Aplicación de técnicas de qPCR para la cuantificación relativa de enterobacterias

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Medicina Preventiva y Salud Pública y Microbiología. Fecha de Lectura: 22-06-2021La colonización por enterobacterias productoras de carbapenemasas (EPCs) durante el ingreso hospitalario es un problema creciente a nivel mundial. Aunque el proceso de colonización es silente, los portadores rectales de EPCs tienen un mayor riesgo de desarrollar infecciones por estas bacterias y por tanto es necesario contener su propagación en los hospitales. Para ello, se implantan medidas higiénicas, la detección de portadores asintomáticos mediante el cultivo de torundas rectales y en ocasiones estrategias de descolonización como los trasplantes fecales, el uso de antibióticos orales no absorbibles o la administración de probióticos. En la práctica habitual, los resultados de colonización o el éxito en la descolonización se expresan como un resultado dicotómico (positivo o negativo). En esta tesis planteamos estudiar la carga intestinal de Klebsiella pneumoniae productora de la carbapenemasa OXA-48 (KpOXA-48) en pacientes colonizados mediante el uso del método 2-ΔΔCt basado en qPCR. Este método nos ha permitido diferenciar aquellos pacientes en los que su microbiota ha sido totalmente desplazada por KpOXA-48 de aquellos en los que estas bacterias suponen una pequeña fracción del total de bacterias intestinales. Además, analizamos aquellos factores que pueden modificar la carga intestinal de estas bacterias y sus posibles consecuencias clínicas. Igualmente, hemos aplicado este método en un ensayo clínico piloto de descolonización rectal de enterobacterias productoras de carbapenemasa OXA-48 mediante la administración de probióticos y prebióticos para determinar los efectos del tratamiento más allá de la descolonización absoluta. Y finalmente abrimos la posibilidad de aplicar esta técnica en otros contextos como el seguimiento de la carga rectal de Serratia marcescens durante un brote en varias unidades de neonatos

Genomics of Serratia marcescens isolates causing outbreaks in the same pediatric unit 47 years apart: Position in an updated phylogeny of the species

The first documented nosocomial outbreak caused by Serratia marcescens in Spain occurred in 1969 at the neonatal intensive care unit (NICU) of the tertiary La Paz Children’s Hospital in Madrid, Spain, and based on the available phenotyping techniques at this time, it was considered as a monoclonal outbreak. Only 47 years later, another S. marcescens outbreak of an equivalent dimension occurred at the same NICU. The aim of the present study was to study isolates from these historical and contemporary outbreaks by phenotypic analysis and whole-genome sequencing techniques and to position these strains along with 444 publicly available S. marcescens genomes, separately comparing core genome and accessory genome contents. Clades inferred by both approaches showed high correlation, indicating that core and accessory genomes seem to evolve in the same manner for S. marcescens. Nine S. marcescens clusters were identified, and isolates were grouped in two of them according to sampling year. One exception was isolate 13F-69, the most genetically distant strain, located in a different cluster. Categorical functions in the annotated accessory genes of both collections were preserved among all isolates. No significant differences in frequency of insertion sequences in historical (0.18–0.20)—excluding the outlier strain—versus contemporary isolates (0.11–0.19) were found despite the expected resting effect. The most dissimilar isolate, 13F-69, contains a highly preserved plasmid previously described in Bordetella bronchiseptica. This strain exhibited a few antibiotic resistance genes not resulting in a resistant phenotype, suggesting the value of gene down expression in adaptation to long-term starvation.CS was supported by “Fundación Mutua Madrileña” grant to RC achieved in 2017 call with reference number AP165902017. MP-A was supported by the Programa Operativo de Empleo Juvenil, cofinanced by the European Social Fund Investing in your future (ESF) and ERDF (PEJD-2018-PRE/BMD-8237). BP-V was funded by H2020 FTIPilot 2016 project no. 730713 “FAST-bact “A novel fast and automated test for antibiotic susceptibility testing for Gram positive and negative bacteria” and co-funded by Instituto de Salud Carlos III and the European Regional Development Fund (ERDF, “A way to achieve Europe”). FB was supported by grants from the Madrid Regional Government (InGEMICS-C; S2017/BMD-3691) and CIBER (CIBER in Epidemiology and Public Health, CIBERESP; CB06/02/0053), co-funded by Instituto de Salud Carlos III and the European Regional Development Fund (ERDF, “A way to achieve Europe”). This work was supported by the Instituto de Salud Carlos III, PI17/00115 (RC), and REIPI (RD16/0016/0011) actions, cofinanced by the European Development Regional Fund “A way to achieve Europe” (ERDF

Genomic epidemiology of SARS-CoV-2 in Madrid, Spain, during the first wave of the pandemic: Fast spread and early dominance by D614G variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Madrid, Spain, on 25 February 2020. It increased in frequency very fast and by the end of May more than 70,000 cases had been confirmed by reverse transcription-polymerase chain reaction (RT-PCR). To study the lineages and the diversity of the viral population during this first epidemic wave in Madrid we sequenced 224 SARS-CoV-2 viral genomes collected from three hospitals from February to May 2020. All the known major lineages were found in this set of samples, though B.1 and B.1.5 were the most frequent ones, accounting for more than 60% of the sequences. In parallel with the B lineages and sublineages, the D614G mutation in the Spike protein sequence was detected soon after the detection of the first coronavirus disease 19 (COVID-19) case in Madrid and in two weeks became dominant, being found in 80% of the samples and remaining at this level during all the study periods. The lineage composition of the viral population found in Madrid was more similar to the European population than to the publicly available Spanish data, underlining the role of Madrid as a national and international transport hub. In agreement with this, phylodynamic analysis suggested multiple independent entries before the national lockdown and air transportation restrictions.This research was partially supported through the European Commission Horizon 2020 Framework Programme (VIRUSCAN, FETPROACT-2016). E.D. was recipient of a Marie Skłodowska- Curie Individual Fellowship (Grant Agreement number 796084). E.V. is supported by the Subprograma Juan Rodés, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain (JR18/00048). R.R. is supported by the Subprograma Río Hortega, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain (CM19/00229)

Intestinal Dominance by <i>Serratia marcescens</i> and <i>Serratia ureilytica</i> among Neonates in the Setting of an Outbreak

(1) Background: We determined the relevance of intestinal dominance by Serratia spp. during a neonatal outbreak over 13 weeks. (2) Methods: Rectal swabs (n = 110) were obtained from 42 neonates. Serratia spp. was cultured from swabs obtained from 13 neonates (Group 1), while the other 29 neonates were culture-negative (Group 2). Total DNA was extracted from rectal swabs, and quantitative PCRs (qPCRs) using Serratia- and 16SrRNA-gene-specific primers were performed. relative intestinal loads (RLs) were determined using ΔΔCt. Clonality was investigated by random amplified polymorphic DNA analysis and whole-genome sequencing. (3) Results: The outbreak was caused by Serratia marcescens during the first eight weeks and Serratia ureilytica during the remaining five weeks. Serratia spp. were detected by qPCR in all Group 1 neonates and eleven Group 2 neonates. RLs of Serratia spp. were higher in Group 1 as compared to Group 2 (6.31% vs. 0.09%, p p Serratia spp.; eight of them were infected. RLs of the patients with extraintestinal spread were higher than the rest (2.79% vs. 0.29%, p Serratia spp. plays a role in outbreaks and extraintestinal spread

Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection.

BackgroundTwo automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated.MethodsOne hundred forty one SARS-CoV-2 positive samples were collected during a period of 10-days. In-house protocols were based on extraction with magnetic beads and designed to be used with either the Opentrons OT-2 (OT-2in-house) liquid handling robot or the MagMAXTM Express-96 system (MMin-house). Both protocols were tested in parallel with a commercial kit that uses the MagMAXTM system (MMkit). Nucleic acid extraction efficiencies were calculated from a SARS-CoV-2 DNA positive control.ResultsNo significant differences were found between both in-house protocols and the commercial kit in their performance to detect positive samples. The MMkit was the most efficient although the MMin-house presented, in average, lower Cts than the other two. In-house protocols allowed to save between 350€ and 400€ for every 96 extracted samples compared to the commercial kit.ConclusionThe protocols described harness the use of easily available reagents and an open-source liquid handling system and are suitable for SARS-CoV-2 detection in high throughput facilities

Influence of a Serratia marcescens outbreak on the gut microbiota establishment process in low-weight preterm neonates.

Adequate gut microbiota establishment is important for lifelong health. The aim was to sequentially analyze the gut microbiota establishment in low-birth-weight preterm neonates admitted to a single neonatal intensive care unit during their first 3 weeks of life, comparing two epidemiological scenarios. Seven control infants were recruited, and another 12 during a severe S. marcescens outbreak. Meconium and feces from days 7, 14, and 21 of life were collected. Gut microbiota composition was determined by 16S rDNA massive sequencing. Cultivable isolates were genotyped by pulsed-field gel electrophoresis, with four S. marcescens submitted for whole-genome sequencing. The expected bacterial ecosystem expansion after birth is delayed, possibly related to antibiotic exposure. The Proteobacteria phylum dominates, although with marked interindividual variability. The outbreak group considerably differed from the control group, with higher densities of Escherichia coli and Serratia to the detriment of Enterococcus and other Firmicutes. Curiously, obligate predators were only detected in meconium and at very low concentrations. Genotyping of cultivable bacteria demonstrated the high bacterial horizontal transmission rate that was confirmed with whole-genome sequencing for S. marcescens. Preterm infants admitted at NICU are initially colonized by homogeneous microbial communities, most of them from the nosocomial environment, which subsequently evolve according to the individual conditions. Our results demonstrate the hospital epidemiology pressure, particularly during outbreak situations, on the gut microbiota establishing process

Usefulness of guideline recommendations for prognosis in patients with candidemia

We aimed to analyze whether the lack of inclusion of specific recommendations for the management of candidemia is an independent risk factor for early and overall mortality. Multicenter study of adult patients with candidemia in 13 hospitals. We assessed the proportion of patients on whom nine specific ESCMID and IDSA guidelines recommendations had been applied, and analyzed its impact on mortality. 455 episodes of candidemia were documented. Patients who died within the first 48 hours were excluded. Sixty-two percent of patients received an appropriate antifungal treatment. Either echinocandin or amphotericin B therapy were administered in 43% of patients presenting septic shock and in 71% of those with neutropenia. Sixty-one percent of patients with breakthrough candidemia underwent a change in antifungal drug class. Venous catheters were removed in 79% of cases. Follow-up blood cultures were performed in 72% of cases. Ophthalmoscopy and echocardiogram were performed in 48% and 50% of patients, respectively. Length of treatment was appropriate in 78% of cases. Early (2-7 days) and overall (2-30 days) mortality were 8% and 27.7%, respectively. Inclusion of less than 50% of the specific recommendations was independently associated with a higher early (HR = 7.02, 95% CI: 2.97-16.57; P &lt; .001) and overall mortality (HR = 3.55, 95% CI: 2.24-5.64; P &lt; .001). In conclusion, ESCMID and IDSA guideline recommendations were not performed on a significant number of patients. Lack of inclusion of these recommendations proved to be an independent risk factor for early and overall mortality

Systematic influenza screening in cardiac intensive care units during the influenza season: A prospective study in Spain

Background: Little is known about the incidence of influenza among admissions to the cardiac intensive care unit (C-ICU), accuracy of clinical suspicion, and influenza vaccination uptake. We evaluated the incidence of influenza at C-ICU admission during the influenza season, potential underdiagnosis, and vaccination uptake. Methods: Prospective study at five C-ICUs during the 2017-2020 influenza seasons. A nasopharyngeal swab was collected at admission from patients who consented (n = 788). Testing was with Xpert®XpressFlu/RSV. Results: Influenza was detected in 43 patients (5.5%) (40 FluA; 3 FluB) and clinically suspected in 27 (62.8%). Compared to patients without influenza, patients with influenza more frequently had heart failure (37.2% vs 22.8%, P = 0.031), previous contact with relatives with influenza-like illnesses (23.3% vs 12.5%, P = 0.042), antimicrobial use (67.4% vs 23.2%, P <0.01), and need for mechanical ventilation (25.6% vs 14.5%, P = 0.048). Patients received oseltamivir promptly. We found no differences in mortality (11.6% vs 5.2%, P = 0.076). Patients with influenza more frequently had myocarditis (9.3% vs 0.9%, P <0.01) and pericarditis (7.0% vs 0.8%, P = 0.01). Overall, 43.0% of patients (339/788) were vaccinated (51.9% of those with a clear indication [303/584]). Conclusion: Influenza seems to be a frequently underdiagnosed underlying condition in admissions to the C-ICU. Influenza should be screened for at C-ICU admission during influenza epidemics