Epidemiological changes in invasive Streptococcus pyogenes infection during the UK alert period: A molecular comparative analysis from a tertiary Spanish hospital in 2023

Objectives: To study the genomic epidemiology of Streptococcus pyogenes causing bloodstream infections (GAS-BSI) in a Spanish tertiary hospital during the United Kingdom invasive S. pyogenes outbreak alert. Methods: Retrospective epidemiological analysis of GAS-BSI during the January–May 2017–2023 period. WGS was performed using Ion torrent GeneStudio™ S5 system for emm typing and identification of superantigen genes in S. pyogenes isolated during the 2022–2023 UK outbreak alert. Results: During 2023, there were more cases of GAS-BSI compared to the same period of previous year with a non-significant increase in children. Fourteen isolates were sequenced. The emm1 (6/14, 42.9%) and emm12 (2/14, 14.3%) types predominated; 5 of 6 (75%) emm1 isolates were from the M1UK clone. The most detected superantigen genes were speG (12/14, 85.7%), speC (10/14, 71.4%), speJ (7/14, 50%), and speA (5/15, 33.3%). speA and speJ were predominant in M1UK clone. Conclusions: Our genomic epidemiology in 2023 is similar to the reported data from the UK outbreak alert in the same period and different from previous national S. pyogenes surveillance reports.(AU)

Objetivos: Estudiar la epidemiología genómica de aislados de Streptococcus pyogenes causantes de bacteriemia (GAS-BSI) en un hospital de tercer nivel español durante la alerta por incremento de infecciones invasivas por S. pyogenes en el Reino Unido. Métodos: Análisis epidemiológico retrospectivo de GAS-BSI durante el periodo enero-mayo 2017-2023. Se realizó una secuenciación de genoma completo con el sistema Ion torrent GeneStudio™ S5 de los aislados obtenidos durante la alerta de brote del Reino Unido 2022-2023 para tipificación emm e identificación de genes de superantígenos. Resultados: Durante el periodo enero-mayo de 2023 hubo más casos de GAS-BSI que en el mismo periodo de años anteriores con un aumento no significativo en niños. Se secuenciaron 14 aislados. Predominaron los tipos emm1 (6/14, 42,9%) y emm12 (2/14, 14,3%); 5 de 6 (75%) aislados emm1 eran del clon M1UK. Los genes de superantígenos más detectados fueron speG (12/14, 85,7%), speC (10/14, 71,4%), speJ (7/14, 50%) y speA (5/15, 33,3%). Los genes speA y speJ predominaron en el clon M1UK. Conclusiones: Nuestra epidemiología genómica de Streptococcus pyogenes causantes de bacteriemia en 2023 es similar a los datos comunicados por el Reino Unido durante el mismo periodo y diferente de los informes nacionales previos de vigilancia.(AU)

Epidemiological changes in invasive Streptococcus pyogenes infection during the UK alert period: A molecular comparative analysis from a tertiary Spanish hospital in 2023.

OBJECTIVES: To study the genomic epidemiology of Streptococcus pyogenes causing bloodstream infections (GAS-BSI) in a Spanish tertiary hospital during the United Kingdom invasive S. pyogenes outbreak alert. METHODS: Retrospective epidemiological analysis of GAS-BSI during the January-May 2017-2023 period. WGS was performed using Ion torrent GeneStudio™ S5 system for emm typing and identification of superantigen genes in S. pyogenes isolated during the 2022-2023 UK outbreak alert. RESULTS: During 2023, there were more cases of GAS-BSI compared to the same period of previous year with a non-significant increase in children. Fourteen isolates were sequenced. The emm1 (6/14, 42.9%) and emm12 (2/14, 14.3%) types predominated; 5 of 6 (75%) emm1 isolates were from the M1UK clone. The most detected superantigen genes were speG (12/14, 85.7%), speC (10/14, 71.4%), speJ (7/14, 50%), and speA (5/15, 33.3%). speA and speJ were predominant in M1UK clone. CONCLUSIONS: Our genomic epidemiology in 2023 is similar to the reported data from the UK outbreak alert in the same period and different from previous national S. pyogenes surveillance reports.

Assessment trial of the effect of enteral insulin on the preterm infant intestinal microbiota.

BACKGROUND: Insulin might be associated with changes in infant gastrointestinal microbiota. The objective of this randomized controlled trial was to assess the efficacy of two doses of recombinant human(rh) enteral insulin administration compared to placebo in intestinal microbiota. METHODS: 19 preterm patients were recruited at the NICU of La Paz University Hospital (Madrid, Spain). Subjects received 2000 µIU of rh enteral insulin/ml(n = 8), 400 µIU of rh enteral insulin/ml(n = 6) or placebo(n = 5) for 28 days administered once per day. Extracted DNA from fecal samples collected at the beginning and end of treatment were analyzed. The 16S rRNA V4 region was amplified and sequenced in a Miseq(Illumina®) sequencer using 2 × 250 bp paired end. Resulting reads were filtered and analyzed using Qiime2 software. Metabolic activity was assessed by GC. RESULTS: Gestational age and birth weight did not differ between groups. At the phylum level, both insulin treated groups increased the relative abundance of Bacillota, while Pseudomonadota decreased. No change was observed in infants receiving placebo. At the genus level, insulin at both doses showed enriching effects on Clostridium. We found a significant increase in concentrations of fecal propionate in both rh insulin treated groups. CONCLUSION: Rh insulin may modify neonatal intestinal microbiota and SCFAs in preterm infants. IMPACT STATEMENT: Decrease of Pseudomonadota (former Proteobacteria phylum) and increase of Bacillota (former Firmicutes phylum) obtained in this study are the changes observed previously in low-risk infants for NEC. The administration of recombinant enteral insulin may modify the microbiota of preterm new-borns and SCFAs. Modulation of the microbiota may be a mechanism whereby insulin contributes to neonatal intestinal maturation and/or protection.

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.

Intestinal loads of extended-spectrum beta-lactamase and Carbapenemase genes in critically ill pediatric patients.

Introduction: Intestinal colonization by Multi-Drug Resistant Organisms (MDROs) can pose a threat on the health of critically ill patients. The extent of colonization by these organisms is related to previous antibiotic treatments and their ability to cause infections among adult patients. The aim of this study is to determine the relationship between the intestinal Relative Loads (RLs) of selected antibiotic resistance genes, antibiotic consumption and extra-intestinal spread among critically ill pediatric patients. Methods: RLs of bla CTX-M-1-Family, bla OXA-1, bla OXA-48 and bla VIM were determined in 382 rectal swabs obtained from 90 pediatric critically ill patients using qPCRs. The RLs were compared to the patients' demographics, antibiotic consumption, and detection of MDROs from extra-intestinal sites. 16SrDNA metagenomic sequencing was performed for 40 samples and clonality analyses were done for representative isolates. Results and discussion: 76 (74.45%) patients from which 340 (89.01%) rectal swabs were collected had at least one swab that was positive for one of the tested genes. Routine cultures did not identify carbapenemases in 32 (45.1%) and 78 (58.2%) swabs that were positive by PCR for bla OXA-48 and blaVIM, respectively. RLs of above 6.5% were associated with extra-intestinal spread of blaOXA-48-harboring MDROs. Consumption of carbapenems, non-carbapenem ß-lactams, and glycopeptides were statistically associated with testing negative for bla CTX-M-1-Family and bla OXA-1 while the consumption of trimethoprim/sulfamethoxazole and aminoglycosides was associated with testing negative for blaOXA-48 (P<0.05). In conclusion, targeted qPCRs can be used to determine the extent of intestinal dominance by antibiotic resistant opportunistic pathogens and their potential to cause extra-intestinal infections among a critically ill pediatric population.

Intestinal Dominance by Multidrug-Resistant Bacteria in Pediatric Liver Transplant Patients.

Pediatric liver transplantation (PLTx) is commonly associated with extensive antibiotic treatments that can produce gut microbiome alterations and open the way to dominance by multidrug-resistant organisms (MDROs). In this study, the relationship between intestinal Relative Loads (RLs) of ß-lactamase genes, antibiotic consumption, microbiome disruption, and the extraintestinal dissemination of MDROs among PLTx patients is investigated. 28 PLTx patients were included, from whom 169 rectal swabs were collected. Total DNA was extracted and blaCTX-M-1-Family, blaOXA-1, blaOXA-48, and blaVIM were quantified via quantitative polymerase chain reaction (qPCR) and normalized to the total bacterial load (16SrRNA) through LogΔΔCt to determine the RLs. 16SrRNA sequencing was performed for 18 samples, and metagenomic sequencing was performed for 2. Patients' clinical data were retrieved from the hospital's database. At least one of the genes tested were detected in all of the patients. The RLs for blaCTX-M-1-Family, blaOXA-1, blaOXA-48, and blaVIM were higher than 1% of the total bacterial population in 67 (80.73%), 56 (78.87%), 57 (77.03%) and 39 (61.9%) samples, respectively. High RLs for blaCTX-M-1-Family, blaOXA-1, and/or blaOXA-48, were positively associated with the consumption of carbapenems with trimethoprim-sulfamethoxazole and coincided with low diversity in the gut microbiome. Low RLs were associated with the consumption of noncarbapenem ß-lactams with aminoglycosides (P < 0.05). Extraintestinal isolates harboring the same gene(s) as those detected intraintestinally were found in 18 samples, and the RLs of the respective swabs were high. We demonstrated a relationship between the consumption of carbapenems with trimethoprim-sulfamethoxazole, intestinal dominance by MDROs and extraintestinal spread of these organisms among PLTx patients. IMPORTANCE In this study, we track the relative intestinal loads of antibiotic resistance genes among pediatric liver transplant patients and determine the relationship between this load, antibiotic consumption, and infections caused by antibiotic-resistant organisms. We demonstrate that the consumption of broad spectrum antibiotics increase this load and decrease the gut microbial diversity among these patients. Moreover, the high loads of resistance genes were related to the extraintestinal spread of multidrug-resistant organisms. Together, our data show that the tracking of the relative intestinal loads of antibiotic resistance genes can be used as a biomarker that has the potential to stop the extraintestinal spread of antibiotic-resistant bacteria via the measurement of the intestinal dominance of these organisms, thereby allowing for the application of preventive measures.

Mycobacterium mageritense Lymphadenitis in Child.

Although human infections caused by Mycobacterium mageritense are rare, there are some case reports involving sinusitis, pneumonia, and hospital-acquired infections in adults. We report a case of lymphadenitis caused by M. mageritense in a child in Spain.

Intestinal Dominance by Serratia marcescens and Serratia ureilytica 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 < 0.05) and in the first swab compared to the last (26.9% vs. 4.37%, p < 0.05). Nine neonates had extraintestinal detection of 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 < 0.05). (4) Conclusions: Intestinal dominance by Serratia spp. plays a role in outbreaks and extraintestinal spread.

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.

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

BACKGROUND: Two automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated. METHODS: One 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. RESULTS: No 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. CONCLUSION: The 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.

Intestinal loads of OXA-48-producing Klebsiella pneumoniae in colonized patients determined from surveillance rectal swabs.

OBJECTIVES: To determine quantitatively the extent of intestinal colonization by OXA-48-producing Klebsiella pneumoniae (KpOXA) in hospitalized patients. METHODS: The load of the OXA-48 ß-lactamase gene in rectal swabs from 147 colonized patients was measured by quantitative PCR. The load was calculated relative to the total bacterial population (represented by the 16S rRNA gene) using the ΔΔCt method and pure cultures of OXA-48-producing K. pneumoniae as reference samples. The relative loads of the epidemic K. pneumoniae clones ST11 and ST405 were also measured. RESULTS: The relative intestinal loads of the OXA-48 ß-lactamase gene, RLOXA-48, in hospitalized patients were high. The median RLOXA-48 was -0.42 (95% confidence interval (CI): -0.60 to -0.16), close to that of a pure culture of OXA-48-producing K. pneumoniae (RLOXA-48 = 0). In those patients colonized by the KpOXA clones ST11 (51/147, 34.7%) and ST405 (14/147, 9.5%), the relative loads of these clones were similarly high (median RLST11 = -1.1, 95% CI: -1.64 to -0.92; median RLST405 = -1.3, 95% CI: -1.76 to -0.96). Patients that had received previous antibiotic treatments and those that developed infections by KpOXA had significantly higher RLOXA-48 values: -0.32 (95% CI: -0.58 to -0.20) vs -1.07 (95% CI: -2.43 to -0.35) and -0.26 (-0.77 to -0.23) vs -0.47 (-0.74 to -0.28), respectively. CONCLUSIONS: Colonization by KpOXA in hospital patients involves intestinal loads much higher than the K. pneumoniae loads reported in the normal microbiota, reaching levels close to those of pure KpOXA cultures in many cases and largely replacing the host microbiota.

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.

Proof-of-concept trial of the combination of lactitol with Bifidobacterium bifidum and Lactobacillus acidophilus for the eradication of intestinal OXA-48-producing Enterobacteriaceae.

BACKGROUND: The major reservoir of carbapenemase-producing Enterobacteriaceae (CPE) is the gastrointestinal tract of colonized patients. Colonization is silent and may last for months, but the risk of infection by CPE in colonized patients is significant. METHODS: Eight long-term intestinal carriers of OXA-48-producing Enterobacteriaceae (OXA-PE) were treated during 3 weeks with daily oral lactitol (Emportal®), Bifidobacterium bifidum and Lactobacillus acidophilus (Infloran®). Weekly stool samples were collected during the treatment period and 6 weeks later. The presence of OXA-PE was investigated by microbiological cultures and qPCR. RESULTS: At the end of treatment (EoT, secondary endpoint 1), four of the subjects had negative OXA-PE cultures. Three weeks later (secondary endpoint 2), six subjects were negative. Six weeks after the EoT (primary endpoint), three subjects had negative OXA-PE cultures. The relative intestinal load of OXA-PE decreased in all the patients during treatment. CONCLUSIONS: The combination of prebiotics and probiotics was well tolerated. A rapid reduction on the OXA-PE intestinal loads was observed. At the EoT, decolonization was achieved in three patients.Clinical Trials Registration: NCT02307383. EudraCT Number: 2014-000449-65.

Metagenomic Detection of Two Vientoviruses in a Human Sputum Sample.

We used metagenomics to analyze one sputum sample from a patient with symptoms of a respiratory infection that yielded negative results for all pathogens tested. We detected two viral genomes that could be assembled and showed sequence similarity to redondoviruses, a recently described group within the CRESS-DNA viruses. One hundred sputum samples were screened for the presence of these viruses using specific primers. One sample was positive for the same two viruses, and another was positive for one of them. These findings raise questions about a possible role of redondoviruses in respiratory infections in humans.

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.