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.

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.

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.

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.

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.

Propensity Score and Desirability of Outcome Ranking Analysis of Ertapenem for Treatment of Nonsevere Bacteremic Urinary Tract Infections Due to Extended-Spectrum-Beta-Lactamase-Producing Enterobacterales in Kidney Transplant Recipients.

There are scarce data on the efficacy of ertapenem in the treatment of bacteremia due to extended-spectrum-beta-lactamase (ESBL)-producing Enterobacterales (ESBL-E) in kidney transplant (KT) recipients. We evaluated the association between treatment with ertapenem or meropenem and clinical cure in KT recipients with nonsevere bacteremic urinary tract infections (B-UTI) caused by ESBL-E. We performed a registered, retrospective, international (29 centers in 14 countries) cohort study (INCREMENT-SOT, NCT02852902). The association between targeted therapy with ertapenem versus meropenem and clinical cure at day 14 (the principal outcome) was studied by logistic regression. Propensity score matching and desirability of outcome ranking (DOOR) analyses were also performed. A total of 201 patients were included; only 1 patient (treated with meropenem) in the cohort died. Clinical cure at day 14 was reached in 45/100 (45%) and 51/101 (50.5%) of patients treated with ertapenem and meropenem, respectively (adjusted OR 1.29; 95% CI 0.51 to 3.22; P = 0.76); the propensity score-matched cohort included 55 pairs (adjusted OR for clinical cure at day 14, 1.18; 95% CI 0.43 to 3.29; P = 0.74). In this cohort, the proportion of cases treated with ertapenem with better DOOR than with meropenem was 49.7% (95% CI, 40.4 to 59.1%) when hospital stay was considered. It ranged from 59 to 67% in different scenarios of a modified (weights-based) DOOR sensitivity analysis when potential ecological advantage or cost was considered in addition to outcome. In conclusion, targeted therapy with ertapenem appears as effective as meropenem to treat nonsevere B-UTI due to ESBL-E in KT recipients and may have some advantages.

Do specific antimicrobial stewardship interventions have an impact on carbapenem resistance in Gram-negative bacilli? A multicentre quasi-experimental ecological study: time-trend analysis and characterization of carbapenemases.

BACKGROUND: Carbapenem-resistant Gram-negative bacilli (CR-GNB) are among the most threatening microorganisms worldwide and carbapenem use facilitates their spread. Antimicrobial stewardship programmes (ASPs) can help to optimize the use of antibiotics. This study evaluates the impact of a multifaceted educational ASP on carbapenem use and on the epidemiology of CR-GNB. METHODS: We conducted a quasi-experimental, time-series study in seven hospitals, from January 2014 to September 2018. The key intervention was composed of educational interviews promoting the appropriate use of carbapenems. The primary endpoints were carbapenem consumption and incidence density (ID) of CR-GNB. All non-duplicated CR-GNB clinical isolates were tested using phenotypic assays and PCR for the presence of carbapenemases. Joinpoint regression and interrupted time-series analyses were used to determine trends. RESULTS: A decrease in carbapenem consumption throughout the study period [average quarterly percentage change (AQPC) -1.5%, P < 0.001] and a -8.170 (-16.064 to -0.277) level change following the intervention were observed. The ID of CR-Acinetobacter baumannii decreased (AQPC -3.5%, P = 0.02) and the overall ID of CR-GNB remained stable (AQPC -0.4%, P = 0.52). CR-GNB, CR-Pseudomonas aeruginosa and CR-A. baumannii IDs per hospital correlated with the local consumption of carbapenems. The most prevalent carbapenem resistance mechanisms were OXA-23 for CR-A. baumannii (76.1%), OXA-48 for CR-Klebsiella pneumoniae (66%) and no carbapenemases for CR-P. aeruginosa (91.7%). The epidemiology of carbapenemases was heterogeneous throughout the study, especially for carbapenemase-producing Enterobacteriaceae. CONCLUSIONS: In conclusion, a multifaceted, educational interview-based ASP targeting carbapenem prescribing reduced carbapenem use and the ID of CR-A. baumannii.

The Impact of Hanseniaspora vineae Fermentation and Ageing on Lees on the Terpenic Aromatic Profile of White Wines of the Albillo Variety.

Hanseniaspora vineae is a non-Saccharomyces yeast that has a powerful impact on the sensory profile of wines. Its effect on the aromatic profile of non-aromatic grape varieties, such as Albillo Mayor (Vitis vinifera, L), during vinification is a useful biotechnology to improve sensory complexity. Fermentation in steel barrels using Hanseniaspora vineae and sequential inoculation with Saccharomyces cerevisiae have been used to study the formation of terpenes and cell lysis in the production of Albillo white wines. The GC-MS analysis profile shows a significant effect of H. vineae fermentation on the contents of terpenes (≈×3), mainly in linalool (>×3), ß-citronellol (>×4), geraniol (>×2) and α-terpineol (≈×2). The contents of several polyoxygenated terpenes and some volatile phenols with a spicy aroma were increased during fermentation. In summary, Hanseniaspora vineae releases a large number of cell wall polysaccharides during fermentation that affect wine palatability and structure. Hanseniaspora vineae is a powerful bio-tool to enhance the fruitiness, floral notes and freshness in non-aromatic white varieties.

Extended-spectrum resistance to ß-lactams/ß-lactamase inhibitors (ESRI) evolved from low-level resistant Escherichia coli.

OBJECTIVES: Escherichia coli is characterized by three resistance patterns to ß-lactams/ß-lactamase inhibitors (BLs/BLIs): (i) resistance to ampicillin/sulbactam and susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (RSS); (ii) resistance to ampicillin/sulbactam and amoxicillin/clavulanic acid, and susceptibility to piperacillin/tazobactam (RRS); and (iii) resistance to ampicillin/sulbactam, amoxicillin/clavulanic acid and piperacillin/tazobactam (RRR). These resistance patterns are acquired consecutively, indicating a potential risk of developing resistance to piperacillin/tazobactam, but the precise mechanism of this process is not completely understood. METHODS: Clinical isolates incrementally pressured by piperacillin/tazobactam selection in vitro and in vivo were used. We determined the MIC of piperacillin/tazobactam in the presence and absence of piperacillin/tazobactam pressure. We deciphered the role of the blaTEM genes in the new concept of extended-spectrum resistance to BLs/BLIs (ESRI) using genomic analysis. The activity of ß-lactamase was quantified in these isolates. RESULTS: We show that piperacillin/tazobactam resistance is induced in E. coli carrying blaTEM genes. This resistance is due to the increase in copy numbers and transcription levels of the blaTEM gene, thus increasing ß-lactamase activity and consequently increasing piperacillin/tazobactam MICs. Genome sequencing of two blaTEM-carrying representative isolates showed that piperacillin/tazobactam treatment produced two types of duplications of blaTEM (8 and 60 copies, respectively). In the clinical setting, piperacillin/tazobactam treatment of patients infected by E. coli carrying blaTEM is associated with a risk of therapeutic failure. CONCLUSIONS: This study describes for the first time the ESRI in E. coli. This new concept is very important in the understanding of the mechanism involved in the acquisition of resistance to BLs/BLIs.

Clinical and prognostic differences between methicillin-resistant and methicillin-susceptible Staphylococcus aureus infective endocarditis.

BACKGROUND: S. aureus (SA) infective endocarditis (IE) has a very high mortality, attributed to the age and comorbidities of patients, inadequate or delayed antibiotic treatment, and methicillin resistance, among other causes. The main study objective was to analyze epidemiological and clinical differences between IE by methicillin-resistant versus methicillin-susceptible SA (MRSA vs. MSSA) and to examine prognostic factors for SA endocarditis, including methicillin resistance and vancomycin minimum inhibitory concentration (MIC) values > 1 µg/mL to MRSA. METHODS: Patients with SA endocarditis were consecutively and prospectively recruited from the Andalusia endocarditis cohort between 1984 and January 2017. RESULTS: We studied 437 patients with SA endocarditis, which was MRSA in 13.5% of cases. A greater likelihood of history of COPD (OR 3.19; 95% CI 1.41-7.23), invasive procedures, or recognized infection focus in the 3 months before IE onset (OR 2.9; 95% CI 1.14-7.65) and of diagnostic delay (OR 3.94; 95% CI 1.64-9.5) was observed in patients with MRSA versus MSSA endocarditis. The one-year mortality rate due to SA endocarditis was 44.3% and associated with decade of endocarditis onset (1985-1999) (OR 8.391; 95% CI (2.82-24.9); 2000-2009 (OR 6.4; 95% CI 2.92-14.06); active neoplasm (OR 6.63; 95% CI 1.7-25.5) and sepsis (OR 2.28; 95% CI 1.053-4.9). Methicillin resistance was not associated with higher IE-related mortality (49.7 vs. 43.1%; p = 0.32). CONCLUSION: MRSA IE is associated with COPD, previous invasive procedure or recognized infection focus, and nosocomial or healthcare-related origin. Methicillin resistance does not appear to be a decisive prognostic factor for SA IE.

Influence of Saccharomyces and non-Saccharomyces Yeasts in the Formation of Pyranoanthocyanins and Polymeric Pigments during Red Wine Making.

Yeast are able to modulate many sensory parameters of wines during red must fermentation. The effect on color and on the formation of derived pigments during fermentation has been studied thoroughly since the 90s. Yeast can increase grape anthocyanin's color by acidification by hyperchromic effect (increase of flavylium molecules). Recent studies with non-Saccharomyces species, as Lachancea thermotolerans, described the intense effect of some strains on anthocyanin's color, and subsequent, stability, by strongly reducing wine's pH during fermentation. Moreover, selected yeast strains of Saccharomyces have been shown to release metabolites such as pyruvic acid or acetaldehyde that promote the formation of vitisin A and B pyranoanthocyanins during must fermentation. Schizosaccharomyces pombe, because of its specific metabolism, can produce higher concentrations of pyruvate, which enhances the formation of vitisin A-type derivatives. The hydroxycinnamate decarboxylase activity that some Saccharomyces strains express during fermentation also promotes the formation of vinylphenolic derivatives. Some non-Saccharomyces species, such as S. pombe or P. guilliermondii can also improve the production of these derivatives compared to selected strains of Saccharomyces cerevisiae. Lastly, some yeasts are also able to modulate the formations of polymeric pigments between grape anthocyanins and flavonoids, such as catechins and procyanidins.

Sonication of Yeast Biomasses to Improve the Ageing on Lees Technique in Red Wines.

Ageing on Lees (AOL) is a technique to improve the aromatic and gustatory complexity of wine, mainly by improving its body and reducing its astringency. However, the autolytic process is slow, resulting in high production costs. This work evaluated the effect of adding sonicated lees and combining it with oak chips, as a new technique to accelerate the AOL process and improve the aromatic quality of aged red wine. Cell disruption due to sonication was verified by optical microscopy. Volatile acidity, total polyphenol index, color intensity, tonality, dissolved oxygen, anthocyanins, and fermentative volatiles were monitored throughout the ageing of the wines. Sensory analysis was performed at the end of the ageing process. Polysaccharides released from the cell walls and the oxygen consumption, was quantified using a hydroalcoholic solution. The results indicated a 20% increase of the polysaccharide content and suggested an increase in the antioxidant capacity of the lees. No significant changes were observed in the fermentative volatile compounds and the total polyphenol index (TPI), except for those wines in contact with wood. The sonication of lees had some protective effect on the total anthocyanins content, however, color intensity was significantly lower in the sonicated treatments. The sonication of the lees did not cause any defect at the sensory level. Therefore, sonication could allow a reduction in the SO2 addition to wine, as well as a shortening of the ageing times.

Use of non-Saccharomyces yeasts and oenological tannin in red winemaking: Influence on colour, aroma and sensorial properties of young wines.

Today, many non-Saccharomyces strains have been verified can be positive for the development of wine anthocyanin and aroma in different fermentation scenarios. Moreover, oenological tannins are widely used in wine industry to improve the colour profile and aroma complexity. The aim of this work is to analyze the fermentation characters of non-Saccharomyces strains and investigate the effects of pre-fermentative addition of oenological tannins on the wine components as well as sensory properties. For this purpose, five selected non-Saccharomyces strains and grape seed tannin were used to carry out the different fermentation trials. As a result, the grape seed tannin were less likely to influence growth kinetics of non-Saccharomyces strains. Schizosaccharomyces pombe has been proved can be effective to reduce the malic acid content while increase the level of vinylphenolic pyranoanthocyanin, which is positive for wine colour stability. Pre-fermentative use of oenological tannin was verified could be beneficial for the wines fermented with non-Saccharomyces regarding the improvement of wine colour, anthocyanin composition and the complexity of volatile compounds. Nevertheless, sensory analysis showed that oenological tannin could be less effective to modify the aroma impression of non-Saccharomyces wines.

Effect of Lachancea thermotolerans on the Formation of Polymeric Pigments during Sequential Fermentation with Schizosaccharosmyces pombe and Saccharomyces cerevisiae.

Anthocyanins in red grape musts may evolve during the winemaking process and wine aging for several different reasons; colour stability and evolution is a complex process that may depend on grape variety, winemaking technology, fermentative yeast selection, co-pigmentation phenomena and polymerization. The condensation of flavanols with anthocyanins may occur either with the flavylium ion or with the hemiacetal formation in order to produce oligomers and polymers. The kinetics of the reaction are enhanced by the presence of metabolic acetaldehyde, promoting the formation of pyranoanthocyanin-type dimers or flavanol-ethyl-anthocyanin structures. The experimental design carried out using white must corrected with the addition of malvidin-3-O-glucoside and flavanols, suggests that non-Saccharomyces yeasts are able to provide increased levels of colour intensity and larger polymeric pigment ratios and polymerization indexes. The selection of non-Saccharomyces genera, in particular Lachancea thermotolerans and Schizosaccharomyces pombe in sequential fermentation, have provided experimental wines with increased fruity esters, as well as producing wines with potential pigment compositions, even though there is an important reduction of total anthocyanins.

The Effects of Pre-Fermentative Addition of Oenological Tannins on Wine Components and Sensorial Qualities of Red Wine.

Today in the wine industry, oenological tannins are widely used to improve wine quality and prevent oxidation in wine aging. With the development of tannin products, new oenological tannins are developed with many specific functions, such as modifying antioxidant effect, colour stabilization and aroma modifications. The aim of this work is to investigate effects of pre-fermentative addition of oenological tannins on wine colour, anthocyanins, volatile compounds and sensorial properties. In this case, Syrah juice was extracted with classic flash thermovinification from fresh must in order to release more colour and tannins. Three types of oenological tannins, which are, respectively, derived from grape skin, seed (Vitis vinifera) and French oak (Quercus robur and Querrus petraea), were selected to carry out the experiments with seven treatments. Results indicated that tannin treatments significantly improved wine aroma complexity and sensorial properties. However, the concentration of some stable pigments such as Vitisin A, Vitisin A-Ac and Vitisin B was negatively affected by tannin additions. Nevertheless, by means of cluster analysis and principal component analysis, it was observed that higher alcohols were significantly promoted by grape seed tannin while most anthocyanins can be improved by addition of grape tannins. In conclusion, low amount of oenological tannin derived from grape seed is a promising method to be applied especially for young red wine making.

Evaluation and optimization of the Sysmex UF1000i system for the screening of urinary tract infection in primary health care elderly patients.

OBJECTIVE: Urinary tract infections (UTIs) are a common problem in the elderly population. Urine culture is still considered the "gold standard" to diagnose infection in this population. However, urine cultures are laborious and costly, and most samples will yield no growth. METHODS: An evaluation was made of the Sysmex UF-1000i flow cytometer as a screening tool for UTI in an elderly population older than 65 years who lived in the community, using 346 urine samples submitted for culture. RESULTS: The Receiver Operating Characteristic (ROC) analysis showed a significant difference (P<0.01) between 0.98 bacteria area under the curve value and 0.82 of white blood cells (WBC). The combination of both counts for screening did not show any improvement in specificity or sensitivity. According to our data, the use of a single cut-off point of 200bacteria/µL is suggested, in which the sensitivity and specificity were 99.11% and 91.59%, respectively, with a NPV of 99.49%. Moreover, this cut-off value could avoid 60.24% of the samples to be cultured, with a minimal false negative results rate of 0.87%. CONCLUSIONS: The stratification of age groups stratification helps in selecting a more adjusted Sysmex UF1000i cut-off limit, leading to an improvement in the screening parameters that would imply a better management of these infections, as well as a high reduction in the workload and cost savings.

Activity of cefepime, carbapenems and new ß-lactam/ß-lactamase inhibitor combinations on Enterobacter cloacae complex and Klebsiella aerogenes in Spain (SMART 2016-2022).

Objectives: To analyse the susceptibility profile to cefepime, carbapenems and new ß-lactam/ß-lactamase inhibitor combinations in Enterobacter cloacae complex and Klebsiella aerogenes isolated from intra-abdominal, urinary, respiratory and bloodstream infections in the SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study in Spain. Methods: The susceptibilities of 759 isolates (473 E. cloacae complex and 286 K. aerogenes) collected in 11 Spanish hospitals from 2016 to 2022 were analysed following the EUCAST 2023 criteria. Molecular characterization looking for ß-lactamase genes was performed through PCR and DNA sequencing analysis. Results: E. cloacae complex showed resistance to third-generation cephalosporins in 25% of the cases, whereas K. aerogenes was resistant in 35%. Regarding cefepime, resistance in E. cloacae was higher (10%) than in K. aerogenes (2%). Carbapenems showed >85% activity in both microorganisms. Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam had good activity against these microorganisms (>95%). In contrast, the activity of ceftolozane/tazobactam was lower (80%). A high proportion of the isolates resistant to new ß-lactam/ß-lactamase inhibitor combinations carried a carbapenemase, mainly OXA-48-like and VIM-1. Conclusions: Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam show high activity against both E. cloacae complex and K. aerogenes isolates recovered in the SMART-Spain study. In contrast, differences have been found in the case of cefepime, showing more activity against K. aerogenes than E. cloacae complex. These results are useful for antimicrobial stewardship programmes and for the implementation of local and national guidelines.

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.

Rapid determination of colistin resistance in clinical strains of Acinetobacter baumannii by use of the micromax assay.

Colistin is an old antibiotic which has been used as a therapeutic option for carbapenem- and multidrug-resistant Gram-negative bacteria, like Acinetobacter baumannii. This pathogen produces life-threatening infections, mainly in patients admitted to intensive care units. Rapid detection of resistance to colistin may improve patient outcomes and prevent the spread of resistance. For this purpose, Micromax technology was evaluated in four isogenic A. baumannii strains with known mechanisms of resistance to colistin and in 66 isolates (50 susceptible and 16 resistant). Two parameters were determined, DNA fragmentation and cell wall damage. To assess DNA fragmentation, cells trapped in a microgel were incubated with a lysing solution to remove the cell wall, and the released nucleoids were visualized under fluorescence microscopy. Fragmented DNA was observed as spots that diffuse from the nucleoid. To assess cell wall integrity, cells were incubated with a lysis solution which removes only weakened cell walls, resulting in nucleoid release exclusively in affected cells. A dose-response relationship was demonstrated between colistin concentrations and the percentages of bacteria with DNA fragmentation and cell wall damage, antibiotic effects that were delayed and less frequent in resistant strains. Receiver operating characteristic (ROC) curves demonstrated that both DNA fragmentation and cell wall damage were excellent parameters for identifying resistant strains. Obtaining ≤11% of bacteria with cell wall damage after incubation with 0.5 µg/ml colistin identified resistant strains of A. baumannii with 100% sensitivity and 96% specificity. Results were obtained in 3 h 30 min. This is a simple, rapid, and accurate assay for detecting colistin resistance in A. baumannii, with strong potential value in critical clinical situations.