Clinical Features and Genomic Epidemiology of Bloodstream Infections due to Enterococcal Species Other Than Enterococcus faecalis or E. faecium in Patients With Cancer.

Background: Non-Enterococcus faecium, non-E. faecalis (NFF) enterococci are a heterogeneous group of clinically pathogenic enterococci that include species with intrinsic low-level vancomycin resistance. Patients with cancer are at increased risk for bacteremia with NFF enterococci, but their clinical and molecular epidemiology have not been extensively described. Methods: We conducted a retrospective review of all patients (n = 70) with NFF bacteremia from 2016 to 2022 at a major cancer center. The main outcomes assessed were 30-day mortality, microbiological failure (positive blood cultures for ≥4 days), and recurrence of bacteremia (positive blood culture <14 days after clearance). Whole-genome sequencing was performed on all available NFF (n = 65). Results: Patients with hematological malignancies made up 56% of the cohort (77% had leukemia). The majority of solid malignancies (87%) were gastrointestinal in origin. The majority of infections (83%) originated from an intra-abdominal source. The most common NFF species were E. gallinarum (50%) and E. casseliflavus (30%). Most (61%) patients received combination therapy. Bacteremia recurred in 4.3% of patients, there was a 30-day mortality of 23%, and 4.3% had microbiological failure. E. gallinarum and E. casseliflavus isolates were genetically diverse with no spatiotemporal clustering to suggest a single strain. Frequencies of ampicillin resistance (4.3%) and daptomycin resistance (1.9%) were low. Patients with hematologic malignancy had infections with NFF enterococci that harbored more resistance genes than patients with solid malignancy (P = .005). Conclusions: NFF bacteremia is caused by a heterogeneous population of isolates and is associated with significant mortality. Hematological malignancy is an important risk factor for infection with NFF resistant to multiple antibiotics.

Membrane Lipids Augment Cell Envelope Stress Signaling via the MadRS System to Defend Against Antimicrobial Peptides and Antibiotics in Enterococcus faecalis.

Enterococci have evolved resistance mechanisms to protect their cell envelopes against bacteriocins and host cationic antimicrobial peptides (CAMPs) produced in the gastrointestinal environment. Activation of the membrane stress response has also been tied to resistance to the lipopeptide antibiotic daptomycin. However, the actual effectors mediating resistance have not been elucidated. Here, we show that the MadRS (formerly YxdJK) membrane antimicrobial peptide defense system controls a network of genes, including a previously uncharacterized three gene operon (madEFG) that protects the E. faecalis cell envelope from antimicrobial peptides. Constitutive activation of the system confers protection against CAMPs and daptomycin in the absence of a functional LiaFSR system and leads to persistence of cardiac microlesions in vivo. Moreover, changes in the lipid cell membrane environment alter CAMP susceptibility and expression of the MadRS system. Thus, we provide a framework supporting a multilayered envelope defense mechanism for resistance and survival coupled to virulence.

Cefiderocol heteroresistance associated with mutations in TonB-dependent receptor genes in Pseudomonas aeruginosa of clinical origin.

The siderophore-cephalosporin cefiderocol(FDC) presents a promising treatment option for carbapenem-resistant (CR) P. aeruginosa (PA). FDC circumvents traditional porin and efflux mediated resistance by utilizing TonB-dependent receptors (TBDRs) to access the periplasmic space. Emerging FDC resistance has been associated with loss of function mutations within TBDR genes or the regulatory genes controlling TBDR expression. Further, difficulties with antimicrobial susceptibility testing (AST) and unexpected negative clinical treatment outcomes have prompted concerns for heteroresistance, where a single lineage isolate contains resistant subpopulations not detectable by standard AST. This study aimed to evaluate the prevalence of TBDR mutations among clinical isolates of P. aeruginosa and the phenotypic effect on FDC susceptibility and heteroresistance. We evaluated the sequence of pirR , pirS , pirA , piuA or piuD from 498 unique isolates collected before the introduction of FDC from 4 clinical sites in Portland, OR (1), Houston, TX (2), and Santiago, Chile (1). At some clinical sites, TBDR mutations were seen in up to 25% of isolates, and insertion, deletion, or frameshift mutations were predicted to impair protein function were seen in 3% of all isolates (n=15). Using population analysis profile testing, we found that P. aeruginosa with major TBDR mutations were enriched for a heteroresistant phenotype and undergo a shift in the susceptibility distribution of the population as compared to susceptible strains with wild type TBDR genes. Our results indicate that mutations in TBDR genes predate the clinical introduction of FDC, and these mutations may predispose to the emergence of FDC resistance.

Membrane Lipids Augment Cell Envelope Stress Signaling and Resistance to Antibiotics and Antimicrobial Peptides in Enterococcus faecalis.

Enterococci have evolved resistance mechanisms to protect their cell envelopes against bacteriocins and host cationic antimicrobial peptides (CAMPs) produced in the gastrointestinal environment. Activation of the membrane stress response has also been tied to resistance to the lipopeptide antibiotic daptomycin. However, the actual effectors mediating resistance have not been elucidated. Here, we show that the MadRS (formerly YxdJK) membrane antimicrobial peptide defense system controls a network of genes, including a previously uncharacterized three gene operon (madEFG) that protects the E. faecalis cell envelope from antimicrobial peptides. Constitutive activation of the system confers protection against CAMPs and daptomycin in the absence of a functional LiaFSR system and leads to persistence of cardiac microlesions in vivo. Moreover, changes in the lipid cell membrane environment alter CAMP susceptibility and expression of the MadRS system. Thus, we provide a framework supporting a multilayered envelope defense mechanism for resistance and survival coupled to virulence.

Evolving landscape of carbapenem-resistant Pseudomonas aeruginosa at a single centre in the USA.

Objectives: The increased identification of carbapenem-resistant Pseudomonas aeruginosa (CR-PA) is an ongoing concern. However, information on the evolving antimicrobial resistance profile and molecular epidemiology of CR-PA over time is scarce. Thus, we conducted a cross-sectional analysis to investigate the phenotypic and genotypic characteristics of CR-PA recovered over different time periods, focusing on the isolates exhibiting a ceftolozane/tazobactam resistance phenotype. Methods: A total of 169 CR-PA isolated from clinical specimens at a single centre in Houston, TX, USA were studied. Among them, 61 isolates collected between 1999 and 2005 were defined as historical strains, and 108 collected between 2017 and 2018 were defined as contemporary strains. Antimicrobial susceptibilities against selected ß-lactams was determined. WGS data were used for the identification of antimicrobial resistance determinants and phylogenetic analysis. Results: Non-susceptibility to ceftolozane/tazobactam and ceftazidime/avibactam increased from 2% (1/59) to 17% (18/108) and from 7% (4/59) to 17% (18/108) from the historical to the contemporary collection, respectively. Carbapenemase genes, which were not identified in the historical collection, were harboured by 4.6% (5/108) of the contemporary strains, and the prevalence of ESBL genes also increased from 3.3% (2/61) to 16% (17/108). Genes encoding acquired ß-lactamases were largely confined to the high-risk clones. Among ceftolozane/tazobactam-resistant isolates, non-susceptibility to ceftazidime/avibactam, imipenem/relebactam and cefiderocol was observed in 94% (15/16), 56% (9/16) and 12.5% (2/16), respectively. Resistance to ceftolozane/tazobactam and imipenem/relebactam was primarily associated with the presence of exogenous ß-lactamases. Conclusions: Acquisition of exogenous carbapenemases and ESBLs may be a worrisome trend in P. aeruginosa.

Clinical characteristics, microbiology and outcomes of a cohort of patients treated with ceftolozane/tazobactam in acute care inpatient facilities, Houston, Texas, USA.

Background: Ceftolozane/tazobactam is a ß-lactam/ß-lactamase inhibitor combination with activity against a variety of Gram-negative bacteria, including MDR Pseudomonas aeruginosa. This agent is approved for hospital-acquired and ventilator-associated bacterial pneumonia. However, most real-world outcome data come from small observational cohorts. Thus, we sought to evaluate the utilization of ceftolozane/tazobactam at multiple tertiary hospitals in Houston, TX, USA. Methods: We conducted a multicentre retrospective study of patients receiving at least 48 h of ceftolozane/tazobactam therapy from January 2016 through to September 2019 at two hospital systems in Houston. Demographic, clinical and microbiological data were collected, including the infecting bacterial isolate, when available. The primary outcome was composite clinical success at hospital discharge. Secondary outcomes included in-hospital mortality and clinical disposition at 14 and 30 days post ceftolozane/tazobactam initiation. Multivariable logistic regression analysis was used to identify predictors of the primary outcome and mortality. Recovered isolates were tested for susceptibility to ceftolozane/tazobactam and underwent WGS. Results: A total of 263 patients were enrolled, and composite clinical success was achieved in 185 patients (70.3%). Severity of illness was the most consistent predictor of clinical success. Combination therapy with ceftolozane/tazobactam and another Gram-negative-active agent was associated with reduced odds of clinical success (OR 0.32, 95% CI 0.16-0.63). Resistance to ceftolozane/tazobactam was noted in 15.4% of isolates available for WGS; mutations in ampC and ftsI were common but did not cluster with a particular ST. Conclusions: Clinical success rate among this patient cohort treated with ceftolozane/tazobactam was similar compared with previous experiences. Ceftolozane/tazobactam remains an alternative agent for treatment of susceptible isolates of P. aeruginosa.

Multisite Detection of Tn1549-Mediated vanB Vancomycin Resistance in Multidrug-Resistant Enterococcus faecalis ST6 in Texas and Florida.

In the United States, vanB-mediated resistance in enterococci is rare. We characterized three sequence type (ST) 6, vancomycin-resistant Enterococcus faecalis isolates causing bacteremia in unique patients in spatiotemporally distinct settings. Isolates were recovered between 2018 and 2020 in two cities in the United States (Houston, TX; Miami, FL). The isolates harbored the vanB operon on a chromosomally located Tn1549 transposon, and epidemiological data suggested multiple introductions of the vanB gene cluster into ST6 E. faecalis.

Multiomics characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolates with heterogeneous intermediate resistance to vancomycin (hVISA) in Latin America.

BACKGROUND: Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) compromise the clinical efficacy of vancomycin. The hVISA isolates spontaneously produce vancomycin-intermediate Staphylococcus aureus (VISA) cells generated by diverse and intriguing mechanisms. OBJECTIVE: To characterize the biomolecular profile of clinical hVISA applying genomic, transcriptomic and metabolomic approaches. METHODS: 39 hVISA and 305 VSSA and their genomes were included. Core genome-based Bayesian phylogenetic reconstructions were built and alterations in predicted proteins in VISA/hVISA were interrogated. Linear discriminant analysis and a Genome-Wide Association Study were performed. Differentially expressed genes were identified in hVISA-VSSA by RNA-sequencing. The undirected profiles of metabolites were determined by liquid chromatography and hydrophilic interaction in six CC5-MRSA. RESULTS: Genomic relatedness of MRSA associated to hVISA phenotype was not detected. The change Try38 → His in Atl (autolysin) was identified in 92% of the hVISA. We identified SNPs and k-mers associated to hVISA in 11 coding regions with predicted functions in virulence, transport systems, carbohydrate metabolism and tRNA synthesis. Further, capABCDE, sdrD, esaA, esaD, essA and ssaA genes were overexpressed in hVISA, while lacABCDEFG genes were downregulated. Additionally, valine, threonine, leucine tyrosine, FAD and NADH were more abundant in VSSA, while arginine, glycine and betaine were more abundant in hVISA. Finally, we observed altered metabolic pathways in hVISA, including purine and pyrimidine pathway, CoA biosynthesis, amino acid metabolism and aminoacyl tRNA biosynthesis. CONCLUSIONS: Our results show that the mechanism of hVISA involves major changes in regulatory systems, expression of virulence factors and reduction in glycolysis via TCA cycle. This work contributes to the understanding of the development of this complex resistance mechanism in regional strains.

Unravelling complex transposable elements surrounding blaGES-16 in a Pseudomonas aeruginosa ExoU strain.

OBJECTIVES: We characterised the complex surrounding regions of blaGES-16 in a Pseudomonas aeruginosa exoU+ strain (P-10.226) in Brazil. METHODS: Species identification was performed by MALDI-TOF MS, and the antimicrobial susceptibility profile was determined by broth microdilution based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The whole genome sequencing (WGS) of P-10.226 strain was performed using both short-read paired-end sequencing on the Illumina MiSeq platform as well as the long-read Oxford Nanopore MinION. RESULTS: WGS analysis showed that P-10.226 carried blaGES-16, which was found as a gene cassette inserted into a novel class I integron, In1992 (aadB-blaOXA-56-blaGES-16-aadB-aadA6c), whose 3'-CS was truncated by a nested transposable element, IS5564::ISPa157. The structure was even more complex since IS6100-ΔIS6100 structure and a TnAs2-like harbouring the operon merRTPADE was found downstream In1992. Fragments of TnAs3 harbouring 25-bp imperfect inverted repeats were identified bordering the intl1 of In1992 and also flanking IS6100-ΔIS6100, which might be genetic marks of its previous presence in the genome. Interestingly, In1992 also shows a distinct cassette array from In581 (blaGES-16-dfrA22-aacA27-aadA1), which was previously reported in Serratia marcescens strains recovered in Brazil. Finally, exoU gene, which encodes a potent cytotoxin of type III secretion systems (T3SS) effector proteins from P. aeruginosa and is associated to severe infections, was also detected. CONCLUSION: We described the novel In1992 carrying blaGES-16 surrounded by complex transposition events in a XDR P. aeruginosa strain. The identification of many sets of direct repeats adjacent to TnAs3 fragments indicates a major past of transposition events that shaped the current genetic environment of In1992.

Accessory Genomes Drive Independent Spread of Carbapenem-Resistant Klebsiella pneumoniae Clonal Groups 258 and 307 in Houston, TX.

Carbapenem-resistant Klebsiella pneumoniae (CRKp) is an urgent public health threat. Worldwide dissemination of CRKp has been largely attributed to clonal group (CG) 258. However, recent evidence indicates the global emergence of a CRKp CG307 lineage. Houston, TX, is the first large city in the United States with detected cocirculation of both CRKp CG307 and CG258. We sought to characterize the genomic and clinical factors contributing to the parallel endemic spread of CG258 and CG307. CRKp isolates were collected as part of the prospective, Consortium on Resistance against Carbapenems in Klebsiella and other Enterobacterales 2 (CRACKLE-2) study. Hybrid short-read and long-read genome assemblies were generated from 119 CRKp isolates (95 originated from Houston hospitals). A comprehensive characterization of phylogenies, gene transfer, and plasmid content with pan-genome analysis was performed on all CRKp isolates. Plasmid mating experiments were performed with CG307 and CG258 isolates of interest. Dissection of the accessory genomes suggested independent evolution and limited horizontal gene transfer between CG307 and CG258 lineages. CG307 contained a diverse repertoire of mobile genetic elements, which were shared with other non-CG258 K. pneumoniae isolates. Three unique clades of Houston CG307 isolates clustered distinctly from other global CG307 isolates, indicating potential selective adaptation of particular CG307 lineages to their respective geographical niches. CG307 strains were often isolated from the urine of hospitalized patients, likely serving as important reservoirs for genes encoding carbapenemases and extended-spectrum ß-lactamases. Our findings suggest parallel cocirculation of high-risk lineages with potentially divergent evolution. IMPORTANCE The prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKp) infections in nosocomial settings remains a public health challenge. High-risk clones such as clonal group 258 (CG258) are particularly concerning due to their association with blaKPC carriage, which can severely complicate antimicrobial treatments. There is a recent emergence of clonal group 307 (CG307) worldwide with little understanding of how this successful clone has been able to adapt while cocirculating with CG258. We provide the first evidence of potentially divergent evolution between CG258 and CG307 with limited sharing of adaptive genes. Houston, TX, is home to the largest medical center in the world, with a large influx of domestic and international patients. Thus, our unique geographical setting, where two pandemic strains of CRKp are circulating, provides an indication of how differential accessory genome content can drive stable, endemic populations of CRKp. Pan-genomic analyses such as these can reveal unique signatures of successful CRKp dissemination, such as the CG307-associated plasmid (pCG307_HTX), and provide invaluable insights into the surveillance of local carbapenem-resistant Enterobacterales (CRE) epidemiology.

Efficacy, Safety, Pharmacokinetics, and Microbiome Changes of Ibezapolstat in Adults with Clostridioides difficile Infection: A Phase 2a Multicenter Clinical Trial.

BACKGROUND: This study was the first human validation of the gram-positive bacterial DNA polymerase IIIC target in patients with Clostridioides difficile infection. The primary objectives were to assess clinical cure rates and adverse events (AEs). Secondary objectives were to evaluate plasma/fecal pharmacokinetics, microbiologic eradication, microbiome and bile acid effects, and sustained clinical cure (SCC) with ibezapolstat. METHODS: This single-arm, open-label, phase 2a study enrolled adults with C. difficile infection at 4 US centers. Patients received ibezapolstat 450 mg orally every 12 hours for 10 days and followed for an additional 28 days to assess study objectives. RESULTS: Ten patients with a mean (standard deviation [SD]) age of 49 [15] years were enrolled. Seven AEs were reported classified as mild-moderate. Plasma levels of ibezapolstat ranged from 233 to 578 ng/mL while mean (SD) fecal levels were 416 (494) µg/g stool by treatment day 3 and >1000 µg/g stool by days 8-10. A rapid increase in alpha diversity in the fecal microbiome was noted after starting ibezapolstat therapy, which was maintained after completion of therapy. A proportional decrease in Bacteroidetes phylum was observed (mean change [SD], -10.0% [4.8%]; P = .04) with a concomitantly increased proportion of Firmicutes phylum (+14.7% [5.4%]; P = .009). Compared with baseline, total primary bile acids decreased by a mean (SD) of 40.1 (9.6) ng/mg stool during therapy (P < .001) and 40.5 (14.1) ng/mg stool after completion of therapy (P = .007). Rates of both initial clinical cure and SCC at 28 days were 100% (10 of 10 patients). CONCLUSIONS: In this phase 2a study, 10 of 10 patients achieved SCC, demonstrated favorable pharmacokinetics, minimal AEs, and beneficial microbiome and bile acids results. These results support continued clinical development.

Contemporary Clinical and Molecular Epidemiology of Vancomycin-Resistant Enterococcal Bacteremia: A Prospective Multicenter Cohort Study (VENOUS I).

BACKGROUND: Vancomycin-resistant enterococci (VRE) are major therapeutic challenges. Prospective contemporary data characterizing the clinical and molecular epidemiology of VRE bloodstream infections (BSIs) are lacking. METHODS: The Vancomycin-Resistant Enterococcal BSI Outcomes Study (VENOUS I) is a prospective observational cohort of adult patients with enterococcal BSI in 11 US hospitals. We included patients with Enterococcus faecalis or Enterococcus faecium BSI with ≥1 follow-up blood culture(s) within 7 days and availability of isolate(s) for further characterization. The primary study outcome was in-hospital mortality. Secondary outcomes were mortality at days 4, 7, 10, 12, and 15 after index blood culture. A desirability of outcome ranking was constructed to assess the association of vancomycin resistance with outcomes. All index isolates were subjected to whole genome sequencing. RESULTS: Forty-two of 232 (18%) patients died in hospital and 39 (17%) exhibited microbiological failure (lack of clearance in the first 4 days). Neutropenia (hazard ratio [HR], 3.13), microbiological failure (HR, 2.4), VRE BSI (HR, 2.13), use of urinary catheter (HR, 1.85), and Pitt BSI score ≥2 (HR, 1.83) were significant predictors of in-hospital mortality. Microbiological failure was the strongest predictor of in-hospital mortality in patients with E faecium bacteremia (HR, 5.03). The impact of vancomycin resistance on mortality in our cohort changed throughout the course of hospitalization. Enterococcus faecalis sequence type 6 was a predominant multidrug-resistant lineage, whereas a heterogeneous genomic population of E faecium was identified. CONCLUSIONS: Failure of early eradication of VRE from the bloodstream is a major factor associated with poor outcomes.

Candida auris Invasive Infections during a COVID-19 Case Surge.

Clinical cases of C. auris noted during a COVID-19 surge led to an epidemiological, clinical, and genomic investigation. Evaluation identified a close genetic relationship but inconclusive epidemiologic link between all cases. Prolonged hospitalization due to critical illness from COVID-19 and use of antimicrobials may have contributed to clinical infections.

Selective digestive decontamination with oral colistin plus gentamicin for persistent bacteraemia caused by non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae in a neutropenic patient.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKp) have become an increasing public health problem worldwide. While most CRKp around the world harbour a carbapenemase enzyme, the clinical relevance of non-carbapenemase-producing CRKp (non-CP-CRKp) is increasingly recognized. Selective digestive decontamination (SDD) has been proven successful as a decolonization strategy for patients colonized with Gram-negatives in the ICU. However, it is not regularly used to treat invasive infections. OBJECTIVES: To report the use of SDD as a useful strategy for managing recalcitrant CRKp bloodstream infections. PATIENTS AND METHODS: We present a neutropenic patient with a recalcitrant bloodstream infection with non-CP-CRKp treated with SDD. Besides, genomic analyses of five isolates of non-CP-CRKp was performed. RESULTS: After 11 days of SDD treatment with oral colistin and gentamicin, bacteraemia was successfully eradicated. Genomic analysis indicates a fully carbapenem-resistant phenotype evolved in vivo and suggests that the mechanism of carbapenem resistance in our strains relates to gene amplification of narrow-spectrum ß-lactamases. CONCLUSIONS: Our report highlights that SDD might be a useful strategy to manage CRKp bloodstream infections, when intestinal translocation is the likely source of the bacteraemia. In addition, the development of a resistant phenotype during therapy is worrisome as therapies directed against these organisms are likely to favour the amplification process.

Genomic analysis of carbapenem-resistant Pseudomonas aeruginosa ST143 clone showing susceptibility to broad-spectrum cephalosporins.

OBJECTIVES: Using whole-genome sequencing (WGS), we aimed to characterise a Pseudomonas aeruginosa ST143 clinical strain (Pb9) that presented resistance to meropenem and imipenem and susceptibility to piperacillin/tazobactam and broad-spectrum cephalosporins. METHODS: The antimicrobial susceptibility profile was confirmed by broth microdilution. WGS was performed using an Illumina MiSeq platform to identify possible genetic determinants of ß-lactam resistance. Transcription levels of chromosomally encoded efflux systems and oprD were evaluated by RT-qPCR. RESULTS: WGS analysis showed that no acquired carbapenemase-encoding gene was found in isolate Pb9, although mutations in the chromosomally encoded ß-lactamase genes blaOXA-488, blaPIB-1 and blaPDC-5 were observed. In addition, we detected a premature stop codon in the major porin-encoding gene oprD coupled with hyperexpression of MexAB-OprM and MexEF-OprN. CONCLUSION: Our results suggest that the ß-lactam resistance phenotype presented by strain Pb9 might be related to an association of OprD loss with hyperexpression of the efflux pump systems MexAB-OprM and MexEF-OprN. However, the contribution of OXA-488, PDC-5 and PIB-1 to this phenotype remains unclear and warrants further investigation.

Evolution of Cefiderocol Non-Susceptibility in Pseudomonas aeruginosa in a Patient Without Previous Exposure to the Antibiotic.

We report the emergence of non-susceptibility to cefiderocol from a subpopulation of Pseudomonas aeruginosa recovered from a patient without history of cefiderocol exposure. Whole genome sequencing identified mutations in major iron transport pathways previously associated with cefiderocol uptake. Susceptibility testing should be performed before therapy with siderophore cephalosporins.

Dynamics of blaKPC-2 Dissemination from Non-CG258 Klebsiella pneumoniae to Other Enterobacterales via IncN Plasmids in an Area of High Endemicity.

Carbapenem-resistant Enterobacterales (CRE) pose a significant threat to global public health. The most important mechanism for carbapenem resistance is the production of carbapenemases. Klebsiella pneumoniae carbapenemase (KPC) represents one of the main carbapenemases worldwide. Complex mechanisms of blaKPC dissemination have been reported in Colombia, a country with a high endemicity of carbapenem resistance. Here, we characterized the dynamics of dissemination of blaKPC gene among CRE infecting and colonizing patients in three hospitals localized in a highly endemic area of Colombia (2013 and 2015). We identified the genomic characteristics of KPC-producing Enterobacterales recovered from patients infected/colonized and reconstructed the dynamics of dissemination of blaKPC-2 using both short and long read sequencing. We found that spread of blaKPC-2 among Enterobacterales in the participating hospitals was due to intra- and interspecies horizontal gene transfer (HGT) mediated by promiscuous plasmids associated with transposable elements that was originated from a multispecies outbreak of KPC-producing Enterobacterales in a neonatal intensive care unit. The plasmids were detected in isolates recovered in other units within the same hospital and nearby hospitals. The gene "epidemic" was driven by IncN-pST15-type plasmids carrying a novel Tn4401b structure and non-Tn4401 elements (NTEKPC) in Klebsiella spp., Escherichia coli, Enterobacter spp., and Citrobacter spp. Of note, mcr-9 was found to coexist with blaKPC-2 in species of the Enterobacter cloacae complex. Our findings suggest that the main mechanism for dissemination of blaKPC-2 is HGT mediated by highly transferable plasmids among species of Enterobacterales in infected/colonized patients, presenting a major challenge for public health interventions in developing countries such as Colombia.

Mechanistic Insights Into the Differential Efficacy of Daptomycin Plus ß-Lactam Combinations Against Daptomycin-Resistant Enterococcus faecium.

BACKGROUND: The combination of daptomycin (DAP) plus ampicillin (AMP), ertapenem (ERT), or ceftaroline has been demonstrated to be efficacious against a DAP-tolerant Enterococcus faecium strain (HOU503). However, the mechanism for the efficacy of these combinations against DAP-resistant (DAP-R) E. faecium strains is unknown. METHODS: We investigated the efficacy of DAP in combination with AMP, ERT, ceftaroline, ceftriaxone, or amoxicillin against DAP-R E. faecium R497 using established in vitro and in vivo models. We evaluated pbp expression, levels of penicillin-binding protein (PBP) 5 (PBP5) and ß-lactam binding affinity in HOU503 versus R497. RESULTS: DAP plus AMP was the only efficacious regimen against DAP-R R497 and prevented emergence of resistance. DAP at 8, 6, and 4 mg/kg in combination with AMP was efficacious but showed delayed killing compared with 10 mg/kg. PBP5 of HOU503 exhibited amino acid substitutions in the penicillin-binding domain relative to R497. No difference in pbp mRNA or PBP5 levels was detected between HOU503 and R497. labeling of PBPs with Bocillin FL, a fluorescent penicillin derivative, showed increased ß-lactam binding affinity of PBP5 of HOU503 compared with that of R497. CONCLUSIONS: Only DAP (10 mg/kg) plus AMP or amoxicillin was efficacious against a DAP-R E. faecium strain, and pbp5 alleles may be important contributors to efficacy of DAP plus ß-lactam therapy.