Evolution of ceftazidime-avibactam and cefiderocol resistance in ST131-H30R1-Escherichia coli isolates with KPC-3 mutants and application of FTIR biotyping.

Ceftazidime-avibactam and cefiderocol represent two of the few alternatives for infections by KPC-producing Enterobacterales. We reported the emergence of resistance to both ceftazidime-avibactam and cefiderocol in a KPC-producing ST131-Escherichia coli (KPC-ST131-Ec) clinical isolate. Antimicrobial susceptibility testing, Fourier-transform infrared (FTIR) spectroscopy, whole-genome sequencing, and cloning experiments were performed. A KPC-49-Ec isolate resistant to ceftazidime-avibactam (MICCZA > 16/4 mg/L) and susceptible to cefiderocol (MICFDC: 2 mg/L) was recovered in a blood sample from an oncologic patient hospitalized in the medical ICU (June 2019) during ceftazidime-avibactam treatment. After 44 days, a KPC-31-Ec resistant to both ceftazidime-avibactam and cefiderocol (MICCZA > 16/4 mg/L, MICFDC: 8 mg/L) was found in a rectal sample during a second cycle of ceftazidime-avibactam treatment. Both KPC-49 (R163S) and KPC-31 (D179Y) were detected in the epidemic ST131-H30R1-Ec high-risk clone and showed a phenotype resembling that of ESBL producers. FTIR spectroscopy managed to differentiate cefiderocol-susceptible and resistant ST131-Ec isolates, and these from others belonging to different clones. After cloning and transformation experiments, KPC-49 and KPC-31 were responsible for ceftazidime-avibactam resistance (MICCZA > 16/4 mg/L) and decreased carbapenem MICs (MICMER ≤ 0.12 mg/L, MICIMI ≤ 1 mg/L). KPC-31 was also shown to be associated with increased MICs of cefiderocol (twofold and threefold dilutions over KPC-3 and KPC-49, respectively). However, mutations in proteins participating in outer membrane stability and integrity, such as TolR, could have a more relevant role in cefiderocol resistance. The effects of ceftazidime-avibactam and cefiderocol co-resistance in clinical isolates of Enterobacterales producing KPC mutants make their identification challenging for clinical laboratories.IMPORTANCEThroughout four admissions in our hospital of a single patient, different KPC-3 variants (KPC-3, KPC-49, and KPC-31) were found in surveillance and clinical ST131-Escherichia coli isolates, after prolonged therapies with meropenem and ceftazidime-avibactam. Different patterns of resistance to cefiderocol and ceftazidime-avibactam emerged, accompanied by restored carbapenem susceptibility. The inability to detect these variants with some phenotypic methods, especially KPC-31 by immunochromatography, and the expression of a phenotype similar to that of ESBL producers, posed challenge to identify these variants in the clinical microbiology laboratory. Molecular methods and whole-genome sequencing are necessary and new techniques able to cluster or differentiate related isolates could also be helpful; this is the case of Fourier-transform infrared spectroscopy, which managed in our study to discriminate isolates by cefiderocol susceptibility within ST131, and those from the non-ST131 ones.

Non-perinatal listeriosis: Changes in frequency, clinical spectrum and prognostic factors.

INTRODUCTION: Listeria monocytogenes infection is a severe disease affecting mainly aged people and patients with immune depression. The incidence of listeriosis seems to be increasing. In the present study cases of listeriosis from two hospitals are analyzed with the aims of studying changes in its incidence, clinical forms of presentation and possible factors associated with mortality. METHODS: Retrospective multicentric study of patients with culture-proven listeriosis in two university hospitals in Madrid between 1977 and 2021. Epidemiological and clinical variables, as well as factors for immune depression, complementary studies and treatments were registered. Factors associated with mortality were analyzed. RESULTS: A total of 194 cases of listeriosis were analyzed. The incidence of listeriosis among in-patients increased through the study period, with a significant drop in the number of cases in 2020. The most common clinical presentations were isolated bacteriemia (37.1%) and central nervous system involvement (CNS) (36.6%). Symptoms of gastroenteritis occurred in 21% of cases. Other focal infections were present in 16.5% of patients, the most frequent were spontaneous bacterial peritonitis (8.2%), cholecystitis (2.1%), respiratory infection (1.5%) and vascular prothesis infection (1.5%). In-hospital mortality was 24.7%. Independent factors associated with mortality at admission were age (Odds Ratio [OR] 1.027, 95% confidence interval [IC95%] 1.003-1.056) and a diagnosis of a solid tumor (OR 3.525, IC95% 1.652-7.524). CONCLUSIONS: This study confirms an increasing incidence of listeriosis in our millieu. The most common clinical presentations were isolated bacteriemia and central nervous system involvement. In-hospital mortality was associated with age and the diagnosis of a solid tumor.

First detection in Spain of NDM-1-producing Pseudomonas aeruginosa in two patients transferred from Ukraine to a university hospital.

OBJECTIVES: Carbapenemase-mediated carbapenem resistance in Pseudomonas aeruginosa is a relevant health problem. We detected for the first time in Spain two clinical NDM-producing P. aeruginosa (NDM-Pa) isolates in two Ukrainian patients admitted to our hospital between April and August 2022. METHODS: Antimicrobial susceptibility was studied by microdilution and MIC gradient strips (EUCAST-2022 criteria). Carbapenemase genes were detected by the Xpert Carba-R and immunochromatography assays. WGS (Illumina and Oxford-Nanopore) was also performed. RESULTS: In May 2022, we detected an NDM-Pa in a sternotomy wound in a patient. In June-2022, a second NDM-Pa along with an OXA-48-Klebsiella pneumoniae (OXA-48-Kp) isolate was detected in a mandibular abscess from an unrelated patient. Moreover, an NDM+OXA-48-K. pneumoniae (NDM+OXA-48-Kp) was also found in a rectal sample of this patient. Both patients had undergone surgery in Ukraine before their transfer to our hospital. NDM-Pa isolates were resistant to all tested antimicrobials with the exception of aztreonam (MIC = 8 mg/L), colistin (MIC =2 mg/L) and cefiderocol (MIC range = 0.75-2 mg/L). WGS confirmed that both P. aeruginosa isolates were NDM-1 producers, belonged to ST773 and shared an identical resistome. blaNDM-1 was located on a ∼117-Kb chromosomally integrated integrative conjugative element (ICE). OXA-48-Kp and NDM+OXA-48-Kp belonged to ST147 and contained blaOXA-48 on an identical ∼300-Kb IncHIB-plasmid. blaNDM-1 was located on a 51-Kb IncFIB-plasmid only found in NDM+OXA-48-Kp. CONCLUSIONS: This is the first description of NDM-Pa in Spain. We highlight the threat of further cross-border dissemination of NDM-1 through P. aeruginosa along with K. pneumoniae high-risk clones also carrying OXA-48, which draws a complex epidemiological scenario.

Molecularly defined extraintestinal pathogenic Escherichia coli status predicts virulence in a murine sepsis model better than does virotype, individual virulence genes, or clonal subset among E. coli ST131 isolates.

BACKGROUND: Escherichia coli ST131, mainly its H30 clade, is the leading cause of extraintestinal E. coli infections but its correlates of virulence are undefined. MATERIALS AND METHODS: We tested in a murine sepsis model 84 ST131 isolates that differed by country of origin (Spain vs. USA), clonal subset, resistance markers, and virulence genes (VGs). Virulence outcomes, including illness severity score (ISS) and "killer" status (>80% mouse lethality), were compared statistically with clonal subset, individual and combined VGs, molecularly defined extraintestinal and uropathogenic E. coli (ExPEC, UPEC) status, and country of origin. RESULTS: Virulence varied widely by strain. Univariable correlates of median ISS and percent "killer" (outcomes if variable present vs. absent) included pap (ISS, 4.4 vs. 3.8; "killer", 71% vs. 46%), kpsMII (4.1 vs. 2.3; 59% vs. 25%), K2/K100 (4.4 vs. 3.2; 77% vs. 41%), ExPEC (4.2 vs. 2.2; 62% vs. 17%), Spanish origin (4.3 vs. 3.1; 65% vs. 36%), and H30R1 subset (2.5 vs. 4.1; 35% vs. 59%). With multivariable adjustment, ExPEC status was the only consistently significantly predictive variable. CONCLUSION: Within ST131 the strongest predictor of experimental virulence was molecularly defined ExPEC status. Clonal subsets seemed to behave differently in the murine sepsis model by country of origin.

Long-term clonal dynamics of Enterococcus faecium strains causing bloodstream infections (1995-2015) in Spain.

OBJECTIVES: To investigate the population structure of Enterococcus faecium causing bloodstream infections (BSIs) in a tertiary Spanish hospital with low glycopeptide resistance, and to enhance our knowledge of the dynamics of emergence and spread of high-risk clonal complexes. METHODS: All available E. faecium causing BSIs (n = 413) in our hospital (January 1995-May 2015) were analysed for antibiotic susceptibility (CLSI), putative virulence traits (PCR, esp, hylEfm) and clonal relationship (SmaI-PFGE, MLST evaluated by goeBURST and BAPS). RESULTS: The increased incidence of BSIs caused by enterococci [2.3‰ of attended patients (inpatients and outpatients) in 1996 to 3.0‰ in 2014] significantly correlated with the increase in BSIs caused by E. faecium (0.33‰ of attended patients in 1996 to 1.3‰ in 2014). The BSIs Enterococcus faecalis:E. faecium ratio changed from 5:1 in 1996 to 1:1 in 2014. During the last decade an increase in E. faecium BSIs episodes in cancer patients (10.9% in 1995-2005 and 37.1% in 2006-15) was detected. Ampicillin-susceptible E. faecium (ASEfm; different STs/BAPS) and ampicillin-resistant E. faecium (AREfm; ST18/ST17-BAPS 3.3a) isolates were recovered throughout the study. Successive waves of BAPS 2.1a-AREfm (ST117, ST203 and ST80) partially replaced ASEfm and ST18-AREfm since 2006. CONCLUSIONS: Different AREfm clones (belonging to BAPS 2.1a and BAPS 3.3a) consistently isolated during the last decade from BSIs might be explained by a continuous and dense colonization (favouring both invasion and cross-transmission) of hospitalized patients. High-density colonization by these clones is probably enhanced in elderly patients by heavy and prolonged antibiotic exposure, particularly in oncological patients.

New Insights into the Enterococcus faecium and Streptococcus gallolyticus subsp. gallolyticus Host Interaction Mechanisms.

Enterococcus faecium and Streptococcus gallolyticus subsp. gallolyticus (S. gallolyticus) were classically clustered into the Lancefield Group D streptococci and despite their taxonomic reclassification still share a similar genetic content and environment. Both species are considered as opportunistic pathogens. E. faecium is often associated with nosocomial bacteraemia, and S. gallolyticus is sporadically found in endocarditis of colorectal cancer patients. In both cases, the source of infection is commonly endogenous with a translocation process that launches through the intestinal barrier. To get new insights into the pathological processes preceding infection development of both organisms, we used an in vitro model with Caco-2 cells to study and compare the adhesion, invasion and translocation inherent abilities of 6 E. faecium and 4 S. gallolyticus well-characterized isolates. Additionally, biofilm formation on polystyrene, collagen I and IV was also explored. Overall results showed that E. faecium translocated more efficiently than S. gallolyticus, inducing a destabilization of the intestinal monolayer. Isolates Efm106, Efm121 and Efm113 (p < .001 compared to Ef222) exhibited the higher translocation ability and were able to adhere 2-3 times higher than S. gallolyticus isolates. Both species preferred the collagen IV coated surfaces to form biofilm but the S. gallolyticus structures were more compact (p = .01). These results may support a relationship between biofilm formation and vegetation establishment in S. gallolyticus endocarditis, whereas the high translocation ability of E. faecium high-risk clones might partially explain the increasing number of bacteraemia.

Emergence and long-lasting persistence of linezolid-resistant Enterococcus faecium-ST117 in an oncohematologic patient after a nine-day course of linezolid.

We report the emergence and long-lasting persistence of linezolid resistance in an ampicillin-resistant Enterococcus faecium strain in the intestine of a neutropenic oncohematologic patient receiving chemotherapy. The patient was first colonized by an epidemic ampicillin-resistant E. faecium (ARE)-ST117 clustering into lineage 78. This clone exhibited resistance to levofloxacin, erythromycin and high-level resistance to streptomycin and gentamicin. After receiving treatment with several broad spectrum antibiotics for febrile neutropenia, a 9-day course of oral linezolid was administered once the patient developed bacteraemia by the same ARE colonizing clone. Linezolid-resistant ARE was detected 17 days later in the follow-up fecal samples and persisted 41 days after suppression of linezolid therapy. Resistance to linezolid was associated with G2576T transversion in the 23S rRNA and the presence of cfr gene was not detected. The persistence of G2576T-ARE strains, especially in oncohematologic patients with injured intestinal membranes, could increase the risk of bacteraemia.