Antimicrobial Stewardship in the Emergency Department Observation Unit: Definition of a New Indicator and Evaluation of Antimicrobial Use and Clinical Outcomes.

We aimed to define a novel indicator for monitoring antimicrobial use specifically in the Emergency Department Observation Unit (EDOU) and to assess the long-term impact of an institutional education-based antimicrobial stewardship program (ASP) on the antimicrobial prescribing pattern and clinical outcomes in this setting. A quasi-experimental interrupted time-series study was performed from 2011 to 2022. An educational ASP was implemented at the EDOU in 2015. To estimate changes in antimicrobial use, we designed an indicator adjusted for patients at risk of antimicrobial prescribing: defined daily doses (DDDs) per 100 patients transferred from the Emergency Department to the Observation Unit (TOs) per quarter. The number of bloodstream infections (BSIs) and the crude all-cause 14-day mortality were assessed as clinical outcomes. Antimicrobial use showed a sustained reduction with a trend change of -1.17 DDD per 100 TO and a relative effect of -45.6% (CI95% -64.5 to -26.7), particularly relevant for meropenem and piperacillin-tazobactam, with relative effects of -80.4% (-115.0 to -45.7) and -67.9% (-93.9 to -41.9), respectively. The incidence density of all BSIs increased significantly during the ASP period, with a relative effect of 123.2% (41.3 to 284.7). The mortality rate remained low and stable throughout the study period, with an absolute effect of -0.7% (-16.0 to 14.7). The regular monitoring of antimicrobial use in the EDOU by using this new quantitative indicator was useful to demonstrate that an institutional education-based ASP successfully achieved a long-term reduction in overall antimicrobial use, with a low and steady BSI mortality rate.

C-terminal deletion of RelA protein is suggested as a possible cause of infective endocarditis recurrence with Enterococcus faecium.

Infective endocarditis (IE) caused by Enterococcus spp. represents the third most common cause of IE, with high rates of relapse compared with other bacteria. Interestingly, late relapses (>6 months) have only been described in Enterococcus faecalis, but here we describe the first reported IE relapse with Enterococcus faecium more than a year (17 months) after the initial endocarditis episode. Firstly, by multi locus sequence typing (MLST), we demonstrated that both isolates (EF646 and EF641) belong to the same sequence type (ST117). Considering that EF641 was able to overcome starvation and antibiotic treatment conditions surviving for a long period of time, we performed bioinformatic analysis in identifying potential genes involved in virulence and stringent response. Our results showed a 13-nucleotide duplication (positions 1638-1650) in the gene relA, resulting in a premature stop codon, with a loss of 167 amino acids from the C-terminal domains of the RelA enzyme. RelA mediates the stringent response in bacteria, modulating levels of the alarmone guanosine tetraphosphate (ppGpp). The relA mutant (EF641) was associated with lower growth capacity, the presence of small colony variants, and higher capacity to produce biofilms (compared with the strain EF646), but without differences in antimicrobial susceptibility patterns according to standard procedures during planktonic growth. Instead, EF641 demonstrated tolerance to high doses of teicoplanin when growing in a biofilm. We conclude that all these events would be closely related to the long-term survival of the E. faecium and the late relapse of the IE. These data represent the first clinical evidence of mutations in the stringent response (relA gene) related with E. faecium IE relapse.

Implementation of a PCR-based strategy to control an outbreak by Serratia marcescens in a Neonatal Intensive Care Unit.

OBJECTIVES: To evaluate the clinical and epidemiological impact of a new molecular surveillance strategy based on qPCR to control an outbreak by Serratia marcescens in a Neonatal Intensive Care Unit (NICU). METHODS: We design a specific qPCR for the detection of S. marcescens in rectal swabs of patients admitted to a NICU. We divided the surveillance study into two periods: (a) the pre-PCR, from the outbreak declaration to the qPCR introduction, and (b) the PCR period, from the introduction of the qPCR until the outbreak was solved. In all cases, S. marcescens isolates were recovered and their clonal relationship was analysed by PFGE. Control measures were implemented during the outbreak. Finally, the number of bloodstream infections (BSI) was investigated in order to evaluate the clinical impact of this molecular strategy. RESULTS: Nineteen patients colonized/infected by S. marcescens were detected in the pre-PCR period (October 2020-April 2021). On the contrary, after the PCR implementation, 16 new patients were detected. The PFGE revealed 24 different pulsotypes belonging to 7 different clonal groups, that were not overlapping at the same time. Regarding the clinical impact, 18 months after the qPCR implementation, no more outbreaks by S. marcescens have been declared in the NICU of our hospital, and only 1 episode of BSI has occurred, compared with 11 BSI episodes declared previously to the outbreak control. CONCLUSIONS: The implementation of this qPCR strategy has proved to be a useful tool to control the nosocomial spread of S. marcescens in the NICU.

BIChromET: A Chromogenic Culture Medium for Detection of Piperacillin/Tazobactam and Cefepime Resistance in Pseudomonas aeruginosa.

OBJECTIVES: The BIChromET selective medium for detecting piperacillin-tazobactam (TZP) and cefepime (FEP) resistant Pseudomonas aeruginosa was developed. METHODS: The performance of this medium was first evaluated using a collection of 100 P. aeruginosa clinical strains (70 TZP-susceptible, 30 TZP-resistant, 58 FEP-susceptible, and 42 FEP-resistant). Then, we performed clinical validation by testing 173 respiratory clinical samples. RESULTS: The BIChromET medium showed excellent sensitivity (TZP (avg. 96.7%); FEP (avg. 92.7%)) and specificity (TZP (avg. 98.9%); FEP (avg. 98%)) in distinguishing the detection limit ranging from 104 to 108 CFU/mL. Then, testing the bronchoalveolar lavage (BAL) and tracheobronchial aspirate (TBA) clinical specimens (N = 173) revealed the excellent performance of the medium with P. aeruginosa, showing 100% and 92.6% of categorical agreements with the results obtained via the broth microdilution methods (BMD) for TZP and FEP, respectively. CONCLUSION: This medium allows for easy and accurate detection of TZP/FEP-resistant isolates regardless of their resistance mechanisms.

The imperative for quality control programs in Monkeypox virus DNA testing by PCR: CIBERINFEC quality control.

To evaluate molecular assays for Mpox diagnosis available in various clinical microbiology services in Spain through a quality control (QC) approach. A total of 14 centers from across Spain participated in the study. The Reference Laboratory dispatched eight serum samples and eight nucleic acid extracts to each participating center. Some samples were spiked with Mpox or Vaccinia virus to mimic positive samples for Mpox or other orthopox viruses. Participating centers provided information on the results obtained, as well as the laboratory methods used. Among the 14 participating centers seven different commercial assays were employed, with the most commonly used kit being LightMix Modular Orthopox/Monkeypox (Mpox) Virus (Roche®). Of the 12 centers conducting Mpox determinations, concordance ranged from 62.5% (n = 1) to 100% (n = 11) for eluates and from 75.0% (n = 1) to 100% (n = 10) for serum. Among the 10 centers performing Orthopoxvirus determinations, a 100% concordance was observed for eluates, while for serum, concordance ranged from 87.5% (n = 6) to 100% (n = 4). Repeatedly, 6 different centers reported a false negative in serum samples for Orthopoxvirus diagnosis, particularly in a sample with borderline Ct = 39. Conversely, one center, using the TaqMan™ Mpox Virus Microbe Detection Assay (Thermo Fisher), reported false positives in Mpox diagnosis for samples spiked with vaccinia virus due to cross-reactions. We observed a positive correlation of various diagnostic assays for Mpox used by the participating centers with the reference values. Our results highlight the significance of standardization, validation, and ongoing QC in the microbiological diagnosis of infectious diseases, which might be particularly relevant for emerging viruses.

Safety of early oral ambulatory treatment of adult patients with bloodstream infections discharged from the emergency department.

This study evaluates the safety of early oral ambulatory treatment of adult patients diagnosed with bacteremia after their discharge from the emergency department. A cohort of 206 febrile ambulatory patients was assessed. Bacteremic low-risk patients were recommended an oral treatment and were compared with matched febrile non-bacteremic outpatients. Rates of 14-day mortality and unplanned re-consultations were similar and below 5% in both cohorts, highlighting the safety of oral therapy of low-risk bacteremia, even from its onset.

An increase in erythromycin resistance in methicillin-susceptible Staphylococcus aureus from blood correlates with the use of macrolide/lincosamide/streptogramin antibiotics. EARS-Net Spain (2004-2020).

Objectives: To describe and analyse erythromycin resistance trends in blood isolates of Staphylococcus aureus (EARS-Net Spain, 2004-2020) and the association of these trends with the consumption of macrolide, lincosamide, and streptogramin B (MLSB) antibiotics. To assess molecular changes that could be involved in erythromycin resistance trends by whole genome analysis of representative isolates. Materials and methods: We collected antibiotic susceptibility data for all first-blood S. aureus isolates in patients from 47 Spanish hospitals according to EARS-Net criteria. MLSB antibiotic consumption was obtained from the Spanish Agency for Medicines and Medical Devices (2008-2020). We sequenced 137 representative isolates for core genome multilocus sequence typing, resistome and virulome analysis. Results: For the 36,612 invasive S. aureus isolates, methicillin resistance decreased from 26.4% in 2004 to 22.4% in 2020. Erythromycin resistance in methicillin-susceptible S. aureus (MSSA) increased from 13.6% in 2004 to 28.9% in 2020 (p < 0.001); however, it decreased from 68.7 to 61.8% (p < 0.0001) in methicillin-resistant S. aureus (MRSA). Total consumption of MLSB antibiotics increased from 2.72 defined daily doses per 1,000 inhabitants per day (DID) in 2014 to 3.24 DID in 2016. By WGS, the macrolide resistance genes detected were erm (59.8%), msrA (46%), and mphC (45.2%). The erm genes were more prevalent in MSSA (44/57, 77.2%) than in MRSA (38/80, 47.5%). Most of the erm genes identified in MSSA after 2013 differed from the predominant ermC gene (17/22, 77.3%), largely because ermT was significantly associated with MSSA after 2013 (11/29, 37.9%). All 13 ermT isolates in this study, except one, belonged to ST398 and came from 10 hospitals and six Spanish provinces. Conclusion: The significant increase in erythromycin resistance in blood MSSA correlated with the consumption of the MLSB antibiotics in Spain. These preliminary data seem support the hypothesis that the human ST398 MSSA clade with ermT-mediated resistance to erythromycin may be involved in this trend.

Role of blaTEM and OmpC in the piperacillin-tazobactam resistance evolution by E. coli in patients with complicated intra-abdominal infection.

Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among Escherichia coli isolates. Although in vitro experiments have suggested that blaTEM gene plays a key role in the P/T-R acquisition, no clinical in vivo study has yet confirmed the role of blaTEM or other genes. Therefore, we aimed to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4 g/0.5 g intravenously. The P/T MIC was tested using broth microdilution, and ß-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of blaTEM and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analyzed using SDS-PAGE, and blaTEM and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). We found a higher copy number of blaTEM gene in P/T-R isolates, generated by three different genetic events: (1) IS26-mediated duplication of the blaTEM gene, (2) generation of a small multicopy plasmid (ColE-like) carrying blaTEM, and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. This study describes the mechanisms involved in the acquisition of P/T-R by E. coli in patients with cIAI. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms.

Enhancing SARS-CoV-2 Surveillance through Regular Genomic Sequencing in Spain: The RELECOV Network.

Millions of SARS-CoV-2 whole genome sequences have been generated to date. However, good quality data and adequate surveillance systems are required to contribute to meaningful surveillance in public health. In this context, the network of Spanish laboratories for coronavirus (RELECOV) was created with the main goal of promoting actions to speed up the detection, analyses, and evaluation of SARS-CoV-2 at a national level, partially structured and financed by an ECDC-HERA-Incubator action (ECDC/GRANT/2021/024). A SARS-CoV-2 sequencing quality control assessment (QCA) was developed to evaluate the network's technical capacity. QCA full panel results showed a lower hit rate for lineage assignment compared to that obtained for variants. Genomic data comprising 48,578 viral genomes were studied and evaluated to monitor SARS-CoV-2. The developed network actions showed a 36% increase in sharing viral sequences. In addition, analysis of lineage/sublineage-defining mutations to track the virus showed characteristic mutation profiles for the Delta and Omicron variants. Further, phylogenetic analyses strongly correlated with different variant clusters, obtaining a robust reference tree. The RELECOV network has made it possible to improve and enhance the genomic surveillance of SARS-CoV-2 in Spain. It has provided and evaluated genomic tools for viral genome monitoring and characterization that make it possible to increase knowledge efficiently and quickly, promoting the genomic surveillance of SARS-CoV-2 in Spain.

Evaluation of the MDR Direct Flow Chip Kit for the Detection of Multiple Antimicrobial Resistance Determinants.

The objective of this study was to evaluate the accuracy of the MDR Direct Flow Chip Kit for the detection of antimicrobial resistance (AMR) determinants from bacterial colonies. Ninety-two clinical isolates with known AMR determinants genotypically characterized were used. The MDR Direct Flow Chip Kit is a microarray-based assay that included 55 AMR determinants for beta-lactams (23), quinolones (13), aminoglycosides (5), macrolides (5), sulfonamides (3), colistin (2), vancomycin (2), chloramphenicol (1), and linezolid (1). The MDR Direct Flow Chip Kit correctly detects 52 of 53 AMR determinants tested. The cfr gene (linezolid resistance) was not detected. The global sensibility, specificity, positive predictive value, and the negative predictive value calculated were 98%, 100%, 100%, and 97%. The Cohen's Kappa coefficient calculated was 0.97 [95% Confidence Interval (0.90-1.03)]. In conclusion, the MDR Direct Flow Chip is an accurate assay for the detection of multiple AMR determinants in one simple reaction.

High-level delafloxacin resistance through the combination of two different mechanisms in Staphylococcus aureus.

Delafloxacin is a new fluoroquinolone indicated for the treatment of complicated bacterial skin infections caused by Staphylococcus aureus. Despite its recent approval by the US Food and Drug Administration, the emergence of S. aureus-resistant strains has been reported. As such, this study aimed to investigate the activity of delafloxacin against a collection of S. aureus, and to determine the mechanisms of resistance. The activity of delafloxacin was measured in 59 S. aureus clinical isolates [40 methicillin-resistant S. aureus (MRSA) and 19 methicillin-susceptible S. aureus (MSSA)]. Whole-genome sequencing (WGS) was performed in the isolates resistant to delafloxacin. The minimum inhibitory concentrations required to inactivate 50% and 90% of the isolates (MIC50 and MIC90, respectively) were higher in MRSA (0.19 mg/L and 0.75 mg/L, respectively) than in MSSA (0.008 mg/L and 0.25 mg/L, respectively). Furthermore, 10 S. aureus clinical isolates (16.9%) were categorized as resistant to delafloxacin. Regarding the WGS data, several mutations were found in the quinolone resistance-determining region. Nevertheless, a mutation in the same position (E84K and E84V) of topoisomerase IV (ParC) was found exclusively in the four high-level delafloxacin-resistant isolates. Interestingly, a plasmid-encoded qacC gene (efflux pump) was found to be harboured by the isolate with the highest delafloxacin MIC value (32 mg/L). The use of a wide-spectrum efflux pump inhibitor revealed an important contribution of this system to the acquisition of delafloxacin resistance. In conclusion, delafloxacin has activity against S. aureus, including MRSA. However, this study showed that mutations in position 84 of ParC and the acquisition of a QacC efflux pump are key factors for the development of delafloxacin resistance in S. aureus.

Rapid Detection of Piperacillin-Tazobactam Resistance in Klebsiella pneumoniae and Escherichia coli.

Rapid determination of susceptibility to piperacillin-tazobactam (TZP) is very important since the development of antibiotic resistance and inadequate treatment could increase the risk of clinical failure in infected patients, especially if such resistance is unknown to the clinician. Therefore, based on color change from orange to yellow of phenol red due to glucose metabolism (bacterial growth) in the presence of an adequate concentration of TZP (10 mg/L piperacillin and 5 mg/L tazobactam), the RapidTZP test has been developed to detect TZP resistance in Escherichia coli and Klebsiella pneumoniae isolates in a maximum of 3 h. A total of 140 isolates, 43 of E. coli and 97 of K. pneumoniae, were used to evaluate the performance of the test, 60 being resistant to TZP. The sensitivity and specificity of the test were 98.24% and 100%, respectively. Additionally, the RapidTZP test was validated by a pellet obtained directly from blood culture bottles. A total of 37 positive blood cultures for E. coli and 43 for K. pneumoniae were used for validation, 8 of them resistant to TZP. The sensitivity and specificity shown in the evaluation were 100% for both parameters. This new test is easy, fast, and accurate, providing results in 3 h. IMPORTANCE TZP is an antibiotic widely used for the empirical treatment of severe infections such as bloodstream infections. However, resistance to TZP in K. pneumoniae and E. coli has been increasing in the last few years. Thus, rapid detection of TZP resistance is critical to optimize the empirical treatment of patients with severe infections. In this study, we developed and evaluated a rapid test (RapidTZP) for the detection of TZP resistance in K. pneumoniae and E. coli directly from positive hemocultures in just 3 h. This rapid test has been validated on 138 K. pneumoniae and E. coli clinical isolates directly from agar plates and 80 K. pneumoniae and E. coli isolates causing bloodstream infections. The results demonstrate that the RapidTZP test has great clinical potential to optimize the empirical treatment of patients with bloodstream infections.

Study protocol for a randomized clinical trial to assess 7 versus 14-days of treatment for Pseudomonas aeruginosa bloodstream infections (SHORTEN-2 trial).

BACKGROUND: Research priorities in Antimicrobial Stewardship (AMS) have rapidly evolved in the last decade. The need for a more efficient use of antimicrobials have fueled plenty of studies to define the optimal duration for antibiotic treatments, and yet, there still are large areas of uncertainty in common clinical scenarios. Pseudomonas aeruginosa has been pointed as a priority for clinical research, but it has been unattended by most randomized trials tackling the effectiveness of short treatments. The study protocol of the SHORTEN-2 trial is presented as a practical example of new ways to approach common obstacles for clinical research in AMS. OBJECTIVE: To determine whether a 7-day course of antibiotics is superior to 14-day schemes for treating bloodstream infections by P. aeruginosa (BSI-PA). METHODS: A superiority, open-label, randomized controlled trial will be performed across 30 Spanish hospitals. Adult patients with uncomplicated BSI-PA will be randomized to receive a 7 versus 14-day course of any active antibiotic. The primary endpoint will be the probability for the 7-day group of achieving better outcomes than the control group, assessing altogether clinical effectiveness, severe adverse events, and antibiotic exposure through a DOOR/RADAR analysis. Main secondary endpoints include treatment failure, BSI-PA relapses, and mortality. A superiority design was set for the primary endpoint and non-inferiority for treatment failure, resulting in a sample size of 304 patients. CONCLUSIONS: SHORTEN-2 trial aligns with some of the priorities for clinical research in AMS. The implementation of several methodological innovations allowed overcoming common obstacles, like feasible sample sizes or measuring the clinical impact and unintended effects. TRIAL REGISTRATION: EudraCt: 2021-003847-10; ClinicalTrials.gov: NCT05210439.

FlowUTI: An interactive web-application for optimizing the use of flow cytometry as a screening tool in urinary tract infections.

Due to the high prevalence of patients attending with urinary tract infection (UTI) symptoms, the use of flow-cytometry as a rapid screening tool to avoid unnecessary cultures is becoming a widely used system in clinical practice. However, the recommended cut-points applied in flow-cytometry systems differ substantially among authors, making it difficult to obtain reliable conclusions. Here, we present FlowUTI, a shiny web-application created to establish optimal cut-off values in flow-cytometry for different UTI markers, such as bacterial or leukocyte counts, in urine from patients with UTI symptoms. This application provides a user-friendly graphical interface to perform robust statistical analysis without a specific training. Two datasets are analyzed in this manuscript: one composed of 204 urine samples from neonates and infants (≤3 months old) attended in the emergency department with suspected UTI; and the second dataset including 1174 urines samples from an elderly population attended at the primary care level. The source code is available on GitHub (https://github.com/GuillermoMG-HUVR/Microbiology-applications/tree/FlowUTI/FlowUTI). The web application can be executed locally from the R console. Alternatively, it can be freely accessed at https://covidiario.shinyapps.io/flowuti/. FlowUTI provides an easy-to-use environment for evaluating the efficiency of the urinary screening process with flow-cytometry, reducing the computational burden associated with this kind of analysis.

Optimización y adecuación de la capacidad diagnóstica para la realización de grandes volúmenes de RT-PCR de SARS-CoV-2 / Optimization and adaptation of the diagnostic capacity for the performance of large volumes of SARS-CoV-2 RT-PCR

Introducción: Este artículo describe la planificación efectuada para adecuar la capacidad diagnóstica de grandes volúmenes de RT-PCR de SARS-CoV-2. Métodos: El análisis y predicción del flujo de trabajo incluyó el número de RT-PCR desde el inicio de la pandemia, con 31.971 registros. La planificación de la capacidad y de las opciones diagnósticas se planteó en base a los posibles escenarios derivados de las predicciones efectuadas. Resultados: De acuerdo con las predicciones obtenidas, se optó por una solución automatizada basada en el empleo de robots OT-2 (Opentrons) para configurar un flujo de trabajo reproducible, que logró una capacidad de procesamiento de 5.640 muestras/día, con un tiempo de respuesta de cuatro horas. Conclusiones: El análisis y predicción del flujo de trabajo, unido al empleo de plataformas basadas en OT-2, proporciona una infraestructura robusta que permite atender con éxito las demandas de pruebas que exige esta pandemia.(AU)

Introduction: In the present manuscript we describe the planning carried out in our hospital to adapt our diagnostic capability to perform large numbers of SARS-CoV-2 RT-PCR. Methods: The analysis and prediction of workflow included the number of RT-PCR per week from the beginning of the pandemic, with a total of 31971 determinations. The planning phase was developed based on the different scenarios previously predicted. Results: According to the predictions obtained, an automated custom solution was chosen, based on the use of the OT-2 open-source liquid-handling robots (Opentrons), to design a reproducible workflow that achieved a production capacity of 5640 samples/day, with a time of response of four hours per procedure. Conclusions: The analysis and prediction of workflow, along with the use of the robotic platforms OT-2, provided a robust structure to deal with the high demand of determinations that this pandemic requires.(AU)

MDR Shigella sonnei in Spain: an ever-evolving emerging threat?

Background: Seven CTX-M-27-producing Shigella sonnei strains were isolated at the University Hospital Virgen del Rocío (Seville, Spain) microbiology service from October to November 2021. Objectives: To offer extensive information on the microbiological and molecular epidemiology results of the seven S. sonnei isolates and compare them with other previously documented CTX-M-27-producing S. sonnei associated with MSM transmission. Methods: S. sonnei isolated from stool samples of patients with acute diarrhoea were identified through biochemical and serological typing. Whole characterization of the seven isolates was performed by sequencing with MinION Mk1C followed by genomic and molecular analysis. Results: All the isolates were resistant to penicillins, cephalosporins, fluoroquinolones, cotrimoxazole and azithromycin. Sequencing showed the presence of several resistance determinants, outstanding bla CTX-M-27, azithromycin resistance genes [ermB and mph(A)], qnrB19 and mutations in the QRDRs. All isolates belonged to the same hierarchical clustering of cgMLST (HierCC) with five allele distance (HC5) scheme v1 from EnteroBase. However, they presented differences in plasmid composition, with all seven isolates harbouring IncFII, IncB/O/K/Z and ColE1-like while SH2, SH6 and SH7 had IncFIB only. Our isolates were closely related to others from Spain (HC5; 98748), Australia (HC5; 98748) and the UK (HC5; 98748), which were also associated with MSM transmission. Nevertheless, the structure of the non-chromosomal genetic elements and the genetic context of bla CTX-M-27 presented a certain variability compared with isolates from other countries and among them. Conclusions: This study confirms the emergence of CTX-M-27-producing S. sonnei (ST152) associated with MSM transmission in Spain, adding it to the Europe outbreak list and reinforcing the necessity of active surveillance and control of this high-risk clone.

Volatile compounds from in vitro metabolism of seven Listeria monocytogenes isolates belonging to different clonal complexes.

Microorganisms produce a wide variety of volatile organic compounds (VOCs) as products of their metabolism and some of them can be specific VOCs linked to the microorganism's identity, which have proved to be helpful for the diagnosis of infection via odour fingerprinting. The aim of this study was to determine the VOCs produced and consumed to characterize the volatile metabolism of seven isolates of different clonal complexes (CCs) of Listeria monocytogenes. For this purpose, dichloromethane extracts from the thioglycolate broth medium were analysed by gas chromatography coupled to mass spectrometry (GC/MS). Also, multivariate analyses were applied to the data obtained. Results showed that all the isolates of L. monocytogenes produced de novo isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 3-(methylthio)-1-propanol, acetic acid, isobutyric acid, butanoic acid, and isovaleric acid. Significant differences were found among isolates for the production amount of these volatiles, which allowed their differentiation. Thus, CC4 (ST-219/CT-3650) and CC87 (ST-87/CT-4557) showed an active volatile compounds metabolism with high consumption nitrogen and sulphur compounds and production of alcohols and acids, and CC8 (ST-8/CT-8813) and CC3 (ST-3/CT-8722) presented a less active volatile metabolism. Moreover, within the VOCs determined, huge differences were found in the production of butanol among the seven isolates analysed, being probably a good biomarker to discriminate among isolates belonging to different CCs. Hence, the analysis of volatile profile generated by the growth of L. monocytogenes in vitro could be a useful tool to differentiate among CCs isolates.

Optimization and adaptation of the diagnostic capacity for the performance of large volumes of SARS-CoV-2 RT-PCR.

INTRODUCTION: In the present manuscript we describe the planning carried out in our hospital to adapt our diagnostic capability to perform large numbers of SARS-CoV-2 RT-PCR. METHODS: The analysis and prediction of workflow included the number of RT-PCR per week from the beginning of the pandemic, with a total of 31971 determinations. The planning phase was developed based on the different scenarios previously predicted. RESULTS: According to the predictions obtained, an automated custom solution was chosen, based on the use of the OT-2 open-source liquid-handling robots (Opentrons), to design a reproducible workflow that achieved a production capacity of 5640 samples/day, with a time of response of four hours per procedure. CONCLUSIONS: The analysis and prediction of workflow, along with the use of the robotic platforms OT-2, provided a robust structure to deal with the high demand of determinations that this pandemic requires.