Imperial College London

MrRobertoFernandez Crespo

Faculty of MedicineDepartment of Surgery & Cancer

Analytics Fellow, BDAU
 
 
 
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Contact

 

roberto.fernandez-crespo13

 
 
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Location

 

1069Queen Elizabeth the Queen Mother Wing (QEQM)St Mary's Campus

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Summary

 

Publications

Publication Type
Year
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10 results found

Clarke J, Flott K, Crespo R, Ashrafian H, Fontana G, Benger J, Darzi A, Elkin Set al., 2021, Assessing the Safety of Home Oximetry for Covid-19: A multi-site retrospective observational study, BMJ Open, Vol: 11, Pages: 1-9, ISSN: 2044-6055

Objectives To determine the safety and effectiveness of home oximetry monitoring pathways safe for Covid-19 patients in the English NHS.Design Retrospective, multi-site, observational study of home oximetry monitoring for patients with suspected or proven Covid-19 Setting This study analysed patient data from four Covid-19 home oximetry pilot sites in England across primary and secondary care settings.Participants A total of 1338 participants were enrolled in a home oximetry programme across four pilot sites. Participants were excluded if primary care data and oxygen saturations are rest at enrolment were not available. Data from 908 participants was included in the analysis. Interventions Home oximetry monitoring was provided to participants with a known or suspected diagnosis of Covid-19. Participants were enrolled following attendance to emergency departments, hospital admission or referral through primary care services. Results Of 908 patients enrolled into four different Covid-19 home oximetry programmes in England, 771 (84.9%) had oxygen saturations at rest of 95% or more, and 320 (35.2%) were under 65 years of age and without comorbidities. 52 (5.7%) presented to hospital and 28 (3.1%) died following enrolment, of which 14 (50%) had Covid-19 as a named cause of death. All-cause mortality was significantly higher in patients enrolled after admission to hospital (OR 8.70 [2.53-29.89]), compared to those enrolled in primary care. Patients enrolled after hospital discharge (OR 0.31 [0.15-0.68]) or emergency department presentation (OR 0.42 [0.20-0.89]) were significantly less likely to present to hospital than those enrolled in primary care. ConclusionsThis study find that home oximetry monitoring can be a safe pathway for Covid-19 patients; and indicates increases in risk to vulnerable groups and patients with oxygen saturations < 95% at enrolment, and in those enrolled on discharge from hospital. Findings from this evaluation have contributed to the national

Journal article

Bosse JT, Li Y, Fernandez Crespo R, Angen O, Holden MTG, Weinert LA, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, Langford PR, Consortium Bet al., 2020, Draft genome sequences of the type strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319), two NAD-dependent bacterial species found in the respiratory tract of pigs, Microbiology Resource Announcements, Vol: 9, Pages: 1-3, ISSN: 2576-098X

We report here the draft genome sequences of the type strains of Actinobacillus indolicus (46K2CT) and Actinobacillus porcinus (NM319T). These NAD-dependent bacterial species are frequently found in the upper respiratory tract of pigs and are occasionally associated with lung pathology.

Journal article

Powell E, Fontanella S, Boakes E, Belgrave D, Shaw A, Cornwell E, Fernandez-Crespo R, Fink C, Custovic A, Kroll JSet al., 2019, Temporal association of the development of oropharyngeal microbiota with early life wheeze in a population-based birth cohort, EBioMedicine, Vol: 46, Pages: 486-498, ISSN: 2352-3964

Background A critical window in infancy has been proposed, during which the microbiota may affect subsequent health. The longitudinal development of the oropharyngeal microbiota is under-studied and may be associated with early-life wheeze. We aimed to investigate the temporal association of the development of the oropharyngeal microbiota with early-life wheeze.Methods A population-based birth cohort based in London, UK was followed for 24 months. We collected oropharyngeal swabs at six time-points. Microbiota was determined using sequencing of the V3-V5 region of the 16S rRNA-encoding gene. Medical records were reviewed for the outcome of doctor diagnosed wheeze. We used a time-varying model to investigate the temporal association between the development of microbiota and doctor-diagnosed wheeze. Findings 159 participants completed the study to 24 months and for 98 there was complete sequencing data at all timepoints and outcome data. Of these, 26 had doctor-diagnosed wheeze. We observed significant increase in the abundance of Neisseria between 9 and 24 months in children who developed wheeze (p=0∙003), while in those without wheezing there was a significant increment in the abundance of Granulicatella (p=0 ∙012) between 9 and 12 months, and of Prevotella (p=0 ∙018) after 18 months. Interpretation A temporal association between the respiratory commensal Granulicatella and also Prevotella with wheeze (negative), and between Neisseria and wheeze (positive) was identified in infants prior to one year of age. This adds to evidence for the proposed role of the microbiota in the development of wheeze.

Journal article

Li Y, da Silva GC, Li Y, Rossi C, Fernandez Crespo R, Williamson S, Langford P, Bazzolli D, Bosse Jet al., 2018, Evidence of illegitimate recombination between two Pasteurellaceae plasmids resulting in a novel multi-resistance replicon, pM3362MDR, in Actinobacillus pleuropneumoniae, Frontiers in Microbiology, Vol: 9, ISSN: 1664-302X

Evidence of plasmids carrying the tetracycline resistance gene, tet(B), was found in the previously reported whole genome sequences of 14 United Kingdom, and 4 Brazilian, isolates of Actinobacillus pleuropneumoniae. Isolation and sequencing of selected plasmids, combined with comparative sequence analysis, indicated that the four Brazilian isolates all harbor plasmids that are nearly identical to pB1001, a plasmid previously found in Pasteurella multocida isolates from Spain. Of the United Kingdom isolates, 13/14 harbor plasmids that are (almost) identical to pTetHS016 from Haemophilus parasuis. The remaining United Kingdom isolate, MIDG3362, harbors a 12666 bp plasmid that shares extensive regions of similarity with pOV from P. multocida (which carries blaROB-1, sul2, and strAB genes), as well as with pTetHS016. The newly identified multi-resistance plasmid, pM3362MDR, appears to have arisen through illegitimate recombination of pTetHS016 into the stop codon of the truncated strB gene in a pOV-like plasmid. All of the tet(B)-carrying plasmids studied were capable of replicating in Escherichia coli, and predicted origins of replication were identified. A putative origin of transfer (oriT) sequence with similar secondary structure and a nic-site almost identical to that of RP4 was also identified in these plasmids, however, attempts to mobilize them from an RP4-encoding E. coli donor strain were not successful, indicating that specific conjugation machinery may be required.

Journal article

Bosse J, Li Y, Fernandez Crespo R, Lacouture S, Gottschalk M, Sarkozi R, Fodor L, Casas Amoribieta M, Angen O, Nedbalcova K, Holden MTG, Duncan MJ, Tucker AW, Wren BW, Rycroft AN, Langford PRet al., 2018, Comparative sequence analysis of the capsular polysaccharide loci of Actinobacillus pleuropneumoniae serovars 1-18, and development of two multiplex PCRs for comprehensive capsule typing, Veterinary Microbiology, Vol: 220, Pages: 83-89, ISSN: 0378-1135

Problems with serological cross-reactivity have led to development of a number of PCRs (individual and multiplex) for molecular typing of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Most of these assays were developed for detection of specific amplicons within capsule biosynthetic genes before the availability of complete sequences for the different serovars. Here we describe comparative analysis of the complete capsular loci for all 18 serovars of A. pleuropneumoniae, and development of two multiplex PCRs for comprehensive capsule typing of this important pig pathogen.

Journal article

Li Y, Li Y, Fernandez Crespo R, Leanse LG, Langford PR, Bosse JTet al., 2017, Characterisation of the Actinobacillus pleuropneumoniae SXT-related Integrative and Conjugative Element ICEApl2, and analysis of the encoded FloR protein: hydrophobic residues in transmembrane domains contribute dynamically to florfenicol and chloramphenicol efflux, Journal of Antimicrobial Chemotherapy, Vol: 73, Pages: 57-65, ISSN: 0305-7453

ObjectivesTo characterize ICEApl2, an SXT-related integrative and conjugative element (ICE) found in a clinical isolate of the porcine pathogen Actinobacillus pleuropneumoniae, and analyse the functional nature of the encoded FloR.MethodsICEApl2 was identified in the genome of A. pleuropneumoniae MIDG3553. Functional analysis was done using conjugal transfer experiments. MIDG3553 was tested for susceptibility to the antimicrobials for which resistance genes are present in ICEApl2. Lack of florfenicol/chloramphenicol resistance conferred by the encoded FloR protein was investigated by cloning and site-directed mutagenesis experiments in Escherichia coli.ResultsICEApl2 is 92 660 bp and contains 89 genes. Comparative sequence analysis indicated that ICEApl2 is a member of the SXT/R391 ICE family. Conjugation experiments showed that, although ICEApl2 is capable of excision from the chromosome, it is not self-transmissible. ICEApl2 encodes the antimicrobial resistance genes floR, strAB, sul2 and dfrA1, and MIDG3553 is resistant to streptomycin, sulfisoxazole and trimethoprim, but not florfenicol or chloramphenicol. Cloning and site-directed mutagenesis of the floR gene revealed the importance of the nature of the hydrophobic amino acid residues at positions 160 and 228 in FloR for determining resistance to florfenicol and chloramphenicol.ConclusionsOur results indicate that the nature of hydrophobic residues at positions 160 and 228 of FloR contribute dynamically to specific efflux of florfenicol and chloramphenicol, although some differences in resistance levels may depend on the bacterial host species. This is also, to our knowledge, the first description of an SXT/R391 ICE in A. pleuropneumoniae or any member of the Pasteurellaceae.

Journal article

Bosse JT, Li Y, Rogers J, Fernandez Crespo R, Li Y, Chaudhuri R, Holden MTG, Maskell DJ, Tucker AW, Wren BW, Rycroft AR, Langford PR, BRaDP1T Consortiumet al., 2017, Whole genome sequencing for surveillance of antimicrobial resistance in Actinobacillus pleuropneumoniae, Frontiers in Microbiology, Vol: 8, ISSN: 1664-302X

The aim of this study was to evaluate the correlation between antimicrobial resistance (AMR) profiles of 96 clinical isolates of Actinobacillus pleuropneumoniae, an important porcine respiratory pathogen, and the identification of AMR genes in whole genome sequence (wgs) data. Susceptibility of the isolates to nine antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, sulfisoxazole, tetracycline, tilmicosin, trimethoprim, and tylosin) was determined by agar dilution susceptibility test. Except for the macrolides tested, elevated MICs were highly correlated to the presence of AMR genes identified in wgs data using ResFinder or BLASTn. Of the isolates tested, 57% were resistant to tetracycline [MIC ≥ 4 mg/L; 94.8% with either tet(B) or tet(H)]; 48% to sulfisoxazole (MIC ≥ 256 mg/L or DD = 6; 100% with sul2), 20% to ampicillin (MIC ≥ 4 mg/L; 100% with blaROB-1), 17% to trimethoprim (MIC ≥ 32 mg/L; 100% with dfrA14), and 6% to enrofloxacin (MIC ≥ 0.25 mg/L; 100% with GyrAS83F). Only 33% of the isolates did not have detectable AMR genes, and were sensitive by MICs for the antimicrobial agents tested. Although 23 isolates had MIC ≥ 32 mg/L for tylosin, all isolates had MIC ≤ 16 mg/L for both erythromycin and tilmicosin, and no macrolide resistance genes or known point mutations were detected. Other than the GyrAS83F mutation, the AMR genes detected were mapped to potential plasmids. In addition to presence on plasmid(s), the tet(B) gene was also found chromosomally either as part of a 56 kb integrative conjugative element (ICEApl1) in 21, or as part of a Tn7 insertion in 15 isolates. Our results indicate that, with the exception of macrolides, wgs data can be used to accurately predict resistance of A. pleuropneumoniae to the tested antimicrobial agents and provides added value for routine surveillance.

Journal article

Bosse JT, Li Y, Fernandez Crespo R, Chaudhuri RR, Rogers J, Holden MTG, Maskell DJ, Tucker AW, Rycroft AN, Langford PRet al., 2016, ICEApl1, an integrative conjugative element related to ICEHin1056, identified in the pig pathogen Actinobacillus pleuropneumoniae, Frontiers in Microbiology, Vol: 7, ISSN: 1664-302X

ICEApl1 was identified in the whole genome sequence of MIDG2331, a tetracycline-resistant (MIC = 8 mg/L) serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. PCR amplification of virB4, one of the core genes involved in conjugation, was used to identify other A. pleuropneumoniae isolates potentially carrying ICEApl1. MICs for tetracycline were determined for virB4 positive isolates, and shotgun whole genome sequence analysis was used to confirm presence of the complete ICEApl1. The sequence of ICEApl1 is 56083 bp long and contains 67 genes including a Tn10 element encoding tetracycline resistance. Comparative sequence analysis was performed with similar integrative conjugative elements (ICEs) found in other members of the Pasteurellaceae. ICEApl1 is most similar to the 59393 bp ICEHin1056, from Haemophilus influenzae strain 1056. Although initially identified only in serovar 8 isolates of A. pleuropneumoniae (31 from the UK and 1 from Cyprus), conjugal transfer of ICEApl1 to representative isolates of other serovars was confirmed. All isolates carrying ICEApl1 had a MIC for tetracycline of 8 mg/L. This is, to our knowledge, the first description of an ICE in A. pleuropneumoniae, and the first report of a member of the ICEHin1056 subfamily in a non-human pathogen. ICEApl1 confers resistance to tetracycline, currently one of the more commonly used antibiotics for treatment and control of porcine pleuropneumonia.

Journal article

Bosse JT, Chaudhiri RR, Li Y, Leanse LG, Fernandez Crespo R, Coupland P, Holden MTG, Bazzolli DM, Tucker AW, Wren BW, Rycroft AN, Langford PRet al., 2016, The complete genome sequence of MIDG2331, a genetically tractable serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, Genome Announcements, Vol: 4, ISSN: 2169-8287

We report here the complete annotated genome sequence of a clinical serovar 8 isolate Actinobacillus pleuropneumoniae MIDG2331. Unlike the serovar 8 reference strain 405, MIDG2331 is amenable to genetic manipulation via natural transformation as well as conjugation, making it ideal for studies of gene function.

Journal article

Bosse J, Li Y, Walker S, Atherton T, Fernandez Crespo R, Williamson SM, Rogers J, Chaudhuri RR, Weinert LA, Oshota O, Holden MTG, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, Langford PR, BRaDP1T Consortiumet al., 2015, Identification of dfrA14 in two distinct plasmids conferring trimethoprimresistance in Actinobacillus pleuropneumoniae, Journal of Antimicrobial Chemotherapy, Vol: 70, Pages: 2217-2222, ISSN: 1460-2091

Objectives The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England.Methods Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids.Results A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene.Conclusions This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial.

Journal article

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