Antibody-Mediated Serum Resistance Protects Pseudomonas aeruginosa During Bloodstream Infections.

BACKGROUND: Pseudomonas aeruginosa is a frequent pathogen isolated from bacterial bloodstream infection (BSI) and is associated with high mortality. To survive in the blood, P aeruginosa must resist the bactericidal action of complement (ie, serum killing). Antibodies usually promote serum killing through the classical complement pathway; however, "cloaking antibodies" (cAbs) have been described, which paradoxically protect bacteria from serum killing. The relevance of cAbs in P aeruginosa BSI is unknown. METHODS: Serum and P aeruginosa were collected from a cohort of 100 patients with BSI. Isolates were tested for sensitivity to healthy control serum (HCS). cAb prevalence was determined in sera. Patient sera were mixed with HCS to determine if killing of the matched isolate was inhibited. RESULTS: Overall, 36 patients had elevated titers of cAbs, and 34 isolates were sensitive to HCS killing. Fifteen patients had cAbs and HCS-sensitive isolates; of these patients, 14 had serum that protected their matched bacteria from HCS killing. Patients with cAbs were less likely to be neutropenic or have comorbidities. CONCLUSIONS: cAbs are prevalent in patients with P aeruginosa BSI and allow survival of otherwise serum-sensitive bacteria in the bloodstream. Generation of cAbs may be a risk factor for the development of BSI.

A machine learning-based risk score for prediction of infective endocarditis among patients with Staphylococcus aureus bacteraemia - The SABIER score.

BACKGROUND: Early risk assessment is needed to stratify Staphylococcus aureus infective endocarditis (SA-IE) risk among Staphylococcus aureus bacteraemia (SAB) patients to guide clinical management. The objective of this study is to develop a novel risk score independent of subjective clinical judgment and can be used early at the time of blood culture positivity. METHODS: We conducted a retrospective big data analysis from territory-wide electronic data and included hospitalized patients with SAB between 2009 and 2019. We applied a random forest risk scoring model to select variables from an array of parameters, according to the statistical importance of each feature in predicting SA-IE outcome. The data was divided into derivation and validation cohorts. The areas under the curve of the receiver operating characteristic (AUCROC) were determined. RESULTS: We identified 15,741 SAB patients, among them 4.18% had SA-IE. The AUCROC was 0.74 (95%CI 0.70-0.76), with a negative predictive value of 0.980 (95%CI 0.977-0.983). The four most discriminatory features were age, history of infective endocarditis, valvular heart disease, and being community-onset. CONCLUSION: We developed a novel risk score with good performance as compared to existing scores and can be used at the time of SAB and prior to subjective clinical judgment.

Clinical and Bacteriological Specificities of Escherichia coli Bloodstream Infections From Biliary Portal of Entries.

BACKGROUND: Escherichia coli is frequently responsible for bloodstream infections (BSIs). Among digestive BSIs, biliary infections appear to be less severe. Respective roles of host factors, bacterial determinants (phylogroups, virulence, and antibiotic resistance), and portal of entry on outcome are unknown. METHODS: Clinical characteristics and prognosis of 770 episodes of E coli BSI were analyzed and isolates sequenced (Illumina technology) comparing phylogroups, multilocus sequence type, virulence, and resistance gene content. BSI isolates were compared with 362 commensal E coli from healthy subjects. RESULTS: Among 770 episodes, 135 were biliary, 156 nonbiliary digestive, and 479 urinary. Compared to urinary infections, BSIs of digestive origin occurred significantly more in men, comorbid, and immunocompromised patients. Digestive portal of entry was significantly associated with septic shock and death. Among digestive infections, patients with biliary infections were less likely to die (P = .032), despite comparable initial severity. Biliary E coli resembled commensals (phylogroup distribution, sequence type, and few virulence-associated genes) whereas nonbiliary digestive and urinary strains carried many virulence-associated genes. CONCLUSIONS: Escherichia coli strains responsible for biliary infections exhibit commensal characteristics and are associated with lower mortality rates, despite similar initial severity, than other digestive BSIs. Biliary drainage in addition to antibiotics in the management of biliary infections may explain improved outcome.

Characterization of gram-negative bloodstream infections in hospitalised Australian children and their clinical outcomes.

BACKGROUND: Gram-negative bloodstream infections (GNBSI) more commonly occur in children with comorbidities and are increasingly associated with antimicrobial resistance. There are few large studies of GNBSI in children that relate the clinical presentation, pathogen characteristics and outcomes. METHODS: A 3-year prospective study of GNBSI in children aged <18 years was conducted in five Australian children's hospitals between 2019-2021. The clinical characteristics, disease severity and outcomes were recorded. Causative pathogens underwent antibiotic susceptibility testing and whole genome sequencing. RESULTS: There were 931 GNBSI episodes involving 818 children. Median age was 3 years (IQR 0.6-8.5). 576/931 episodes (62%) were community onset though 661/931 (71%) occurred in children with comorbidities and a central venous catheter (CVC) was present in 558/931 (60%). CVC (145/931) and urinary tract (149/931) were the most common sources (16% each). 100/931 (11%) children required Intensive Care Unit (ICU) admission and a further 11% (105/931) developed GNBSI in ICU. 659/927 (71%) isolates were Enterobacterales of which 22% (138/630) were third generation cephalosporin resistant (3GCR). Extended spectrum beta-lactamase genes (ESBL) were confirmed in 65/138 (47%) 3GCR-Enterobacterales. Most common ESBL genes were blaCTX-M-15 (34/94, 36%) and blaSHV-12 (10/94, 11%). There were 48 deaths overall and 30-day in-hospital mortality was 3% (32/931). Infections with 3GCR Enterobacterales were independently associated with higher mortality (adjusted OR 3.2, 95%CI 1.6-6.4). CONCLUSION: GNBSI in children are frequently healthcare-associated and affect children under 5 years. Infections with 3GCR Enterobacterales were associated with worse outcomes. These findings will inform optimal management guidelines and help prioritise future antimicrobial clinical trials.

Emulating a Target Trial of Shorter Compared to Longer Course of Antibiotic Therapy for Gram-Negative Bacteremia.

BACKGROUND: Despite the availability of antimicrobial therapies, gram-negative bacteremia remains a significant cause of morbidity and mortality on a global level. Recent randomized controlled trials support shorter antibiotic treatment duration for individuals with uncomplicated gram-negative bacteremia. The target trial framework using the cloning approach utilizes real-world data but eliminates the issue of immortal time bias seen in observational studies by emulating the analysis of randomized trials with full adherence. METHOD: A hypothetical target trial allocating individuals with gram-negative bacteremia to either short antibiotic treatment duration (5-7 days) or longer antibiotic treatment duration (8-14 days) was specified and emulated using the cloning, censoring, and weighting approach. The primary outcome was 90-day all-cause mortality. Secondary outcome was a composite endpoint of clinical and microbiological relapse. The emulated trial included individuals from four hospitals in Copenhagen from 2018 through 2021. RESULTS: In sum, 1040 individuals were included. The median age of the cohort was 76 years, the majority were male (54%), had community-acquired gram-negative bacteremia (86%), urinary tract infection as the source of the infection (78%), and Escherichia coli as the pathogen of the infection (73%). The adjusted 90-day risk difference in all-cause mortality was 1.3% (95% confidence interval [CI]: -.7, 3.3), and the risk ratio was 1.12 (95% CI: .89, 1.37). The adjusted 90-day risk difference in relapse was 0.7% (95% CI: -2.3, 3.8), and the risk ratio was 1.07 (95% CI: .71, 1.45). CONCLUSIONS: We found comparative outcomes for shorter treatment duration compared to longer treatment duration in patients with gram-negative bacteremia.

Win Ratio Analyses of Piperacillin-Tazobactam Versus Meropenem for Ceftriaxone-Nonsusceptible Escherichia coli or Klebsiella pneumoniae Bloodstream Infections: Post Hoc Insights From the MERINO Trial.

BACKGROUND: Clinical trials of treatments for serious infections commonly use the primary endpoint of all-cause mortality. However, many trial participants survive their infection and this endpoint may not truly reflect important benefits and risks of therapy. The win ratio uses a hierarchical composite endpoint that can incorporate and prioritize outcome measures by relative clinical importance. METHODS: The win ratio methodology was applied post hoc to outcomes observed in the MERINO trial, which compared piperacillin-tazobactam with meropenem. We quantified the win ratio with a primary hierarchical composite endpoint, including all-cause mortality, microbiological relapse, and secondary infection. A win ratio of 1 would correspond to no difference between the 2 antibiotics, while a ratio <1 favors meropenem. Further analyses were performed to calculate the win odds and to introduce a continuous outcome variable in order to reduce ties. RESULTS: With the hierarchy of all-cause mortality, microbiological relapse, and secondary infection, the win ratio estimate was 0.40 (95% confidence interval [CI], .22-.71]; P = .002), favoring meropenem over piperacillin-tazobactam. However, 73.4% of the pairs were tied due to the small proportion of events. The win odds, a modification of the win ratio accounting for ties, was 0.79 (95% CI, .68-.92). The addition of length of stay to the primary composite greatly minimized the number of ties (4.6%) with a win ratio estimate of 0.77 (95% CI, .60-.99; P = .04). CONCLUSIONS: The application of the win ratio methodology to the MERINO trial data illustrates its utility and feasibility for use in antimicrobial trials.

Association between infectious diseases consultation and mortality in hospitalized patients with Gram-negative bloodstream infection: a retrospective population-wide cohort study.

OBJECTIVES: Data supporting routine infectious diseases (ID) consultation in Gram-negative bloodstream infection (GN-BSI) are limited. We evaluated the association between ID consultation and mortality in patients with GN-BSI in a retrospective population-wide cohort study in Ontario using linked health administrative databases. METHODS: Hospitalized adult patients with GN-BSI between April 2017 and December 2021 were included. The primary outcome was time to all-cause mortality censored at 30 days, analyzed using a mixed effects Cox proportional hazards model with hospital as a random effect. ID consultation 1-10 days after the first positive blood culture was treated as a time-varying exposure. RESULTS: Of 30,159 patients with GN-BSI across 53 hospitals, 11,013 (36.5%) received ID consultation. Median prevalence of ID consultation for patients with GN-BSI across hospitals was 35.0% with wide variability (range 2.7-76.1%, interquartile range 19.6-41.1%). 1041 (9.5%) patients who received ID consultation died within 30 days, compared to 1797 (9.4%) patients without ID consultation. In the fully-adjusted multivariable model, ID consultation was associated with mortality benefit (adjusted HR 0.82, 95% CI 0.77-0.88, p < 0.0001; translating to absolute risk reduction of -3.8% or NNT of 27). Exploratory subgroup analyses of the primary outcome showed that ID consultation could have greater benefit in patients with high-risk features (nosocomial infection, polymicrobial or non-Enterobacterales infection, antimicrobial resistance, or non-urinary tract source). CONCLUSIONS: Early ID consultation was associated with reduced mortality in patients with GN-BSI. If resources permit, routine ID consultation for this patient population should be considered to improve patient outcomes.

Intestinal mucositis, systemic inflammation and bloodstream infections following high-dose methotrexate treatment in childhood acute lymphoblastic leukaemia: Comparison between the NOPHO ALL 2008 protocol and the ALLTogether1 protocol.

Severe intestinal mucositis (IM) increases the risk of bloodstream infections (BSI) and inflammatory toxicity during acute lymphoblastic leukaemia (ALL) induction treatment. However, the implications of IM in subsequent ALL therapy phases after achieving remission remain unknown. This study investigated the relationship between IM (measured by plasma citrulline and the chemokine CCL20) and the development of BSI and systemic inflammation (reflected by C-reactive protein, CRP) in children with ALL during high-dose methotrexate (HDMTX) treatment, an important part of ALL consolidation therapy. The study compared patients treated according to the NOPHO ALL 2008 protocol (n = 52) and the ALLTogether1 protocol (n = 42), both with identical HDMTX procedures but different scheduling. One week post-HDMTX, citrulline dropped to median levels of 14.5 and 16.9 µM for patients treated according to the NOPHO ALL 2008 and ALLTogether1 protocols, respectively (p = 0.11). In a protocol and neutrophil count-adjusted analysis, hypocitrullinaemia (<10 µmol/L) was associated with increased odds of BSI within 3 weeks from HDMTX (OR = 26.2, p = 0.0074). Patients treated according to the NOPHO ALL 2008 protocol exhibited increased mucosal- and systemic inflammation post-HDMTX compared to patients treated according to ALLTogether1, with increased CCL20 (14.6 vs. 3.7 pg/mL, p < 0.0001) and CRP levels (10.0 vs. 1.0 mg/L, p < 0.0001). Both citrulline and CCL20 correlated with CRP for these patients (rs = -0.44, p = 0.0016 and rs = 0.35, p = 0.016, respectively). These results suggest that hypocitrullinaemia following HDMTX increases the risk of BSI, confirming previous observations from more intensive treatments. Moreover, these data indicate that the patients' vulnerability to mucositis and inflammatory toxicity after chemotherapy varies with treatment protocol.

Genomic investigation and clinical correlates of the in vitro ß-lactam: NaHCO3 responsiveness phenotype among methicillin-resistant Staphylococcus aureus isolates from a randomized clinical trial.

NaHCO3 responsiveness is a novel phenotype where some methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibit significantly lower minimal inhibitory concentrations (MIC) to oxacillin and/or cefazolin in the presence of NaHCO3. NaHCO3 responsiveness correlated with treatment response to ß-lactams in an endocarditis animal model. We investigated whether treatment of NaHCO3-responsive strains with ß-lactams was associated with faster clearance of bacteremia. The CAMERA2 trial (Combination Antibiotics for Methicillin-Resistant Staphylococcus aureus) randomly assigned participants with MRSA bloodstream infections to standard therapy, or to standard therapy plus an anti-staphylococcal ß-lactam (combination therapy). For 117 CAMERA2 MRSA isolates, we determined by broth microdilution the MIC of cefazolin and oxacillin, with and without 44 mM of NaHCO3. Isolates exhibiting ≥4-fold decrease in the MIC to cefazolin or oxacillin in the presence of NaHCO3 were considered "NaHCO3-responsive" to that agent. We compared the rate of persistent bacteremia among participants who had infections caused by NaHCO3-responsive and non-responsive strains, and that were assigned to combination treatment with a ß-lactam. Thirty-one percent (36/117) and 25% (21/85) of MRSA isolates were NaHCO3-responsive to cefazolin and oxacillin, respectively. The NaHCO3-responsive phenotype was significantly associated with sequence type 93, SCCmec type IVa, and mecA alleles with substitutions in positions -7 and -38 in the regulatory region. Among participants treated with a ß-lactam, there was no association between the NaHCO3-responsive phenotype and persistent bacteremia (cefazolin, P = 0.82; oxacillin, P = 0.81). In patients from a randomized clinical trial with MRSA bloodstream infection, isolates with an in vitro ß-lactam-NaHCO3-responsive phenotype were associated with distinctive genetic signatures, but not with a shorter duration of bacteremia among those treated with a ß-lactam.

Comparison of three rapid diagnostic tests for bloodstream infections using Benefit-risk Evaluation Framework (BED-FRAME).

Rapid diagnostic tests (RDTs) for bloodstream infections have the potential to reduce time to appropriate antimicrobial therapy and improve patient outcomes. Previously, an in-house, lipid-based, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) method, Fast Lipid Analysis Technique (FLAT MS), has shown promise as a rapid pathogen identification method. In this study, FLAT MS for direct from blood culture identification was evaluated and compared to FDA-cleared identification methods using the Benefit-risk Evaluation Framework (BED-FRAME) analysis. FLAT MS was evaluated and compared to Bruker Sepsityper and bioMérieux BioFire FilmArray BCID2 using results from a previous study. For this study, 301 positive blood cultures were collected from the University of Maryland Medical Center. The RDTs were compared by their sensitivities, time-to-results, hands-on time, and BED-FRAME analysis. The overall sensitivity of all platforms compared to culture results from monomicrobial-positive blood cultures was 88.3%. However, the three RDTs differed in their accuracy for identifying Gram-positive bacteria, Gram-negative bacteria, and yeast. Time-to-results for FLAT MS, Sepsityper, and BioFire BCID2 were all approximately one hour. Hands-on times for FLAT MS, Sepsityper, and BioFire BCID2 were 10 (±1.3), 40 (±2.8), and 5 (±0.25) minutes, respectively. BED-FRAME demonstrated that each RDT had utility at different pathogen prevalence and relative importance. BED-FRAME is a useful tool that can used to determine which RDT is best for a healthcare center.

Culture-independent identification of bloodstream infections from whole blood: prospective evaluation in specimens of known infection status.

Sepsis caused by bloodstream infection (BSI) is a major healthcare burden and a leading cause of morbidity and mortality worldwide. Timely diagnosis is critical to optimize clinical outcome, as mortality rates rise every hour treatment is delayed. Blood culture remains the "gold standard" for diagnosis but is limited by its long turnaround time (1-7 days depending on the organism) and its potential to provide false-negative results due to interference by antimicrobial therapy or the presence of mixed (i.e., polymicrobial) infections. In this paper, we evaluated the performance of resistance and pathogen ID/BSI, a direct-from-specimen molecular assay. To reduce the false-positivity rate common with molecular methods, this assay isolates and detects genomic material only from viable microorganisms in the blood by incorporating a novel precursor step to selectively lyse host and non-viable microbial cells and remove cell-free genomic material prior to lysis and analysis of microbial cells. Here, we demonstrate that the assay is free of interference from host immune cells and common antimicrobial agents at elevated concentrations. We also demonstrate the accuracy of this technology in a prospective cohort pilot study of individuals with known sepsis/BSI status, including samples from both positive and negative individuals. IMPORTANCE: Blood culture remains the "gold standard" for the diagnosis of sepsis/bloodstream infection (BSI) but has many limitations which may lead to a delay in appropriate and accurate treatment in patients. Molecular diagnostic methods have the potential for markedly improving the management of such patients through faster turnaround times and increased accuracy. But molecular diagnostic methods have not been widely adopted for the identification of BSIs. By incorporating a precursor step of selective lysis of host and non-viable microorganisms, our resistance and pathogen ID (RaPID)/BSI molecular assay addresses many limitations of blood culture and other molecular assay. The RaPID/BSI assay has an approximate turnaround time of 4 hours, thereby significantly reducing the time to appropriate and accurate diagnosis of causative microorganisms in such patients. The short turnaround time also allows for close to real-time tracking of pathogenic clearance of microorganisms from the blood of these patients or if a change of antimicrobial regimen is required. Thus, the RaPID/BSI molecular assay helps with optimization of antimicrobial stewardship; prompt and accurate diagnosis of sepsis/BSI could help target timely treatment and reduce mortality and morbidity in such patients.

Performance evaluation of machine-assisted interpretation of Gram stains from positive blood cultures.

Manual microscopy of Gram stains from positive blood cultures (PBCs) is crucial for diagnosing bloodstream infections but remains labor intensive, time consuming, and subjective. This study aimed to evaluate a scan and analysis system that combines fully automated digital microscopy with deep convolutional neural networks (CNNs) to assist the interpretation of Gram stains from PBCs for routine laboratory use. The CNN was trained to classify images of Gram stains based on staining and morphology into seven different classes: background/false-positive, Gram-positive cocci in clusters (GPCCL), Gram-positive cocci in pairs (GPCP), Gram-positive cocci in chains (GPCC), rod-shaped bacilli (RSB), yeasts, and polymicrobial specimens. A total of 1,555 Gram-stained slides of PBCs were scanned, pre-classified, and reviewed by medical professionals. The results of assisted Gram stain interpretation were compared to those of manual microscopy and cultural species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The comparison of assisted Gram stain interpretation and manual microscopy yielded positive/negative percent agreement values of 95.8%/98.0% (GPCCL), 87.6%/99.3% (GPCP/GPCC), 97.4%/97.8% (RSB), 83.3%/99.3% (yeasts), and 87.0%/98.5% (negative/false positive). The assisted Gram stain interpretation, when compared to MALDI-TOF MS species identification, also yielded similar results. During the analytical performance study, assisted interpretation showed excellent reproducibility and repeatability. Any microorganism in PBCs should be detectable at the determined limit of detection of 105 CFU/mL. Although the CNN-based interpretation of Gram stains from PBCs is not yet ready for clinical implementation, it has potential for future integration and advancement.

Etiology of nosocomial infections in intensive care patients in German hospitals: An analysis of trends between 2008 and 2022.

PURPOSE: Data from the intensive care component of the German hospital infection surveillance system (KISS) was used to investigate the epidemiology of pathogens responsible for the most frequent device-associated infections and their development over time. METHOD: The 10 most common pathogens were identified for ventilator-associated lower respiratory tract infections (VALRTI), catheter associated urinary tract infections (CAUTI), and central venous catheter associated bloodstream infections (CVC-BSI). The development over time was analyzed based on three five-year time periods: 2008-2012, 2013-2017, 2018-2022. RESULTS: Data from 1425 ICUs were included together with 121,762 device-associated infections with 138,299 isolated pathogens. A remarkable and significant increase in the frequency of Klebsiella spp. was found for VALRTI, that was almost twice as high during 2018-2022 compared to 2008-2012. For CAUTI, there was a significant increase of all Enterobacterales with the most prominent increase in Klebsiella spp. With regard to CVC-BSI, the situation for coagulase-negative staphylococci and E. coli was relatively stable; while there was a significant increase in Enterococcus spp. and Klebsiella spp. and a decrease in S. aureus. CONCLUSION: Knowledge about the current frequency of pathogens responsible for nosocomial infections in intensive care units is important for guiding empirical antimicrobial therapy. Data from national nosocomial infection surveillance systems can provide relevant information about the development of pathogens.

Neighborhood Income Is Associated with Health Care Use in Pediatric Short Bowel Syndrome.

OBJECTIVE: To evaluate associations between neighborhood income and burden of hospitalizations for children with short bowel syndrome (SBS). STUDY DESIGN: We used the Pediatric Health Information System (PHIS) database to evaluate associations between neighborhood income and hospital readmissions, readmissions for central line-associated bloodstream infections (CLABSI), and hospital length of stay (LOS) for patients <18 years with SBS hospitalized between January 1, 2006, and October 1, 2015. We analyzed readmissions with recurrent event analysis and analyzed LOS with linear mixed effects modeling. We used a conceptual model to guide our multivariable analyses, adjusting for race, ethnicity, and insurance status. RESULTS: We included 4289 children with 16 347 hospitalizations from 43 institutions. Fifty-seven percent of the children were male, 21% were Black, 19% were Hispanic, and 67% had public insurance. In univariable analysis, children from low-income neighborhoods had a 38% increased risk for all-cause hospitalizations (rate ratio [RR] 1.38, 95% CI 1.10-1.72, P = .01), an 83% increased risk for CLABSI hospitalizations (RR 1.83, 95% CI 1.37-2.44, P < .001), and increased hospital LOS (ß 0.15, 95% CI 0.01-0.29, P = .04). In multivariable analysis, the association between low-income neighborhoods and elevated risk for CLABSI hospitalizations persisted (RR 1.70, 95% CI 1.23-2.35, P < .01, respectively). CONCLUSIONS: Children with SBS from low-income neighborhoods are at increased risk for hospitalizations due to CLABSI. Examination of specific household- and neighborhood-level factors contributing to this disparity may inform equity-based interventions.

A multisite validation of a two hours antibiotic susceptibility flow cytometry assay directly from positive blood cultures.

BACKGROUND: Rapid antimicrobial susceptibility testing (AST) is urgently needed to provide safer treatment to counteract antimicrobial resistance. This is critical in septic patients, because resistance increases empiric therapy uncertainty and the risk of a poor outcome. We validate a novel 2h flow cytometry AST assay directly from positive blood cultures (PBC) by using a room temperature stable FASTgramneg and FASTgrampos kits (FASTinov® Porto, Portugal) in three sites: FASTinov (site-1), Hospital Ramon y Cajal, Madrid, Spain (site-2) and Centro Hospitalar S. João, Porto, Portugal (site-3). A total of 670 PBC were included: 333 spiked (site-1) and 337 clinical PBC (151 site-2 and 186 site-3): 367 gram-negative and 303 gram-positive. Manufacturer instructions were followed for sample preparation, panel inoculation, incubation (1h/37ºC) and flow cytometry analysis using CytoFlex (Site-1 and -2) or DxFlex (site-3) both instruments from Beckman-Coulter, USA. RESULTS: A proprietary software (bioFAST) was used to immediately generate a susceptibility report in less than 2 h. In parallel, samples were processed according to reference AST methods (disk diffusion and/or microdilution) and interpreted with EUCAST and CLSI criteria. Additionally, ten samples were spiked in all sites for inter-laboratory reproducibility. Sensitivity and specificity were >95% for all antimicrobials. Reproducibility was 96.8%/95.0% for FASTgramneg and 95.1%/95.1% for FASTgrampos regarding EUCAST/CLSI criteria, respectively. CONCLUSION: FASTinov® kits consistently provide ultra-rapid AST in 2h with high accuracy and reproducibility on both Gram-negative and Gram-positive bacteria. This technology creates a new paradigm in bacterial infection management and holds the potential to significantly impact septic patient outcomes and antimicrobial stewardship.

Retrospective analysis of molecular characteristics, risk factors, and outcomes in carbapenem-resistant Klebsiella pneumoniae bloodstream infections.

BACKGROUND: Klebsiella pneumoniae (KP) is the second most prevalent Gram-negative bacterium causing bloodstream infections (BSIs). In recent years, the management of BSIs caused by KP has become increasingly complex due to the emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP). Although numerous studies have explored the risk factors for the development of CRKP-BSIs, the mortality of patients with KP-BSIs, and the molecular epidemiological characteristics of CRKP, the variability in data across different populations, countries, and hospitals has led to inconsistent conclusions. In this single-center retrospective observational study, we utilized logistic regression analyses to identify independent risk factors for CRKP-BSIs and factors associated with mortality in KP-BSI patients. Furthermore, a risk factor-based prediction model was developed. CRKP isolates underwent whole-genome sequencing (WGS), followed by an evaluation of microbiological characteristics, including antimicrobial resistance and virulence genes, as well as epidemiological characteristics and phylogenetic analysis. RESULTS: Our study included a total of 134 patients with KP-BSIs, comprising 50 individuals infected with CRKP and 84 with carbapenem-susceptible Klebsiella pneumoniae (CSKP). The independent risk factors for CRKP-BSIs were identified as gastric catheterization (OR = 9.143; CI = 1.357-61.618; P = 0.023), prior ICU hospitalization (OR = 4.642; CI = 1.312-16.422; P = 0.017), and detection of CRKP in non-blood sites (OR = 8.112; CI = 2.130-30.894; P = 0.002). Multivariate analysis revealed that microbiologic eradication after 6 days (OR = 3.569; CI = 1.119-11.387; P = 0.032), high Pitt bacteremia score (OR = 1.609; CI = 1.226-2.111; P = 0.001), and inappropriate empirical treatment after BSIs (OR = 6.756; CI = 1.922-23.753; P = 0.003) were independent risk factors for the 28-day mortality in KP-BSIs. The prediction model confirmed that microbiologic eradication after 6.5 days and a Pitt bacteremia score of 4.5 or higher were significant predictors of the 28-day mortality. Bioinformatics analysis identified ST11 as the predominant CRKP sequence type, with blaKPC-2 as the most prevalent gene variant. CRKP stains carried multiple plasmid-mediated resistance genes along with some virulence genes. Phylogenetic analysis indicated the presence of nosocomial transmission of ST11 CRKP within the ICU. CONCLUSIONS: The analysis of risk factors for developing CRKP-BSIs and the association between KP-BSIs and 28-day mortality, along with the development of a risk factor-based prediction model and the characterization of CRKP strains, enhances clinicians' understanding of the pathogens responsible for BSIs. This understanding may help in the timely administration of antibiotic therapy for patients with suspected KP-BSIs, potentially improving outcomes.

High prevalence of ST5-SCCmec II-t311 clone of methicillin-resistant Staphylococcus aureus isolated from bloodstream infections in East China.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is a challenging global health threat, resulting in significant morbidity and mortality worldwide. This study aims to determine the molecular characteristics and antimicrobial susceptibility of 263 MRSA isolates in Zhejiang Province, east China. METHODS: From 2014 to 2019, a total of 263 MRSA isolates from bloodstream infections (BSIs) were collected from 6 hospitals in 4 cities in Zhejiang province, east China. Antimicrobial susceptibility tests were conducted according to the guidelines set forth by the Clinical and Laboratory Standards Institute (CLSI). To characterize and analyze these isolates, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) typing and virulence genes gene profiles were performed. RESULTS: The most predominant clone was ST5-SCCmec II-t311, which accounted for 41.8% (110/263), followed by ST59 (44/263, 16.7%). Compared with non-ST5-II-t311 isolates, ST5-II-t311 isolates were more resistant to erythromycin, tetracycline, levofloxacin, moxifloxacin, and ciprofloxacin, but more susceptible to clindamycin. Moreover, the rates of multidrug resistance were higher in ST5-II-t311 isolates compared to the non-ST5-II-t311 isolates. In comparison to the non-ST5-II-t311 isolates, ST5-II-t311 isolates showed no significant difference in virulence genes detected. CONCLUSIONS: MRSA ST5-II-t311 clone has become the most predominant clone in Zhejiang Province, east China and has higher rates of multidrug resistance than other isolates, that should be kept in mind when treating BSI. Moreover, MRSA ST59 clone shows an upward trend and has begun to spread into hospitals. Our findings highlight the importance of epidemiological studies of S. aureus carriage in the eastern region.

Whole genome sequencing and molecular epidemiology of methicillin-resistant Staphylococcus aureus isolated from patients with bacteraemia in Slovenia.

PURPOSE: Data on the molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates from patients with bacteraemia in Slovenia are lacking. The aim of this study was to phenotypically and genotypically investigate 82 MRSA strains isolated from patients with bloodstream infections in central Slovenia between 2019 and 2022. METHODS: Whole-genome sequencing of selected strains was performed to characterize the strains based on sequence typing, antimicrobial resistance, toxin, and virulence factors genes. RESULTS: Most MRSA carried SCCmec II (63.4%), followed by SCCmec IV (34.1%) and SCCmec V (2.5%). A high proportion of strains belonging to the ST225 lineage (45.1%) was observed, followed by ST97 (18.3%), ST2883 (15.9%), ST22 (9.8%), ST5 (3.7%), and the ST1, ST398 and ST45 lineages (2.4% each). Sixteen different spa types were identified, predominantly ST225-t003 (31.7%), ST97-t359 (15.9%), and ST2883-t4336 (14.6%). None of the strains carried Panton-Valentine leukocidin, exfoliative toxins, or toxic shock toxin. All MRSA strains were susceptible to linezolid, rifampicin, vancomycin, teicoplanin, and trimethoprim-sulfamethoxazole. MRSA strains were resistant to erythromycin, clindamycin, tetracycline and gentamicin, with a frequency of 74.4%, 74.4%, 8.5%, and 1.2%, respectively. CONCLUSION: This study demonstrates that bacteraemia in central Slovenia is caused by diverse MRSA lineages. Identification of newly emerged lineages should be followed in the future to detect changes in the molecular epidemiology of MRSA in our country.

Population-based genomic surveillance of Pseudomonas aeruginosa causing bloodstream infections in a large Canadian health region.

PURPOSE: Population-based surveillance was undertaken to determine clinical factors, susceptibility patterns, and incidence rates (IR) of Pseudomonas aeruginosa causing bloodstream infections (BSIs) in a Canadian region (2010-2018). METHODS: We combined clinical data with genomics to characterize P. aeruginosa (BSIs) (n = 167) in a well-defined Canadian (Calgary) human population over a 9-year period (2010-2018). RESULTS: The annual population IR per 100,000 patient years increased from 3.4/100,000 in 2010 to 5.9/100,000 in 2018, with the highest IRs in elderly males from the hospital setting. Over a quarter of patients presented with febrile neutropenia, followed by urinary tract infections and pneumonia. Antimicrobial resistance (AMR) rates and determinants were rare. The P. aeruginosa population was polyclonal consisting of three dominant sequence types (STs), namely ST244, ST111, and ST17. Antimicrobial-susceptible ST244 was the most common clone and belonged to three clades (A, B, C). The ST244 IR/100,000 increased over time due to the expansion of clade C. Multidrug-resistant ST111 was the second most common clone and IR/100,000 decreased over time. ST111 belonged to three clades (A, B, C) with clade C containing blaVIM-2. Different serotypes were linked to various STs. The IR/100,000 of P. aeruginosa that belonged to serotypes O6 increased significantly over time. CONCLUSION: An effective multivalent vaccine consisting of five serotypes (O1, O3, O5, O6, O11) would confer protection to > 70% of Calgary residents with P. aeruginosa BSIs. This study has provided a unique perspective of the population dynamics over time of P. aeruginosa STs, clades, and serotypes responsible for BSIs.

Clinical evaluation of a multiplex droplet digital PCR for pathogen detection in critically ill COVID-19 patients with bloodstream infections.

BACKGROUND: Nosocomial bloodstream infections (nBSI) have emerged as a clinical concern for physicians treating COVID-19 patients. In this study, we aimed to evaluate the effectiveness of a multiplex ddPCR in detecting bacterial pathogens in the blood of COVID-19 critically ill patients. METHODS: This prospective diagnostic study included RT-PCR-confirmed COVID-19 patients admitted to our hospital from December 2022 to February 2023. A multiplex ddPCR assay was used to detect common bacterial pathogens and AMR genes in blood samples of the patients, along with antimicrobial susceptibility testing (AST). The diagnostic performance of the ddPCR assay was evaluated by comparing the results with those obtained through blood culture and clinical diagnosis. Additionally, the ability of ddPCR in detecting bacterial resistance was compared with the AST results. RESULTS: Of the 200 blood samples collected from 184 patients, 45 (22.5%) were positive using blood culture, while 113 (56.5%) were positive for bacterial targets using the ddPCR assay. The ddPCR assay outperformed blood culture in pathogen detection rate, mixed infection detection rate, and fungal detection rate. Acinetobacter baumannii and Klebsiella pneumoniae were the most commonly detected pathogens in COVID-19 critically ill patients, followed by Enterococcus and Streptococcus. Compared to blood culture, ddPCR achieved a sensitivity of 75.5%, specificity of 51.0%, PPV of 30.9%, and NPV of 87.8%, respectively. However, there were significant differences in sensitivity among different bacterial species, where Gram-negative bacteria have the highest sensitivity of 90.3%. When evaluated on the ground of clinical diagnosis, the sensitivity, specificity, PPV and NPV of ddPCR were 78.1%, 90.5%, 94.7%, and 65.5%, respectively. In addition, the ddPCR assay detected 23 cases of blaKPC, which shown a better consistent with clinical test results than other detected AMR genes. Compared to blaKPC, there were few other AMR genes detected, indicating that the application of other AMR gene detection in the COVID-19 critically ill patients was limited. CONCLUSION: The multiplex ddPCR assay had a significantly higher pathogen detection positivity than the blood culture, which could be an effective diagnostic tool for BSIs in COVID-19 patients and to improve patient outcomes and reduce the burden of sepsis on the healthcare system, though there is room for optimization of the panels used.- Adjusting the targets to include E. faecalis and E. faecium as well as Candida albicans and Candida glabrata could improve the ddPCR' s effectiveness. However, further research is needed to explore the potential of ddPCR in predicting bacterial resistance through AMR gene detection.