Guillermo Martín-Gutiérrez

The primary obstacle in detecting bloodstream infections is their often extremely low bacterial concentration. To accurately detect such infections, we developed a quantitative PCR assay, named LowLoad-qPCR, based on the combination of increased input DNA by random priming amplification method and the use of multiple probes sharing a single fluorophore. Here, we detail the key improvements over conventional qPCR and evaluate its performance using blood samples.

Background: To assess the impact of the rapid diagnosis and treatment of severe infections (rDTSI) program on diagnostic and clinical outcomes in patients with severe infections. Method: We conducted a pre–post quasi-experimental study evaluating patients with severe pneumonia or sepsis before (October 2019–February 2020) and after (March 2022–March 2023) rDTSI implementation. The program integrated rapid molecular diagnostics, a 24/7 laboratory workflow, and multidisciplinary training. Primary outcomes included time from clinical diagnosis to pathogen-directed therapy and targeted therapy within 48 h. Secondary outcomes assessed antimicrobial appropriateness (DOOR-MAT), length of stay, and mortality. Results: The rDTSI program significantly reduced the median time to pathogen-directed therapy in pneumonia (48.8 vs 23.6 h, P < .001) and increased targeted therapy within 48 h (36.17% to 58.14%, P = .049). Hospital stays decreased (38.9 to 22.2 days, P < .001). In sepsis, diagnostic times (19.4 vs 18.1 h, P = .028) and DOOR-MAT scores (80.4 vs 88.0, P = .024) improved, while other clinical outcomes remained unchanged. Conclusions: The rDTSI program accelerated microbiological diagnosis, optimized antimicrobial therapy, and improved hospital efficiency in severe infections. These findings support integrating rapid diagnostics into antimicrobial stewardship programs to enhance severe infection management.

Pseudomonas aeruginosa is a critical priority pathogen due to its high resistance rates and limited treatment options. Rapid antimicrobial susceptibility testing (rAST) is essential to optimize therapy, particularly because resistance mechanisms in P. aeruginosa often arise from adaptive genetic expression rather than specific resistance genes. FASTinov, a flow cytometry-based rAST system, was developed to address this challenge by providing phenotypic susceptibility results within hours. In this study, we assessed FASTinov’s performance for rapid antimicrobial susceptibility testing (rAST) in 100 P. aeruginosa strains, using broth microdilution as the reference method. For clinical relevance, we also included the MicroScan WalkAway system as a comparator. FASTinov demonstrated a categorical agreement of 99% across the tested antibiotics, with particularly strong agreement for cephalosporins. Notably, FASTinov significantly reduced the time to results, delivering susceptibility data in an average of 3.15 h (CI 95% 3.13–3.17), compared to 28.88 h (CI 95% 20.71–37.04) for standard methods. Additionally, the system exhibited lower rates of major and very major errors compared to MicroScan, further reinforcing its reliability. These findings highlight FASTinov as a promising rAST system capable of providing rapid and accurate susceptibility profiles directly from bacterial colonies. Further research is warranted to evaluate its clinical impact on patient outcomes and cost-effectiveness.

Background/Objectives: Meropenem is widely used to treat Pseudomonas aeruginosa infections; however, the pathogen’s increasing resistance compromises its efficacy. In this study, we aimed to develop a selective culture medium for detecting the presence of meropenem-resistant Pseudomonas aeruginosa in respiratory specimens within 24 h. Methods: The medium’s performance was challenged using a collection of 130 clinical Pseudomonas aeruginosa strains (of which 85 were meropenem-susceptible, 14 were meropenem-intermediate, and 21 were meropenem-resistant). Subsequently, clinical validation was carried out using 130 respiratory samples. Results: The selective medium demonstrated excellent sensitivity (average 98.7%) and specificity (average 90%) across bacterial concentrations ranging from 1 × 10⁴ to 1 × 10⁸ CFU/mL, and a high negative predictive value (average 99.2%) compared to the broth microdilution (BMD) method. Clinical validation with bronchoalveolar lavage (BAL) and tracheobronchial aspirate (TBA) clinical specimens (N = 130) revealed a strong performance, with 92,3% categorical agreement. Conclusions: This method accelerates susceptibility testing, is user-friendly, and delivers reliable results, contributing to the optimization of empirical treatment for respiratory tract infections.

Background/Objectives: Meropenem (MER) is widely used for the treatment of Pseudomonas aeruginosa infections; however, increasing resistance compromises its efficacy. This study aimed to develop a selective culture medium for the detection of meropenem-resistant P. aeruginosa from respiratory specimens within 24 hours. Methods: The performance of the medium was challenged using a collection of 130 clinical P. aeruginosa strains (85 MER-susceptible, 14 MER-intermediate, and 21 MER-resistant). Subsequently, clinical validation was carried out using 130 respiratory samples. Results: The selective medium demonstrated excellent sensitivity (avg. 98.7%) and specificity (avg. 90%) across bacterial concentrations ranging from 1 × 10⁴ to 1 × 10⁸ CFU/mL, and a high negative predictive value (avg. 99.2%) compared to the broth microdilution (BMD) method. Clinical validation with bronchoalveolar lavage (BAL) and tracheobronchial aspirate (TBA) clinical specimens (N = 130) showed high performance, with 92,3% categorical agreement. Conclusions: This method accelerates susceptibility testing, is user-friendly, and delivers reliable results, contributing to the optimization of empirical treatment for respiratory tract infections.

Multiple studies have demonstrated the utility of sonication to improve culture yield in patients with cardiovascular implantable electronic device (CIED) infections. To analyze the usefulness of sonication in the microbiological diagnosis of CIED infections in comparison with traditional cultures. Systematic database searches were performed to identify studies that provided enough data concerning both sensitivity and specificity of traditional (non-sonicated) and sonicated cultures from CIED samples. The diagnostic accuracy measures were obtained by three different statistical approaches: (i) The univariate model; (ii) The bivariate random; and (iii) The Bayesian bivariate hierarchical model. Heterogeneity was assessed using meta-regression. Nine studies met the criteria for inclusion in the meta-analysis (1684 cultures). The summary estimates of sensitivity were higher for sonicated cultures (0.756) in comparison with non-sonicated cultures (0.446). On meta-regression, sonication of CIEDs significantly increased the sensitivity (p = 0.001) as well as the rates of false positive results (p = 0.003). The final model also showed that the studies that used a threshold for positivity were associated with lower rates of false positive results (p < 0.001). Our results suggest that sonication improves the microbiological diagnosis of CIED infections in comparison with traditional cultures, but a standardization of processes is necessary.

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.

Different factors, including antimicrobial resistance, may diminish the effectiveness of antibiotic therapy, challenging the management of post-transplant urinary tract infection (UTI). The association of acidic urine pH with microbiological and clinical outcomes was evaluated after fosfomycin or ciprofloxacin therapy in 184 kidney transplant recipients (KTRs) with UTI episodes by Escherichia coli (N = 115) and Klebsiella pneumoniae (N = 69). Initial urine pH, antimicrobial therapy, and clinical and microbiological outcomes, and one- and six-month follow-up were assessed. Fosfomycin was prescribed in 88 (76.5%) E. coli and 46 (66.7%) K. pneumoniae UTI episodes in the total cohort. When the urine pH ≤ 6, fosfomycin was prescribed in 60 (52.2%) E. coli and 29 (42.0%) K. pneumoniae. Initial urine pH ≤ 6 in E. coli UTI was associated with symptomatic episodes (8/60 vs. 0/55, p = 0.04) at one-month follow-up, with a similar trend in those patients receiving fosfomycin (7/47 vs. 0/41, p = 0.09). Acidic urine pH was not associated with microbiological or clinical cure in K. pneumoniae UTI. At pH 5, the ciprofloxacin MIC₉₀ increased from 8 to >8 mg/L in E. coli and from 4 to >8 mg/L in K. pneumoniae. At pH 5, the fosfomycin MIC₉₀ decreased from 8 to 4 mg/L in E. coli and from 512 to 128 mg/L in K. pneumoniae. Acidic urine is not associated with the microbiological efficacy of fosfomycin and ciprofloxacin in KTRs with UTI, but it is associated with symptomatic UTI episodes at one-month follow-up in E. coli episodes.

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.

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.