Differences in clinical outcomes of bloodstream infections caused by Klebsiella aerogenes, Klebsiella pneumoniae and Enterobacter cloacae: a multicentre cohort study.

BACKGROUND: Klebsiella aerogenes has been reclassified from Enterobacter to Klebsiella genus due to its phenotypic and genotypic similarities with Klebsiella pneumoniae. It is unclear if clinical outcomes are also more similar. This study aims to assess clinical outcomes of bloodstreams infections (BSI) caused by K. aerogenes, K. pneumoniae and Enterobacter cloacae, through secondary data analysis, nested in PRO-BAC cohort study. METHODS: Hospitalized patients between October 2016 and March 2017 with monomicrobial BSI due to K. aerogenes, K. pneumoniae or E. cloacae were included. Primary outcome was a composite clinical outcome including all-cause mortality or recurrence until 30 days follow-up. Secondary outcomes were fever ≥ 72 h, persistent bacteraemia, and secondary device infection. Multilevel mixed-effect Poisson regression was used to estimate the association between microorganisms and outcome. RESULTS: Overall, 29 K. aerogenes, 77 E. cloacae and 337 K. pneumoniae BSI episodes were included. Mortality or recurrence was less frequent in K. aerogenes (6.9%) than in E. cloacae (20.8%) or K. pneumoniae (19.0%), but statistical difference was not observed (rate ratio (RR) 0.35, 95% CI 0.08 to 1.55; RR 0.42, 95% CI 0.10 to 1.71, respectively). Fever ≥ 72 h and device infection were more common in K. aerogenes group. In the multivariate analysis, adjusted for confounders (age, sex, BSI source, hospital ward, Charlson score and active antibiotic therapy), the estimates and direction of effect were similar to crude results. CONCLUSIONS: Results suggest that BSI caused by K. aerogenes may have a better prognosis than E. cloacae or K. pneumoniae BSI.

Simultaneous and divergent evolution of resistance to cephalosporin/ß-lactamase inhibitor combinations and imipenem/relebactam following ceftazidime/avibactam treatment of MDR Pseudomonas aeruginosa infections.

OBJECTIVES: To describe and characterize the emergence of resistance to ceftolozane/tazobactam, ceftazidime/avibactam and imipenem/relebactam in a patient receiving ceftazidime/avibactam treatment for an MDR Pseudomonas aeruginosa CNS infection. METHODS: One baseline (PA1) and two post-exposure (PA2 and PA3) isolates obtained before and during treatment of a nosocomial P. aeruginosa meningoventriculitis were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. The impact on ß-lactam resistance of mutations in blaPDC and mexR was determined through cloning experiments and complementation assays. RESULTS: Isolate PA1 showed baseline resistance mutations in DacB (I354A) and OprD (N142fs) conferring resistance to conventional antipseudomonals but susceptibility to ceftazidime/avibactam, ceftolozane/tazobactam and imipenem/relebactam. Post-exposure isolates showed two divergent ceftazidime/avibactam-resistant phenotypes associated with distinctive mutations affecting the intrinsic P PDC ß-lactamase (S254Ins) (PA2: ceftolozane/tazobactam and ceftazidime/avibactam-resistant) or MexAB-OprM negative regulator MexR in combination with modification of PBP3 (PA3: ceftazidime/avibactam and imipenem/relebactam-relebactam-resistant). Cloning experiments demonstrated the role of PDC modification in resistance to ceftolozane/tazobactam and ceftazidime/avibactam. Complementation with a functional copy of the mexR gene in isolate PA3 restored imipenem/relebactam susceptibility. CONCLUSIONS: We demonstrated how P. aeruginosa may simultaneously develop resistance and compromise the activity of new ß-lactam/ß-lactamase inhibitor combinations when exposed to ceftazidime/avibactam through selection of mutations leading to PDC modification and up-regulation of MexAB-OprM-mediated efflux.

Activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam against ceftolozane/tazobactam- and ceftazidime/avibactam-resistant Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam against a clinical and laboratory collection of ceftolozane/tazobactam- and ceftazidime/avibactam-resistant Pseudomonas aeruginosa ß-lactamase mutants. METHODS: The activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam, cefepime/zidebactam and comparators was evaluated against a collection of 30 molecularly characterized ceftolozane/tazobactam- and/or ceftazidime/avibactam-resistant P. aeruginosa isolates from patients previously treated with cephalosporins. To evaluate how the different ß-lactamases in the clinical isolates affected the resistance to these agents, a copy of each blaPDC, blaOXA-2 and blaOXA-10 ancestral and mutant allele from the clinical isolates was cloned in pUCp24 and expressed in dual blaPDC-oprD (for blaPDC-like genes) or single oprD (for blaOXA-2-like and blaOXA-10-like genes) PAO1 knockout mutants. MICs were determined using reference methodologies. RESULTS: For all isolates, MICs were higher than 4 and/or 8 mg/L for ceftolozane/tazobactam and ceftazidime/avibactam, respectively. Cefiderocol was the most active agent, showing activity against all isolates, except one clinical isolate that carried an R504C substitution in PBP3 (MIC = 16 mg/L). Imipenem/relebactam was highly active against all isolates, except two clinical isolates that carried the VIM-20 carbapenemase. Cefepime/zidebactam and cefepime/taniborbactam displayed activity against most of the isolates, but resistance was observed in some strains with PBP3 amino acid substitutions or that overexpressed mexAB-oprM or mexXY efflux pumps. Evaluation of transformants revealed that OXA-2 and OXA-10 extended-spectrum variants cause a 2-fold increase in the MIC of cefiderocol relative to parental enzymes. CONCLUSIONS: Cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam show promising and complementary in vitro activity against ceftolozane/tazobactam- and ceftazidime/avibactam-resistant P. aeruginosa. These agents may represent potential therapeutic options for ceftolozane/tazobactam- and ceftazidime/avibactam-resistant P. aeruginosa infections.

Transmission of toxigenic Clostridiodes difficile between a pet dog with diarrhea and a 10-month-old infant.

We describe a case of interspecies transmission of toxigenic Clostridioides difficile involving a female and her dog, both with diarrhea without another diagnosis. Genomic analysis showed that isolates were grouped into MLST clade I, closely related to ribotype 020 and shared identical genotypes.

Molecular mechanisms driving the in vivo development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR Pseudomonas aeruginosa infections.

BACKGROUND: The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning. OBJECTIVES: Characterization of the mechanisms leading to the development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR P. aeruginosa infections. METHODS: Four paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of ß-lactamases were determined by genotypic methods, including WGS. The OXA-10 variants were cloned in PAO1 to assess their impact on resistance. Models for the OXA-10 derivatives were constructed to evaluate the structural impact of the amino acid changes. RESULTS: The same XDR ST253 P. aeruginosa clone was detected in all four cases evaluated. All initial isolates showed OprD deficiency, produced an OXA-10 enzyme and were susceptible to ceftazidime, ceftolozane/tazobactam, ceftazidime/avibactam and colistin. During treatment, the isolates developed resistance to all cephalosporins. Comparative genomic analysis revealed that the evolved resistant isolates had acquired mutations in the OXA-10 enzyme: OXA-14 (Gly157Asp), OXA-794 (Trp154Cys), OXA-795 (ΔPhe153-Trp154) and OXA-824 (Asn143Lys). PAO1 transformants producing the evolved OXA-10 derivatives showed enhanced ceftolozane/tazobactam and ceftazidime/avibactam resistance but decreased meropenem MICs in a PAO1 background. Imipenem/relebactam retained activity against all strains. Homology models revealed important changes in regions adjacent to the active site of the OXA-10 enzyme. The blaOXA-10 gene was plasmid borne and acquired due to transposition of Tn6746 in the pHUPM plasmid scaffold. CONCLUSIONS: Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/ß-lactamase inhibitor combinations in P. aeruginosa.

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.

Sterility assurance of technetium-99m radiopharmaceuticals: no specific air conditions are required for their preparation, storage and dispensation.

BACKGROUND: The aim of this study was to ascertain whether the extemporaneous technetium-99m radiopharmaceuticals - prepared by closed procedures - maintain or not sterility throughout their lifespan regardless the quality of the environmental air where they are prepared, stored and dispensed, provided that the basic aseptic rules for closed procedures are followed. METHODS: Three different types of assays were performed in this study: 1) sterility tests of each and every one of the vials with the remains of technetium-99m radiopharmaceuticals, which have been prepared in our hospital radiopharmacy during three years without any special environmental air conditions; 2) integrity tests of punctured rubber plug closures using both microbial challenge testing and dye ingress testing; and 3) simulation of the dispensing process using a liquid growth medium. RESULTS: Sterility tests of more than 6,000 vials with the remains of technetium-99m radiopharmaceuticals - prepared without any special ambient air conditions - were performed, all of them with a negative result (no growth of microorganisms occurs). The integrity of punctured rubber plugs closure was sufficiently proven under extreme conditions by two different methods. The maintenance of sterility during the dispensing process of technetium-99m radiopharmaceuticals was also proven by simulation of the dispensing process using a liquid growth medium. CONCLUSIONS: This study strongly supports that it is not necessary any special ambient air conditions to prepare, store and dispense extemporaneous technetium-99m radiopharmaceuticals in order to preserve their sterility when the basic aseptic rules for closed procedures are followed.

Molecular and biochemical insights into the in vivo evolution of AmpC-mediated resistance to ceftolozane/tazobactam during treatment of an MDR Pseudomonas aeruginosa infection.

BACKGROUND: Pseudomonas aeruginosa may develop resistance to novel cephalosporin/ß-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC. OBJECTIVES: To describe the molecular and biochemical mechanisms involved in the development of resistance to ceftolozane/tazobactam in vivo through the selection and overproduction of a novel AmpC variant, designated PDC-315. METHODS: Paired susceptible/resistant isolates obtained before and during ceftolozane/tazobactam treatment were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. Characterization of the novel PDC-315 variant was performed through genotypic and biochemical studies. The effects at the molecular level of the Asp245Asn change were analysed by molecular dynamics simulations using Amber. RESULTS: WGS identified mutations leading to modification (Asp245Asn) and overproduction of AmpC. Susceptibility testing revealed that PAOΔC producing PDC-315 displayed increased MICs of ceftolozane/tazobactam, decreased MICs of piperacillin/tazobactam and imipenem and similar susceptibility to ceftazidime/avibactam compared with WT PDCs. The catalytic efficiency of PDC-315 for ceftolozane was 10-fold higher in relation to the WT PDCs, but 3.5- and 5-fold lower for piperacillin and imipenem. IC50 values indicated strong inhibition of PDC-315 by avibactam, but resistance to cloxacillin inhibition. Analysis at the atomic level explained that the particular behaviour of PDC-315 is linked to conformational changes in the H10 helix that favour the approximation of key catalytic residues to the active site. CONCLUSIONS: We deciphered the precise mechanisms that led to the in vivo emergence of resistance to ceftolozane/tazobactam in P. aeruginosa through the selection of the novel PDC-315 enzyme. The characterization of this new variant expands our knowledge about AmpC-mediated resistance to cephalosporin/ß-lactamase inhibitors in P. aeruginosa.

Challenging Antimicrobial Susceptibility and Evolution of Resistance (OXA-681) during Treatment of a Long-Term Nosocomial Infection Caused by a Pseudomonas aeruginosa ST175 Clone.

Selection of extended-spectrum mutations in narrow-spectrum oxacillinases (e.g., OXA-2 and OXA-10) is an emerging mechanism for development of in vivo resistance to ceftolozane-tazobactam and ceftazidime-avibactam in Pseudomonas aeruginosa Detection of these challenging enzymes therefore seems essential to prevent clinical failure, but the complex phenotypic plasticity exhibited by this species may often lead to their underestimation. The underlying resistance mechanisms of two sequence type 175 (ST175) P. aeruginosa isolates showing multidrug-resistant phenotypes and recovered at early and late stages of a long-term nosocomial infection were evaluated. Whole-genome sequencing (WGS) was used to investigate resistance genomics, whereas molecular and biochemical methods were used for characterization of a novel extended-spectrum OXA-2 variant selected during therapy. The metallo-ß-lactamase blaVIM-20 and the narrow-spectrum oxacillinase blaOXA-2 were present in both isolates, although they differed by an inactivating mutation in the mexB subunit, present only in the early isolate, and in a mutation in the blaOXA-2 ß-lactamase, present only in the final isolate. The new OXA-2 variant, designated OXA-681, conferred elevated MICs of the novel cephalosporin-ß-lactamase inhibitor combinations in a PAO1 background. Compared to OXA-2, kinetic parameters of the OXA-681 enzyme revealed a substantial increase in the hydrolysis of cephalosporins, including ceftolozane. We describe the emergence of the novel variant OXA-681 during treatment of a nosocomial infection caused by a Pseudomonas aeruginosa ST175 high-risk clone. The ability of OXA-681 to confer cross-resistance to ceftolozane-tazobactam and ceftazidime-avibactam together with the complex antimicrobial resistance profiles exhibited by the clinical strains harboring this new enzyme argue for maintaining active surveillance on emerging broad-spectrum resistance in P. aeruginosa.

A prospective survey of Aspergillus spp. in respiratory tract samples: Species identification and susceptibility patterns.

We analyzed the species distribution and susceptibility patterns of 433 strains of Aspergillus spp. isolated from respiratory samples of 419 in-patients included in multicenter prospective study (FUNGAE-IFI) between July 2014 and October 2015. Identification was carried out by conventional methods at each participating center and by molecular sequencing of a portion of the ß-tubulin gene at one of the centers. In vitro susceptibility was evaluated by broth microdilution methods and using the E-test (for cryptic species). Species identified included 249 A. fumigatus sensu stricto, 60 A. terreus sensu stricto, 47 A. flavus sensu stricto, 44 A. tubingensis, 18 A. niger sensu stricto , five A. nidulans sensu stricto, three A. tamarii, two A. calidoustus, two A. carneus, one A. acuelatus, one A. carbonarius, and one A. sydowii. Cryptic species were found in 12.5% of isolates (n = 54). The frequency of non-wild-type isolates for amphotericin B was 3.4% (n = 15) of the isolates tested and for azoles 3% (n = 10). None of the Aspergillus spp. were non-wild type to echinocandins. Of the 54 cryptic species only two strains were non-wild-type strains by microdilution method (3.7%) (two A. tubingensis, one to amphotericin B and another one to voriconazole) and by E-test method five strains of A. tubingensis showed high minimal inhibitory concentration (MIC) to amphotericin B (11.4%) and five to azoles (12.1%), one A. calidoustus strain showed high MICs for three azoles (50%), A. carneus to itraconazole (100%) and A. sydowii to amphotericin B and itraconazole (100%). These results provide relevant information on susceptibility patterns, frequency, and epidemiology of species involved in respiratory tract samples and of the incidence of recently described cryptic species.

[Application of mass spectrometry to bacterial identification]. / Aplicación de la espectrometría de masas en la identificación de bacterias.

Correct and rapid identification of bacteria is essential for the correct diagnosis and treatment of infected patients. Until a few years ago, biochemical, colorimetric or even antibiotic sensitivity tests were used to identify genera and species. The main limitations of these methods were the time needed for their performance and the difficulty of distinguishing between microorganisms that were little reactive, highly similar, or difficult to culture. Many of these problems have been solved by the introduction of mass spectrometry (MS) in the laboratory with the use of MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight). Knowledge of the strengths and weaknesses of this technology is essential to be able to take maximum advantage of this technique. Not all microorganisms can be identified with the same ease and reliability by MALDI-TOF and microbiologists need to know how to interpret the results obtained with this technique and the available alternatives in order to identify the microorganisms causing the most problems. This article aims to summarise the available information on the correct identification of the main human pathogenic bacteria through the use of MALDI-TOF MS, focusing on Gram-negative, Grampositive and anaerobic microorganisms. The main factors that must be taken into account for the reliable identification of any bacterium are the conditions for culture, sample preparation with the ideal extraction method and especially the use of a correct and updated database.

Candida nivariensis as a New Emergent Agent of Vulvovaginal Candidiasis: Description of Cases and Review of Published Studies.

Candida nivariensis is a new emergent agent related to human infections in the vaginal tract and other localizations, but the phenotypic characteristics are very similar to Candida glabrata and can be misidentified and underdiagnosed. We described four cases of vulvovaginitis identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and confirmed the results with PCR amplification and sequencing of the entire ITS genomic region (ITS1, ITS2 and 5.8 rRNA). We reinforce the need for new diagnostic tools for the correct identification of yeast infections.

Candida galli as a cause of tinea unguium-molecular characterization of a rare clinical fungal entity.

We present the first clinical report of an infection caused by Candida galli, an anamorphic yeast species in the Yarrowia clade. C. galli has been described in the literature only four times, but never before it has been isolated from clinical samples. The colony morphology on Sabouraud medium and morphotype on CHROMagar Candida medium were similar to C. lipolytica as well as the carbon assimilation profile. The phenotypic differences with C. lipolytica were the non-assimilation of N-acetyl glucosamine, the absence of urease activity, growth in 10 % NaCl with 5 % glucose and in vitamin-free medium. MALDI-TOF MS could not generate reliable identification of the strain. Molecular analysis based on amplification of the ITS1-5.8S-ITS2 rDNA regions confirmed the identity as C. galli. Antifungal susceptibility test clearly demonstrated high MICs to 5-fluorocytosine, amphotericin B and fluconazole, as in the species belonging to the Yarrowia clade.

Molecular Detection of Cryptosporidium spp. and Microsporidia in Human and Animal Stool Samples.

Cryptosporidium spp. and Microsporidia are opportunistic microorganisms with remarkable zoonotic transmission potential due to their capacity to infect humans and animals. The aim of this study was to evaluate the prevalence of these microorganisms in stool samples of animal and human origin. In total, 369 stool samples (205 from human patients with diarrhea and 164 of animal origin) were included in the study. Cryptosporidium spp. and Microsporidia presence were determined by using multiplex nested PCR. Positive results were analyzed by using Sanger sequencing of the amplicon, utilizing BLASTN and ClustalX software to confirm identification. Cryptosporidium spp. were found in 0.97% and 4.26% of human and animal samples, respectively. Enterocytozoon bieneusi was detected in human and animal stools in 6.82% and 3.05% of the samples, respectively. No associations were found when analyzing the presence of Cryptosporidium spp. and E. bieneusi and the demographic and clinical variables of patients and animals. This study demonstrates the presence of these microorganisms in human and animal samples from different species, and the most interesting findings are the detection of Cryptosporidium spp. in pets (e.g., rodents) that are not usually included in this type of study, and the identification of E. bieneusi in patients with diarrhea without underlying disease.

Antibiotic use and outcome in patients with negative blood cultures, a new target population for antimicrobial stewardship interventions: A prospective multicentre cohort (NO-BACT).

OBJECTIVES: To evaluate the appropriateness of antimicrobial treatment and the risk factors for mortality in patients with negative blood cultures (BC), in order to evaluate whether this population would be a suitable target for antimicrobial stewardship (AMS) interventions. METHODS: A multicentre prospective cohort study of patients with negative BC in three Spanish hospitals between October 2018 and July 2019 was performed. The main endpoints were the appropriateness of antimicrobial treatment (evaluated by two investigators according to local guidelines) and 30-day mortality. Cox-regression was performed to estimate the association between variables and 30-day mortality. RESULTS: Of 1011 patients in whom BC was obtained, these were negative in 803 (79%) and were included; 30-day mortality was 9% (70 patients); antibiotic treatment was considered inappropriate in 299 (40%) of 747 patients evaluated at day 2, and in 266 (46%) of 573 at day 5-7. The variables independently associated with increased risk of 30-day mortality were higher age (HR 1.05; 95% CI 1.03-1.07), neoplasia (HR 2.73; 95% CI 1.64-4.56), antibiotic treatment in the 48 h prior to BC extraction (HR 2.06; 95% CI 1.23-3.43) and insufficient antibiotic coverage at day 2 after BC obtainment (HR 2.35; 95% CI 1.39-4.00). Urinary, catheter and biliary sources of infection were associated with lower risk (HR 0.40; 95% CI 0.20-0.81). CONCLUSIONS: Antimicrobial treatment is frequently inappropriate among patients with negative BC; insufficient antibiotic coverage at day 2 was associated with mortality. These results suggest that patients with negative BC are a suitable population for AS interventions. SUMMARY: Antimicrobial treatment in patients with negative blood culture was frequently inappropriate, and inappropriate coverage at day 2 was associated with increased risk of death. These data support the consideration of this population as a potential target for antimicrobial stewardship interventions.

Association of microbiological factors with mortality in Escherichia coli bacteraemia presenting with sepsis/septic shock: a prospective cohort study.

OBJECTIVES: This study aimed to determine the association of Escherichia coli microbiological factors with 30-day mortality in patients with bloodstream infection (BSI) presenting with a dysregulated response to infection (i.e. sepsis or septic shock). METHODS: Whole-genome sequencing was performed on 224 E coli isolates of patients with sepsis/septic shock, from 22 Spanish hospitals. Phylogroup, sequence type, virulence, antibiotic resistance, and pathogenicity islands were assessed. A multivariable model for 30-day mortality including clinical and epidemiological variables was built, to which microbiological variables were hierarchically added. The predictive capacity of the models was estimated by the area under the receiver operating characteristic curve (AUROC) with 95% confidence intervals (CI). RESULTS: Mortality at day 30 was 31% (69 patients). The clinical model for mortality included (adjusted OR; 95% CI) age (1.04; 1.02-1.07), Charlson index ≥3 (1.78; 0.95-3.32), urinary BSI source (0.30; 0.16-0.57), and active empirical treatment (0.36; 0.11-1.14) with an AUROC of 0.73 (95% CI, 0.67-0.80). Addition of microbiological factors selected clone ST95 (3.64; 0.94-14.04), eilA gene (2.62; 1.14-6.02), and astA gene (2.39; 0.87-6.59) as associated with mortality, with an AUROC of 0.76 (0.69-0.82). DISCUSSION: Despite having a modest overall contribution, some microbiological factors were associated with increased odds of death and deserve to be studied as potential therapeutic or preventive targets.

Whole-genome characterisation of Escherichia coli isolates from patients with bacteraemia presenting with sepsis or septic shock in Spain: a multicentre cross-sectional study.

BACKGROUND: Escherichia coli is the most frequent cause of bloodstream infections (BSIs). About one-third of patients with BSIs due to E coli develop sepsis or shock. The objective of this study is to characterise the microbiological features of E coli blood isolates causing sepsis or septic shock to provide exploratory information for future diagnostic, preventive, or therapeutic interventions. METHODS: E coli blood isolates from a multicentre cross-sectional study of patients older than 14 years presenting with sepsis or septic shock (according to the Third International Consensus Definitions for Sepsis and Septic Shock criteria) from hospitals in Spain between Oct 4, 2016, and Oct 15, 2017, were studied by whole-genome sequencing. Phylogroups, sequence types (STs), serotype, FimH types, antimicrobial resistance (AMR) genes, pathogenicity islands, and virulence factors were identified. Susceptibility testing was performed by broth microdilution. The main outcome of this study was the characterisation of the E coli blood isolates in terms of population structure by phylogroups, groups (group 1: phylogroups B2, F, and G; group 2: A, B1, and C; group 3: D), and STs and distribution by geographical location and bloodstream infection source. Other outcomes were virulence score and prevalence of virulence-associated genes, pathogenicity islands, AMR, and AMR-associated genes. Frequencies were compared using χ² or Fisher's exact tests, and continuous variables using the Mann-Whitney test, with Bonferroni correction for multiple comparisons. FINDINGS: We analysed 224 isolates: 140 isolates (63%) were included in phylogenetic group 1, 52 (23%) in group 2, and 32 (14%) in group 3. 85 STs were identified, with four comprising 44% (n=98) of the isolates: ST131 (38 [17%]), ST73 (25 [11%]), ST69 (23 [10%]), and ST95 (12 [5%]). No significant differences in phylogroup or ST distribution were found according to geographical areas or source of bloodstream infection, except for ST95, which was more frequent in urinary tract infections than in other sources (11 [9%] of 116 vs 1 [1%] of 108, p=0·0045). Median virulence score was higher in group 1 (median 25·0 [IQR 20·5-29·0) than in group 2 (median 14·5 [9·0-20·0]; p<0·0001) and group 3 (median 21 [16·5-23·0]; p<0·0001); prevalence of several pathogenicity islands was higher in group 1. No significant differences were found between phylogenetic groups in proportions of resistance to antibiotics. ST73 had higher median virulence score (32 [IQR 29-35]) than the other predominant clones (median range 21-28). Some virulence genes and pathogenicity islands were significantly associated with each ST. ST131 isolates had higher prevalence of AMR and a higher proportion of AMR genes, notably blaCTX-M-15 and blaOXA-1. INTERPRETATION: In this exploratory study, the population structure of E coli causing sepsis or shock was similar to previous studies that included all bacteraemic isolates. Virulence genes, pathogenicity islands, and AMR genes were not randomly distributed among phylogroups or STs. These results provide a comprehensive characterisation of invasive E coli isolates causing severe response syndrome. Future studies are required to determine the contribution of these microbiological factors to severe clinical presentation and worse outcomes in patients with E coli bloodstream infection. FUNDING: Instituto de Salud Carlos III.

Molecular characterization of consecutive isolates of OXA-48-producing Klebsiella pneumoniae: changes in the virulome using next-generation sequencing (NGS).

Little is known about the clonality of consecutive OXA-48 producing-Klebsiella pneumoniae isolates from the same patient and the possibility of changes in their virulomes over time. We studied the molecular characteristics of twenty OXA-48-producing K. pneumoniae consecutive isolates from six patients using whole-genome sequencing. The genomes were screened for antimicrobial resistance and virulence factor genes and for replicon groups. MLST and SNPs analysis was performed. MLST analysis found 3 STs: ST11 (n = 13; 65.0%); ST4975 (n = 5, 25.0%); ST307 (n = 2; 10.0%). AcrAb efflux pump, siderophore enterobactin and rcsAB capsule synthesis regulator were detected in all sequenced isolates. The regulator of mucoid phenotype A (rmpA) and rmpA2 were not detected. Isolates also carried type 3 fimbriae (n = 19; 95.0%), yersiniabactin (n = 15; 75.0%) and type 1 fimbriae (7; 35.0%). Type 3 fimbriae and yersiniabactin were lost and recovered in consecutive isolates of two patients, probably acquired by horizontal gene transfer. Our findings reveal that recurrent infections are due to the same isolate, with an average of 2.69 SNPs per month, with different virulence profiles, and that the acquisition of virulence factor genes over time is possible.

Enteroaggregative Escherichia coli as etiological agent of endemic diarrhea in Spain: A prospective multicenter prevalence study with molecular characterization of isolates.

Background: Enteroaggregative Escherichia coli (EAEC) is increasingly associated with domestically acquired diarrheal episodes in high-income countries, particularly among children. However, its specific role in endemic diarrhea in this setting remains under-recognized and information on molecular characteristics of such EAEC strains is limited. We aimed to investigate the occurrence of EAEC in patients with non-travel related diarrhea in Spain and molecularly characterize EAEC strains associated with illness acquired in this high-income setting. Methods: In a prospective multicenter study, stool samples from diarrheal patients with no history of recent travel abroad (n = 1,769) were collected and processed for detection of EAEC and other diarrheagenic E. coli (DEC) pathotypes by PCR. An additional case-control study was conducted among children ≤5 years old. Whole-genome sequences (WGS) of the resulting EAEC isolates were obtained. Results: Detection of DEC in the study population. DEC was detected in 23.2% of patients aged from 0 to 102 years, with EAEC being one of the most prevalent pathotypes (7.8%) and found in significantly more patients ≤5 years old (9.8% vs. 3.4%, p < 0.001). Although not statistically significant, EAEC was more frequent in cases than in controls. WGS-derived characterization of EAEC isolates. Sequence type (ST) 34, ST200, ST40, and ST10 were the predominant STs. O126:H27, O111:H21, and O92:H33 were the predominant serogenotypes. Evidence of a known variant of aggregative adherence fimbriae (AAF) was found in 89.2% of isolates, with AAF/V being the most frequent. Ten percent of isolates were additionally classified as presumptive extraintestinal pathogenic E. coli (ExPEC), uropathogenic E. coli (UPEC), or both, and belonged to clonal lineages that could be specifically associated with extraintestinal infections. Conclusion: EAEC was the only bacterial enteric pathogen detected in a significant proportion of cases of endemic diarrhea in Spain, especially in children ≤5 years old. In particular, O126:H27-ST200, O111:H21-ST40, and O92:H33-ST34 were the most important subtypes, with all of them infecting both patients and asymptomatic individuals. Apart from this role as an enteric pathogen, a subset of these domestically acquired EAEC strains revealed an additional urinary/systemic pathogenic potential.

Conventional Hospitalization versus Sequential Outpatient Parenteral Antibiotic Therapy for Staphylococcus aureus Bacteremia: Post-Hoc Analysis of a Multicenter Observational Cohort.

It is not known whether sequential outpatient parenteral antimicrobial (OPAT) is as safe and effective as conventional hospitalization in patients with S. aureus bacteremia (SAB). A post-hoc analysis of the comparative effectiveness of conventional hospitalization versus sequential OPAT was performed in two prospective Spanish cohorts of patients with S. aureus bacteremia. The PROBAC cohort is a national, multicenter, prospective observational cohort of patients diagnosed in 22 Spanish hospitals between October 2016 and March 2017. The DOMUS OPAT cohort is a prospective observational cohort including patients from two university hospitals in Seville, Spain from 2012 to 2021. Multivariate regression was performed, including a propensity score (PS) for receiving OPAT, stratified analysis according to PS quartiles, and matched pair analyses based on PS. Four hundred and thirteen patients were included in the analysis: 150 in sequential OPAT and 263 in the full hospitalization therapy group. In multivariate analysis, including PS and center effect as covariates, 60-day treatment failure was lower in the OPAT group than in the full hospitalization group (p < 0.001; OR 0.275, 95%CI 0.129−0.584). In the PS-based matched analyses, sequential treatment under OPAT was not associated with higher 60-day treatment failure (p = 0.253; adjusted OR 0.660; % CI 0.324−1.345). OPAT is a safe and effective alternative to conventional in-patient therapy for completion of treatment in well-selected patients with SAB, mainly those associated with a low-risk source and without end-stage kidney disease.