Controlling diarrheagenic E. coli with bacteriophages: facts and challenges

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine, causing severe diarrhoea in humans and animals. The rise of antibiotic resistances and limitation on their use demands news strategies to tackle this pathology. Bacteriophages (phages), viruses specifically infecting bacteria and harmless to animals and plants, are a promising antibacterial tool. Although studies support their ability to efficiently overcome ETEC infections, they have been shown to be highly strain-specific. If this can be associated with the presence of anti-phage defense systems (APDS) in ETEC genomes, it is also true that phages can counter-evolve to escape APDS. This work aimed to define phage cocktail with broader lytic spectra, capable of overcoming APDS of ETEC, enhancing phage efficacy. We firstly sequenced 29 ETEC strains from our collection to search for the presence of APDS in their genomes. Then, we performed phage isolation and the subsequent in vitro and genomic characterization: i) evaluation of lytic spectra against ETEC collection; ii) whole-genome sequencing and phage safety evaluation (absence of undesirable genes) iii)presence of proteins responsible for escaping the main APDS. We were able to identify distinct mechanisms supporting APDS. Bacterial proteins that prevent the entry of DNA from phages (CRISPR-Cas-related proteins) or that enable the cut of phage nucleic acids (restriction-modification enzymes) were detected, however, most of them were related with the induction of abortive infection events (e.g. toxin-antitoxin systems). We also isolated 3 phages, SUS35, SUS42 and SUS65, which proved to be safe for therapy and to encode proteins enabling to escape APDS, inclusively against abortive infection. Phage-host interaction mechanisms must be considered when preparing phage- based products for therapy. This work clearly indicates that a strict selection of phages, or the construction of synthetic phages with desired traits will be a turning point in their versatility to fight against ETEC infections.info:eu-repo/semantics/publishedVersio

Whole Genome Sequencing and Characteristics of mcr-1–Harboring Plasmids of Porcine Escherichia coli Isolates Belonging to the High-Risk Clone O25b:H4-ST131 Clade B

Porcine Escherichia coli ST131 isolates are scarcely documented. Here, whole genome sequencing and core genome (CG) and plasmidome analysis of seven isolates collected from diarrheic piglets and four from pork meat were performed. All of the 11 ST131 isolates belonged to serotype O25b:H4 and clade B and showed fimH22 allele or mutational derivatives. The 11 porcine isolates possessed virulence traits that classified the isolates as avian pathogenic, uropathogenic, and extraintestinal pathogenic E. coli–like (APEC-, UPEC-, and ExPEC-like) and constituted virotype D. The CG was performed for all porcine isolates in addition to 73 ST131 reference isolates from different origins. Within clade B, the CG showed nine subclusters, allowing us to describe five new subclades (B6, B6-like, B7, B8, and B9). There was an association between subclade B6, PST43, virotype D2, and food origin, whereas subclade B7 included PST9 isolates with virotype D5 from diarrheic piglets (p = 0.007). The distance between human and porcine isolates from subclades B6 and B7 had an average of 20 and 15 SNP/Mb, respectively. [F2:A-:B1]-IncF, ColE1-like, and IncX plasmids were the most prevalent. Besides, IncF plasmids harbored a ColV region frequent among APEC isolates. Antimicrobial resistance genes conferring resistance to penicillin, tetracycline, quinolones, and colistin were the most common. The mcr-1.1 gene was detected in 5 of 11 porcine isolates, integrated into the chromosome of one isolate and into plasmids in the remainder isolates (two MOBH11/IncHI2-ST4, one MOBP3/IncX4, and one MOBF12/IncF [F2:A-:B1] supposedly cointegrated with an IncHI2). The surrounding environments of the mcr-1 cassette showed variability. However, there were conserved structures within the same plasmid family. In conclusion, CG analysis defined five new subclades. The ST131 porcine isolates belonged to new subclades B6 and B7. Moreover, porcine and clinical human isolates were strongly related. The 11 porcine ST131 isolates harbored a wide variety of plasmids, virulence, and resistance genes. Furthermore, epidemic plasmids IncX4 and IncHI2 are responsible for the acquisition of mcr-1.1 gene. We hypothesize that the APEC-IncF plasmid acquired the mcr-1.1 gene via cointegrating an IncHI2 plasmid, which is worrying due to combination of virulence and resistance attributes in a single mobile genetic elementS-CF-S acknowledges the FPU programme for her grant (FPU15/02644) from the Secretaría General de Universidades, Spanish Ministerio de Educación, Cultura y Deporte. IG-M and VG acknowledge the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for his predoctoral grant (ED481A-2015/149) and her postdoctoral grant (ED481B2018/018), respectively. AM acknowledges the Ministerio de Educación, Cultura y Deporte (Spain) for the mobility grant PRX16/00023 for teachers and researchers from the Programa Estatal de Promoción del Talento y su Empleabilidad, Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016S

Genomic characterization of prevalent mcr-1, mcr-4, and mcr-5 Escherichia coli within swine enteric colibacillosis in Spain

Antimicrobial agents are crucial for the treatment of many bacterial diseases in pigs, however, the massive use of critically important antibiotics such as colistin, fluoroquinolones and 3rd–4th-generation cephalosporins often selects for co-resistance. Based on a comprehensive characterization of 35 colistin-resistant Escherichia coli from swine enteric colibacillosis, belonging to prevalent Spanish lineages, the aims of the present study were to investigate the characteristics of E. coli clones successfully spread in swine and to assess the correlation of the in vitro results with in silico predictions from WGS data. The resistome analysis showed six different mcr variants: mcr-1.1; mcr-1.10; mcr-4.1; mcr-4.2; mcr-4.5; and mcr-5.1. Additionally, blaCTX–M–14, blaCTX–M–32 and blaSHV–12 genes were present in seven genomes. PlasmidFinder revealed that mcr-1.1 genes located mainly on IncHI2 and IncX4 types, and mcr-4 on ColE10-like plasmids. Twenty-eight genomes showed a gyrA S83L substitution, and 12 of those 28 harbored double-serine mutations gyrA S83L and parC S80I, correlating with in vitro quinolone-resistances. Notably, 16 of the 35 mcr-bearing genomes showed mutations in the PmrA (S39I) and PmrB (V161G) proteins. The summative presence of mechanisms, associated with high-level of resistance to quinolones/fluoroquinolones and colistin, could be conferring adaptive advantages to prevalent pig E. coli lineages, such as the ST10-A (CH11-24), as presumed for ST131. SerotypeFinder allowed the H-antigen identification of in vitro non-mobile (HNM) isolates, revealing that 15 of the 21 HNM E. coli analyzed were H39. Since the H39 is associated with the most prevalent O antigens worldwide within swine colibacillosis, such as O108 and O157, it would be probably playing a role in porcine colibacillosis to be considered as a valuable subunit antigen in the formulation of a broadly protective Enterotoxigenic E. coli (ETEC) vaccine. Our data show common features with other European countries in relation to a prevalent clonal group (CC10), serotypes (O108:H39, O138:H10, O139:H1, O141:H4), high plasmid content within the isolates and mcr location, which would support global alternatives to the use of antibiotics in pigs. Here, we report for first time a rare finding so far, which is the co-occurrence of double colistin-resistance mechanisms in a significant number of E. coli isolatesThis study was supported by projects PI16/01477 from Plan Estatal de I+D+I 2013–2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación, and FEDER; AGL2016-79343-R from the Agencia Estatal de Investigación (AEI, Spain) and FEDER; ED431C 2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia) and FEDER. IG-M and VG acknowledge the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for their pre-doctoral and post-doctoral grants (Grant Numbers ED481A-2015/149 and ED481B-2018/018, respectively). SF-S acknowledges the FPU programme from the Secretaría General de Universidades, Ministerio de Educación, Cultura y Deporte, Gobierno de España (Grant Number FPU15/02644)S

Occurrence and Genomic Characterization of Clone ST1193 Clonotype 14-64 in Uncomplicated Urinary Tract Infections Caused by Escherichia coli in Spain

We conducted a prospective, multicenter, specific pilot study on uncomplicated urinary tract infections (uUTI). One-hundred non-duplicated uropathogenic Escherichia coli (UPEC) from uUTI occurred in 2020 in women attending 15 primary care centers of a single health region of northern Spain were characterized using a clonal diagnosis approach. Among the high genetic diversity showed by 59 different phylogroup-clonotype combinations, 11 clones accounted for 46% of the isolates: B2-ST73 (CH24-30); B2-ST73 (CH24-103); B2-ST131 (CH40-30); B2-ST141 (CH52-5); B2-ST372 (CH103-9); B2-ST404 (CH14-27); B2-ST404 (CH14-807); B2-ST1193 (CH14-64); D-ST69 (CH35-27); D-ST349 (CH36-54), and F-ST59 (CH32-41). The screening of the UPEC status found that 69% of isolates carried ≥ 3 of chuA, fyuA, vat, and yfcV genes. Multidrug resistance to at least one antibiotic of ≥ 3 antimicrobial categories were exhibited by 30% of the isolates, with the highest rates of resistance against ampicillin/amoxicillin (48%), trimethoprim (35%), norfloxacin (28%), amoxicillin-clavulanic acid (26%), and trimethoprim-sulfamethoxazole (24%). None extended-spectrum beta-lactamase/carbapenemase producer was recovered. According to our results, fosfomycin and nitrofurantoin should be considered as empirical treatment of choice for uUTI by E. coli (resistance rates 4% and 2%, respectively). We uncover the high prevalence of the pandemic fluoroquinolone-resistant ST1193 clone (6%) in uUTI, which represents the first report in Spain in this pathology. The genomic analysis showed similar key traits than those ST1193 clones disseminated worldwide. Through the SNP comparison based on the core genome, the Spanish ST1193 clustered with isolates retrieved from the Enterobase, showing high genomic similarity than the global ST1193 described in the United States, Canada and Australia. IMPORTANCE Analyzing the clonal structure and antimicrobial resistance of E. coli isolates implicated in uncomplicated urinary tract infections, one of the most frequent visits managed in primary health care, is of interest for clinicians to detect changes in the dynamics of emerging uropathogenic clones associated with the spread of fluoroquinolone resistance. It can also provide consensus concerning optimal control and antibiotic prescribingThis study was supported by the projects and funds PID2019-104439RB-C21/AEI/10.13039/501100011033 from the Agencia Estatal de Investigación (AEI, Spain), co-funded by the European Regional Development Fund of the European Union: A Way to Make Europe (ERDF); FIS PI17-00728 (Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain), co-funded by ERDF; GRUPIN IDI/2022/000033 by the Regional Ministry of Science of Asturias (IDI/2022/000033). ED431C 2021/11 from the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia) and ERDF. I.G-M. and V.G. acknowledge the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for their post-doctoral grants (Grant Number ED481B-2021-006 and ED481-B2018/018, respectively). The research stay of I.G-M at the Hospital Universitario Central de Asturias was funded by a grant from the Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC). L.L. acknowledges the Ministry of Education of Spain for her predoctoral grant FPU19/01127.S

Studies on the interaction of three lytic bacteriophages with a wide collection of Escherichia coli strains implicated in swine enteric colibacillosis

bioRxiv - the Preprint Server for BiologyThe misuse of antibiotics in the swine industry and their on-going restriction requires alternatives to control enterotoxigenic and shiga toxin-producing Escherichia coli (ETEC and STEC, respectively). This study evaluates the potential of three coliphages, vB_EcoM_FJ1, vB_EcoM_FN and vB_EcoM_SP1 against 104 ETEC, STEC and ETEC/STEC strains isolated from pig colibacillosis in Portuguese (2018-2020) and Spanish farms (2006-2016), encompassing 71.2% mcr-positive strains (33.7% with mcr-1, 1.9% mcr-2, 35.6% mcr-4 and 2.9% mcr-5) and 18.3% positive strains for TEM (1%), SHV (6.7%), and CTX-M (11.5%) extended-spectrum beta-lactamase-encoding genes. In general, all bacteriophages presented a narrow lytic spectrum (up to 2.9%) against the 104 ETEC, STEC and ETEC/STEC. Bacteriophages shared >80% overall nucleotide identity with E. coli phage T4 (Tevenvirinae subfamily), but a particular look at the distal part of the long tail fiber (gp38) revealed no homology. All bacteriophages recognize lipopolysaccharides as receptors, and additionally, FN binds to an outer membrane protein A. Bacteriophage-insensitive mutants of vB_EcoM_FJ1 (90%) and vB_EcoM_FN (100%) were shown to be more susceptible to pig serum inactivation comparatively to the parental strain and furthermore, their adhesion capacity to porcine intestinal cells was diminished by, approximately, 90%. Contrariwise, vB_EcoM_SP1 insensitive variants did not display phenotypic differences comparing to the wild-type strain. This study demonstrates that besides being T4-like, these bacteriophages revealed a narrow lytic spectrum against diarrhoeagenic E. coli strains and that the acquisition of novel bacteriophage-encoded adhesins (gp38) seems to be determinant for such results.info:eu-repo/semantics/publishedVersio

Genomic characterization of escherichia coli isolates belonging to a new hybrid aepec/expec pathotype o153:H10-a-st10 eae-beta1 occurred in meat, poultry, wildlife and human diarrheagenic samples

Different surveillance studies (2005–2015) in northwest Spain revealed the presence of eae-positive isolates of Escherichia coli O153:H10 in meat for human consumption, poultry farm, wildlife and human diarrheagenic samples. The aim of this study was to explore the genetic and genomic relatedness between human and animal/meat isolates, as well as the mechanism of its persistence. We also wanted to know whether it was a geographically restricted lineage, or whether it was also reported elsewhere. Conventional typing showed that 32 isolates were O153:H10-A-ST10 fimH54, fimAvMT78, traT and eae-beta1. Amongst these, 21 were CTX-M-32 or SHV-12 producers. The PFGE XbaI-macrorestriction comparison showed high similarity (>85%). The plasmidome analysis revealed a stable combination of IncF (F2:A-:B-), IncI1 (STunknown) and IncX1 plasmid types, together with non-conjugative Col-like plasmids. The core genome investigation based on the cgMLST scheme from EnteroBase proved close relatedness between isolates of human and animal origin. Our results demonstrate that a hybrid MDR aEPEC/ExPEC of the clonal group O153:H10-A-ST10 (CH11-54) is circulating in our region within different hosts, including wildlife. It seems implicated in human diarrhea via meat transmission, and in the spreading of ESBL genes (mainly of CTX-M-32 type). We found genomic evidence of a related hybrid aEPEC/ExPEC in at least one other countryThis study was supported by projects: AGL2013-47852-R from the Ministerio de Economía y Competitividad (MINECO, Spain) and Fondo Europeo de Desarrollo Regional (FEDER); AGL2016-79343-R from the Agencia Estatal de Investigación (AEI, Spain) and FEDER; PI16/01477 from Plan Estatal de I+D+I 2013-2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación and FEDER; and ED431C2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria of Xunta de Galicia and FEDERS

Swine enteric colibacillosis in Spain: pathogenic potential of mcr-1 ST10 and ST131 E. coli Isolates

This is a wide epidemiological study of 499 E. coli isolates recovered from 179 outbreaks of enteric colibacillosis from pig production farms in Spain during a period of 10 years. Most samples were of diarrheagenic cases occurred during the post-wean period (PWD) which showed to be significantly associated with ETEC (67%) followed by aEPEC (21.7%). On the contrary, aEPEC was more prevalent (60.3%) among diarrheas of suckling piglets, followed by ETEC (38.8%). STEC/ETEC or STEC were recovered in 11.3 and 0.9% of PWD and neonatal diarrhea, respectively. Detection of the F4 colonization factor was not significantly different between isolates recovered from neonatal pigs and those recovered post wean (40.5 versus 27.7%) while F18 was only present among PWD isolates (51.5% of ETEC, STEC, and STEC/ETEC isolates). We also found a high prevalence of resistance to colistin related to the presence of the mcr-1 gene (25.6% of the diarreagenic isolates). The characterization of 65 representative mcr-1 isolates showed that all were phenotypically resistant to colistin (>2 μg/ml), and most (61 of 65) multidrug-resistant (MDR). Six ETEC and one STEC mcr-1 isolates were also carriers of ESBL genes. In addition, other seven mcr-1 isolates harbored mcr-4 (three ETEC) and mcr-5 (two ETEC and two aEPEC) genes. In the phylogenetic analysis of the 65 mcr-1 diarrheagenic isolates we found that more than 50% (38 out of 65) belonged to A-ST10 Cplx and from those, 29 isolates showed the clonotype CH11-24. In this study, we also recovered 18 ST131 isolates including seven mcr-1 carriers. To the best of our knowledge, this would be the first report of ST131 mcr-1 isolation in pigs. Worryingly, the swine mcr-1 ST131 carriers also showed MDR, including to trimethoprim-sulfamethoxazole, tobramycin, gentamicin and ciprofloxacin. In the PFGE-macrorestriction comparison of clinical swine and human ST131, we found high similarities (≥85%) between two pig and two human ST131 isolates of virotype D5. Acquisition of mcr-1 by this specific clone means an increased risk due to its special feature of congregating virulence and resistance traits, together with its spread capability. Here we show a potential zoonotic swine source of ST131This study was supported by projects AGL2016-79343-R from the Agencia Estatal de Investigación (AEI, Spain) and FEDER; PI16/01477 from Plan Estatal de I C D C I 2013–2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación, and FEDER; CN2012/303 andED431C 2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia), and FEDER IG-M acknowledges the Conselleria de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for his grant (Ref. ED481A-2015/149) “Axudas de apoio á etapa predoutoral do Plan galego de investigación, innovación e crecemento 2011-2015S

Clonal Structure, Virulence Factor-encoding Genes and Antibiotic Resistance of Escherichia coli, Causing Urinary Tract Infections and Other Extraintestinal Infections in Humans in Spain and France during 2016

Escherichia coli is the main pathogen responsible for extraintestinal infections. A total of 196 clinical E. coli consecutively isolated during 2016 in Spain (100 from Lucus Augusti hospital in Lugo) and France (96 from Beaujon hospital in Clichy) were characterized. Phylogroups, clonotypes, sequence types (STs), O:H serotypes, virulence factor (VF)-encoding genes and antibiotic resistance were determined. Approximately 10% of the infections were caused by ST131 isolates in both hospitals and approximately 60% of these infections were caused by isolates belonging to only 10 STs (ST10, ST12, ST58, ST69, ST73, ST88, ST95, ST127, ST131, ST141). ST88 isolates were frequent, especially in Spain, while ST141 isolates significantly predominated in France. The 23 ST131 isolates displayed four clonotypes: CH40-30, CH40-41, CH40-22 and CH40-298. Only 13 (6.6%) isolates were carriers of extended-spectrum beta-lactamase (ESBL) enzymes. However, 37.2% of the isolates were multidrug-resistant (MDR). Approximately 40% of the MDR isolates belonged to only four of the dominant clones (B2-CH40-30-ST131, B2-CH40-41-ST131, C-CH4-39-ST88 and D-CH35-27-ST69). Among the remaining MDR isolates, two isolates belonged to B2-CH14-64-ST1193, i.e., the new global emergent MDR clone. Moreover, a hybrid extraintestinal pathogenic E.coli (ExPEC)/enteroaggregative isolate belonging to the A-CH11-54-ST10 clone was identifiedThis study was supported by projects: PI16/01477 from Plan Estatal de I+D+I 2013-2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación, Ministerio de Economía y Competitividad (Gobierno de España) and Fondo Europeo de Desarrollo Regional (FEDER); and ED431C2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria, (Xunta de Galicia) and FEDERS

Characterisation, antimicrobial resistance and diversity of atypical EPEC and STEC isolated from COW’S milk, cheese and dairy cattle farm environments

25 p.This study was carried out to determine the occurrence and characteristics of enteropathogenic 18 Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) strains in cow’s milk, cheese 19 and dairy cattle farm environments, and to estimate distribution of antimicrobial resistance. A 20 collection of 18 atypical EPEC -aEPEC, 14 STEC, and one E. albertii was obtained and 21 characterized from 502 samples. Occurrence of aEPEC in cow’s milk was high (>6%) whereas non- 22 O157 STEC was isolated in ca. 2% of milk samples. Detection of these diarrheagenic E. coli was absent in more than 100 cheese samples obtained from raw milk. This is the first 23 report identifying 24 E. albertii (O69:HNM) in a dairy cattle farm. Nearly one-third of aEPEC strains showed 25 antimicrobial resistance, mostly presenting a multidrug resistance pattern. One clonal complex 26 (ST20 Cplx) containing aEPEC strains from milk and faecal samples was determined. Two STEC 27 strains belonged to serotypes with importance in human disease (O91:H21 and O55:H8) and were 28 isolated from air samples which suggests a high dissemination potential. Spanish bulk tank cow’s 29 milk can constitute an important source of aEPEC strains besides STEC, bearing multiple 30 antimicrobial resistance and with high diversity of both serotypes and genetic features linked to 31 potential human infectionS

High Prevalence and Diversity of Cephalosporin-Resistant Enterobacteriaceae Including Extraintestinal Pathogenic E. coli CC648 Lineage in Rural and Urban Dogs in Northwest Spain

The aim of this work was to assess the prevalence of extended spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae in fecal samples recovered from rural and urban healthy dogs in Northwest Spain (Galicia) to identify potential high-risk clones and to molecularly characterize positive isolates regarding the genes coding for ESBL/pAmpC resistance and virulence. Thirty-five (19.6%) out of 179 dogs were positive for cephalosporin-resistant Enterobacteriaceae, including Escherichiacoli and Klebsiella pneumoniae (39 and three isolates, respectively). All the isolates were multidrug resistant, with high rates of resistance to different drugs, including ciprofloxacin (71.4%). A wide diversity of ESBL/pAmpC enzymes, as well as E. coli phylogroups (A, B1, C, D, E, F and clade I) were found. The eight isolates (20.5%) found to conform to the ExPEC status, belonged to clones O1:H45-clade I-ST770 (CH11-552), O18:H11-A-ST93-CC168 (CH11-neg), O23:H16-B1-ST453-CC86 (CH6-31), and O83:H42-F-ST1485-CC648 (CH231-58), with the latter also complying the uropathogenic (UPEC) status. The three K. pneumoniae recovered produced CTX-M-15 and belonged to the ST307, a clone previously reported in human clinical isolates. Our study highlights the potential role of both rural and urban dogs as a reservoir of high-risk Enterobacteriaceae clones, such as the CC648 of E. coli and antimicrobial resistance traits. Within a One-Health approach, their surveillance should be a priority in the fight against antimicrobial resistanceThis research was funded project FIS PI17-00728 (Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain), cofunded by the European Regional Development Fund of the European Union: a Way to Making Europe (FEDER); Project PID2019-104439RB-C21/AEI/10.13039/501100011033 and FEDER; ED431C 2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia and FEDER; and by the Strategic Researcher Cluster BioReDeS funded by the Regional Government Xunta de Galicia under the project no. ED431E 2018/09. D. Díaz-Jiménez and I. García-Meniño acknowledge the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for their pre-doctoral grants (ED481A-2019/022 and ED481A-2015/149, respectively). The Research stay of I. García-Meniño at the Hospital Universitario Central de Asturias was funded by a grant from the Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC)S