). In addition to these pathogens, detection of colistin-resistant Enterobacteriaceae was included due its relevance as being the antibiotic of choice to treat infections caused by multidrug resistant bacteria (MDR). From the total of 80 analyzed samples, 100% of the wastewater treatment plants (WWTPs) and collectors (from hospitals and slaughterhouses) and 96.4% of the rivers, carried antibiotic resistant bacteria (ARB) against the tested antibiotics. Fifty-five (17.7%) of the isolates were identified as target microorganisms (high and critical priority pathogens of WHO list) and 58.2% (n = 32) of them came from WWTPs and collectors. Phenotypic and genotypic characterization showed that 96.4% were MDR and resistance to penicillins/cephalosporins was the most widespread. The presence of bla genes, KPC-type carbapenemases, mcr-1 and vanB genes has been confirmed. In summary, the presence of clinically relevant MDR bacteria in the studied aquatic environments demonstrates the need to improve surveillance and treatments of wastewaters from slaughterhouses, hospitals and WWTPs, in order to minimize the dispersion of resistance through the effluents of these areas.
Autores: Pérez Etayo, Lara; González Fernández, David; Vitas Pemán, Ana Isabel
Revista: MICROORGANISMS
ISSN 2076-2607  Vol. 8  Nº 4  2018  págs. 568
One of the main public health problems nowadays is the increase of antimicrobial resistance, both in the hospital environment and outside it (animal environment, food and aquatic ecosystems, among others). It is necessary to investigate the virulence-associated factors and the ability of horizontal gene transfer among bacteria for a better understanding of the pathogenicity and the mechanisms of dissemination of resistant bacteria. Therefore, the objective of this work was to detect several virulence factors genes (fimA, papC, papG III, cnf1, hlyA and aer) and to determine the conjugative capacity in a wide collection of extended-spectrum ß-lactamases-producing E. coli isolated from different sources (human, food, farms, rivers, and wastewater treatment plants). Regarding virulence genes, fimA, papC, and aer were distributed throughout all the studied environments, papG III was mostly related to clinical strains and wastewater is a route of dissemination for cnf1 and hlyA. Strains isolated from aquatic environments showed an average conjugation frequencies of 1.15 × 10-1 ± 5 × 10-1, being significantly higher than those observed in strains isolated from farms and food (p < 0.05), with frequencies of 1.53 × 10-4 ± 2.85 × 10-4 and 9.61 × 10-4 ± 1.96 × 10-3, respectively. The reported data suggest the importance that the aquatic environment (especially WWTPs) acquires for the exchange of genes and the dispersion of resistance. Therefore, specific surveillance programs of AMR indicators in wastewaters from animal or human origin are needed, in order to apply sanitation measures to reduce the burden of resistant bacteria arriving to risky environments as WWTPs.
Autores: Pérez Etayo, Lara (Autor de correspondencia); Berzosa Suñer, Melibea; González Fernández, David; et al.
Revista: INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH
ISSN 1660-4601  Vol. 15  Nº 10  2018  págs. E2308
Mobile genetic elements play an important role in the dissemination of antibiotic resistant bacteria among human and environmental sources. Therefore, the aim of this study was to determine the occurrence and patterns of integrons and insertion sequences of extended-spectrum -lactamase (ESBL)-producing Escherichia coli isolated from different sources in Navarra, northern Spain. A total of 150 isolates coming from food products, farms and feeds, aquatic environments, and humans (healthy people and hospital inpatients), were analyzed. PCRs were applied for the study of class 1, 2, and 3 integrons (intI1, intI2, and intI3), as well as for the determination of insertion sequences (IS26, ISEcp1, ISCR1, and IS903). Results show the wide presence and dissemination of intI1 (92%), while intI3 was not detected. It is remarkable, the prevalence of intI2 among food isolates, as well as the co-existence of class 1 and class 2 (8% of isolates). The majority of isolates have two or three IS elements, with the most common being IS26 (99.4%). The genetic pattern IS26-ISEcp1 (related with the pathogen clone ST131) was present in the 22% of isolates (including human isolates). In addition, the combination ISEcp1-IS26-IS903-ISCR1 was detected in 11 isolates being, to our knowledge, the first study that describes this genetic complex. Due to the wide variability observed, no relationship was determined among these mobile genetic elements and beta-lactam resistance. More investigations regarding the genetic composition of these elements are needed to understand the role of multiple types of integrons and insertion sequences on the dissemination of antimicrobial resistance genes among different environments.
Autores: Vitas Pemán, Ana Isabel (Autor de correspondencia); Naik, D.; Pérez Etayo, Lara; et al.
Revista: INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY
ISSN 0168-1605  Vol. 269  2018  págs. 80 - 86
The aim of this study was to determine the occurrence and patterns of resistance of extended-spectrum beta-plactamase (ESBL) producing Enterobacteriaceae in food products purchased in Navarra, northern Spain. A total of 174 samples of fish and chicken were analyzed from September 2015 to September 2016, including raw and ready-to-eat products: trout (n = 25), salmon (n = 28), panga (n = 13), chicken nuggets and chicken scalopes (n = 32), sushi (n = 31) and sliced cooked poultry (n = 45). Cefpodoxime-resistant strains were isolated on ChromlD ESBL agar and further phenotypic (antimicrobial study on MicroScan(C) NM37 panel) and genotypic characterization (multiplex PCR, sequencing and multi-locus sequence typing, MLST) was performed to confirm and characterize ESBL producers. Raw chicken and sushi have been determined as the most risky products regarding transmission of ESBL-producing Enterobacteriaceae (occurrence 53.1% and 19.4%, respectively), while sliced cooked poultry products appear to be a safe product in this aspect. With regard to raw fish, prevalence in salmon was lower (3.6%) than in trout and panga (16.0%). Ninety-eight per cent of ESBL isolates (n = 50) show multidrug-resistant profiles, highlighting the high resistances against quinolones and tetracyclines observed in chicken isolates, as well as against ertapenem and chloramphenicol in sushi strains. Predominant 13-lactamase type was SHV-12 (50.1%), followed by TEM-type (24.5%) and CTX-M (20.8%). In addition, CTX-M type was only detected in chicken products. The phylogenetic study showed the prevalence of groups A (35%), F (25%) and B1 (15%), usually related to nonvirulent strains. MIST E. coil isolates (n = 20) were grouped into 5 clonal complexes (CC) and 15 sequence types (ST), showing high clonal diversity. ST117 was the prevalent sequence type, while the human pathogen ST131 was not detected in this study. The high prevalence of ESBL-producing multidrug-resistant Enterobacteriaceae detected in products of widespread consumption such as chicken and sushi, increases the concern regarding human exposure to superbugs and encourages the need to improve surveillance of this public health issue.
Autores: Ojer Usoz, Elena; González Fernández, David; Vitas Pemán, Ana Isabel
Revista: INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH
ISSN 1660-4601  Vol. 14  Nº 7  2017  págs. 676
This study presents a comprehensive approach of a clonal diversity analysis of 448 Extended-spectrum beta-lactamase (ESBL)-producing E. coli isolated from environmental, human and food samples in Spain. The phenotypic confirmation of ESBL production was performed by disc diffusion and microdilution methods, while Polymerase Chain Reaction (PCR) and sequencing were used for the molecular characterization of beta-lactamase genes (bla(CTX-M), bla(SHV), bla(TEM), bla(OXA)). Clonal relationship of isolates was determined by multi-locus sequence typing (MLST). Multidrug resistant strains were present in all the studied niches, with percentages above 50.0%. The most prevalent beta-lactamase genes were bla(CTXM-14) (26%) and bla(CTXM-1) (21.4%), followed by bla(SHV-12), bla(CTX-M-15) and bla(TEM-42). MLST isolates were grouped into 26 clonal complexes (CC) and 177 different sequence types (ST) were detected. Despite the high clonal diversity observed, CC10 was the prevalent and the only CC detected in all niches, while other complexes as CC131 were mainly associated to human isolates. The observed prevalence and diversity of these resistant bacteria across the different environments encourages a One Health approach to prevent and control ESBL dissemination between environment and consumers.
Autores: Vitas Pemán, Ana Isabel; Díez Leturia, María; Tabar, L.; et al.
Revista: INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY
ISSN 0168-1605  Vol. 184  2014  págs. 109 - 112
Controlling Listeria monocytogenes in smoked salmon is still a challenge for food business operators. The reported lowlevels and uneven distribution of the pathogenmake it necessary to apply good sampling plans and analytical procedures in order to detect contaminated batches. However, analyses are expensive and due to current economic situations, usually only one sample per lot is checked instead of the n = 5 samples established in Regulation (EC) No 2073/2005. This work proposes the investigation of L. monocytogenes by pooling pre-enriched Half Fraser from several units from a batch (n = 6). The results obtained from a preliminary in-house validation (n = 51 pools) showed that the performance characteristics (sensitivity and specificity) are very high (N95%). This high reliability, together with reduced costs (nearly half), suggests that the wet pooling test could be a good cost-effective approach for investigating L.monocytogenes in smoked salmon. Additional research is needed before this approach can be used for other foodmatrices, such as determining the maximumnumber of units per batch that can be pooled with effective detection of L. monocytogenes.
Autores: Pérez-Rodríguez, F.; Saiz-Abajo, M.J.; Garcia-Gimeno, R.M.; et al.
Revista: INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY
ISSN 0168-1605  Vol. 184  2014  págs. 86 - 91
Fecal pathogen distributions in fresh-cut leafy vegetables are essential to develop suitable sampling plans so as to detect pathogen contaminations. In this study, a typical fresh-cut lettuce process was reproduced at pilot scale with different initial inoculum levels of Salmonella on lettuce (6-7,4 and 1 log CFU/g). The pathogen was determined in all processed lettuce samples (n >= 50) and obtained count data were used to fit different probability distributions. The study showed that Salmonella is homogenously distributed on fresh-cut leafy vegetables as a result of processing (mainly washing) at all contamination levels. Negative binomial and Poisson-lognormal distributions were suitable to describe pathogen distribution at the high and medium levels, coefficient of variation modified (CV*) indicated no overdispersion (i.e. clustering). Nevertheless, further research will be needed to assess the effect of using disinfectants in washing water on the final distribution pattern of pathogens in processed fresh-cut leafy vegetables.
Autores: Ojer Usoz, E.; González Fernández, David; García Jalón de la Lama, Isabel; et al.
Revista: WATER RESEARCH
ISSN 0043-1354  Vol. 56  2014  págs. 37 - 47
Water environments play an important role in the dissemination of antibiotic-resistant bacteria among humans, animals and agricultural sources. In order to assess the spread of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, we analyzed 279 effluent samples from 21 wastewater treatment plants in Navarra (northern Spain). A total of 185 cefpodoxime-resistant bacteria were isolated on ChromID ESBL agar plates, with high predominance of Escherichia coli among isolated species (73%). ESBL production was determined by different methods, concluding its presence in 86.5% of the isolates by the combination disk test, 75.7% by double-disk synergy test and 73.5% by MicroScan® NM37 automated system. PCR and sequencing analysis showed that the predominant ß-lactamases (bla) genes were blaCTx-M (67.4%) followed by blaTEM (47%), blaSHV (17.4%) and blaOxA (8.3%); furthermore, two or more ß-lactamases genes were found in 34.9% of the isolates. The results demonstrate the high prevalence of ESBL-producing Enterobacteriaceae in effluent water from wastewater treatment plants and confirm the need to optimize current disinfection procedures and to improve management of wastewater in an effort to minimize reservoirs of resistant bacteria. Further studies are needed for examining the presence of these bacteria in other environments and for determining the potential dissemination routes of these resistances as well as their impact on human health.
Autores: González Fernández, David; Vitas Pemán, Ana Isabel; Díez Leturia, María; et al.
Revista: FOOD MICROBIOLOGY
ISSN 0740-0020  Vol. 36  Nº 2  2013  págs. 374 - 378
The aim of this study was to obtain data from refrigerated ready-to-eat seafood products at retail in Spain (young eels, crabstick and smoked salmon), regarding prevalence and levels of Listeria monocytogenes, storage temperatures and the impact of transport conditions (type of bag) on the temperature of the product. The one-year surveillance period was carried out according to the EC Regulation No. 2073/2005, taking 5 units/batch and analyzing 250 samples following ISO 11290-1/A1 and ISO 11290-2/A methodologies. Low prevalence of L. monocytogenes was observed in surimi products, while 4.8% of smoked salmon samples were positive for Listeria with low levels (<10 cfu/g) and uneven pathogen distribution. A single company was responsible for 80% of the positive lots. All purchased products showed values higher than 4 C at retail and an average increase of 2.5 C or up to 6.2 C was recorded when isothermal or plastic shopping bags were used for transport, respectively. To avoid noncompliance of the Food Safety Objective for L. monocytogenes in seafood RTE products more efforts from all stakeholders are needed, with special attention so as to improve control and maintenance of refrigerators at retail and to enhance consumer education regarding food safety practices.
Autores: Ojer Usoz, E.; González Fernández, David; Vitas Pemán, Ana Isabel; et al.
Revista: MEAT SCIENCE
ISSN 0309-1740  Vol. 93  Nº 2  2013  págs. 316 - 321
Patterns of resistance in ß-lactamase-producing Enterobacteriaceae family were investigated in isolates from 141 meat products (beef, poultry and pork) purchased in Spain. The strains that grow in ChromID ESBL agar plates were confirmed using the paired disk diffusion method. Resistance to amoxicillin/clavulanic acid, ceftazidime, ceftriaxone, aztreonam, cefpodoxime, gentamicin, doxycycline, cotrimoxazol, norfloxacin, piperacillin/tazobactam, fosfomycin and cefoxitin were tested following CLSI recommendations. Minimum inhibitory concentrations were determined by the MicroScan® NM37 panel and ß-lactamase genes were detected using multiplex PCR and sequencing. Results show poultry as the meat product having the highest prevalence (84%), with Escherichia coli being the predominant bacteria (71.3%). Predominant ß-lactamase types were CTX-M (37.8%), followed by CTX-M+TEM combination (20.7%), TEM (17%), SHV (12.2%), TEM+SHV combination (10.9%) and OXA (1.2%). 93.9% of the strains were resistant to one or more ß-lactam antibiotics. Results indicate a widespread distribution of ESBL-producing Enterobacteriaceae in meat products, with a high rate of ß-lactam resistance and a low rate of AmpC cephalosporinase-producing strains.
Autores: Elizondo, E.; Sala, S.; Imbuluzqueta Iturburua, Edurne; et al.
Revista: PHARMACEUTICAL RESEARCH
ISSN 0724-8741  Vol. 28  Nº 2  2011  págs. 309 - 321
PURPOSE: To investigate, for the first time, the viability of compressed antisolvent methodologies for the preparation of drug-loaded particles of the biodegradable and bioadhesive polymer poly (methyl vinyl ether-co-maleic anhydride) (PVM/MA), utilizing gentamicin (Gm) as a model drug. METHODS: Precipitation with a Compressed Antisolvent (PCA) method was used for the preparation of PVM/MA particles loaded with gentamicin. Before encapsulation, gentamicin was modified into a hydrophobic complex, GmAOT, by exchanging its sulphate ions with an anionic surfactant. GmAOT:PVM/MA composites were fully characterized in terms of size, morphology, composition, drug distribution, phase composition, in vitro activity and drug release. RESULTS: Homogeneous nanostructured microparticles of PVM/MA loaded with high and uniformly distributed quantities of GmAOT were obtained by PCA. The drug loading factors could be tuned at will, improving up to ten times the loadings obtained by other precipitation techniques. Gentamicin retained its bioactivity after being processed, and, according to its release profiles, after an initial burst it experienced a sustained release over 30 days. CONCLUSIONS: Compressed antisolvent methods are suitable technologies for the one-step preparation of highly loaded nanostructured PVM/MA matrices with promising application in the delivery of low bioavailable drugs.
Autores: Estella Hermoso de Mendoza, Ander; Imbuluzqueta Iturburua, Izaskun; Campanero Martínez, Miguel Ángel; et al.
Revista: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
ISSN 1570-0232  Vol. 879  Nº 30  2011  págs. 3490-6
An ultra high performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed and validated for the quantitation of LBH589, a novel histone deacetylase inhibitor (HDACi), in mouse plasma and tissues (liver, spleen, kidney and lung). Tobramycin was employed as the internal standard. Separation was performed on an Acquity UPLC¿ BEH column, with a mobile phase consisting of 10% water (with 0.1% of trifluoroacetic acid) and 90% methanol (with 0.1% trifluoroacetic acid). LBH589 and tobramycin were determined using an electrospray ionization (ESI) interface. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of LBH589 at m/z 349.42¿157.95 and of tobramycin at 468.2¿163. Calibration curves for the UHPLC method (0.0025-1 ¿g/mL for plasma and tissue homogenates, equivalent to 0.0357-14.2857 ¿g/g for tissue samples) showed a linear range of detector responses (r>0.998). Intra-batch and inter-batch precision expressed as coefficient of variation (CV) ranged from 0.92 to 8.40%. Accuracy expressed as bias, ranged from -2.41 to 2.62%. The lower limit of quantitation (LLOQ) was 0.0025 ¿g/mL for both plasma and tissue homogenate samples, equivalent to 0.0357 ¿g/g tissue. This method was successfully applied to quantify LBH589 in plasma and tissue samples obtained after the intraperitoneal administration of a single dose of 20 mg/kg of LBH589 in BALB/c mice.
Autores: Ojer Usoz, Elena; González Fernández, David; Vitas Pemán, Ana Isabel; et al.
Libro:  2nd workshop on food safety. Technologies and innovations applied to food safety
2012  págs. 285-289
Autores: Vitas Pemán, Ana Isabel; González Fernández, David; García Jalón de la Lama, Isabel; et al.
Libro:  2nd workshop on food safety. Technologies and innovations applied to food safety
2012  págs. 290 - 295