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.

A Theoretical Analysis of Interaction Energies and Intermolecular Interactions between Amphotericin B and Potential Bioconjugates Used in the Modification of Nanocarriers for Drug Delivery.

Amphotericin B (AmB) is an antibiotic with a wide spectrum of action and low multidrug resistance, although it exhibits self-aggregation, low specificity, and solubility in aqueous media. An alternative for its oral administration is its encapsulation in polymers modified with bioconjugates. The aim of the present computational research is to determine the affinity between AmB and six bioconjugates to define which one could be more suitable. The CAM-B3LYP-D3/6-31+G(d,p) method was used for all computational calculations. The dimerization enthalpy of the most stable and abundant systems at pH = 7 allows obtaining this affinity order: AmB_1,2-distearoyl-sn-glycerol-3-phosphorylethanolamine (DSPE) > AmB_γ-cyclodextrin > AmB_DSPEc > AmB_retinol > AmB_cholesterol > AmB_dodecanol, where DSPEc is a DSPE analog. Quantum theory of atoms in molecules, the non-covalent interactions index, and natural bond orbital analysis revealed the highest abundance of noncovalent interactions for AmB-DSPE (51), about twice the number of interactions of the other dimers. Depending on the interactions' strength and abundance of the AmB-DSPE dimer, these are classified as strong: O-H---O (2), N-H---O (3) and weak: C-H---O (25), H---H (18), C-H---C (3). Although the C-H---O hydrogen bond is weak, the number of interactions involved in all dimers cannot be underestimated. Thus, non-covalent interactions drive the stabilization of copolymers, and from our analysis, the most promising candidates for encapsulating are DSPE and γ-cyclodextrin.

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.

Intracerebral and subarachnoid hemorrhage after suicidal "near-hanging".

Hanging is one of the most common methods of attempting suicide with a fast occurring death and a high lethality rate. Depending on how fast the hanged individuals are found, they may be rescued, which is referred to as "near-hanging." Whilst hypoxic and ischemic brain damage are common findings in hanging, intracranial hemorrhages seem to be a rare occurrence. To the authors' knowledge, this is the first autopsy case report of an intracerebral and subarachnoid hemorrhage in a delayed death after incomplete hanging. We hypothesize that the combination of two mechanisms is involved in the development of such hemorrhages. First, the persistent venous hypertension during incomplete hanging causing endothelial damage resulting in vascular leakage. Second, the reperfusion of the brain due to recovery of venous blood return after the release form the rope. Whereas intracranial hemorrhage is not a common finding in daily autopsy cases of fatal hanging, it might be a severe complication in rescued cases.

Antimicrobial sensing coupled with cell membrane remodeling mediates antibiotic resistance and virulence in Enterococcus faecalis.

Bacteria have developed several evolutionary strategies to protect their cell membranes (CMs) from the attack of antibiotics and antimicrobial peptides (AMPs) produced by the innate immune system, including remodeling of phospholipid content and localization. Multidrug-resistant Enterococcus faecalis, an opportunistic human pathogen, evolves resistance to the lipopeptide daptomycin and AMPs by diverting the antibiotic away from critical septal targets using CM anionic phospholipid redistribution. The LiaFSR stress response system regulates this CM remodeling via the LiaR response regulator by a previously unknown mechanism. Here, we characterize a LiaR-regulated protein, LiaX, that senses daptomycin or AMPs and triggers protective CM remodeling. LiaX is surface exposed, and in daptomycin-resistant clinical strains, both LiaX and the N-terminal domain alone are released into the extracellular milieu. The N-terminal domain of LiaX binds daptomycin and AMPs (such as human LL-37) and functions as an extracellular sentinel that activates the cell envelope stress response. The C-terminal domain of LiaX plays a role in inhibiting the LiaFSR system, and when this domain is absent, it leads to activation of anionic phospholipid redistribution. Strains that exhibit LiaX-mediated CM remodeling and AMP resistance show enhanced virulence in the Caenorhabditis elegans model, an effect that is abolished in animals lacking an innate immune pathway crucial for producing AMPs. In conclusion, we report a mechanism of antibiotic and AMP resistance that couples bacterial stress sensing to major changes in CM architecture, ultimately also affecting host-pathogen interactions.

Effectiveness of a case management model for people with multimorbidity: Mixed methods study.

AIMS: The aim was to determine the impact of a case management model on indicators of health service utilization, polypharmacy, quality of life and dependency of patients with multimorbidity, and family caregiver overload in a group of patients insured with two insurance companies in the city of Bogotá (Colombia). DESIGN: This was a mixed methods study, which integrated a quantitative and qualitative component. METHODS: The study was conducted between July 2019 and March 2020. A quantitative component is based on a pre-experimental study with a single group and pre- and post-test measurements. Patients with multimorbidity with a medium or high level of complexity were included in the study. A sample of 317 patients and their caregivers was estimated. Following the completion of the intervention, a descriptive study that explored the perspective of nurses, patients and caregivers was developed to better understand the process and results from their own words and experience. A total of 17 dyads of patients and caregivers were interviewed, as well as six nurse managers. The integration strategy was developed based on a comparison made from the perspective of multiple stakeholders. RESULTS: The model's impact on quality of life, particularly in terms of social functioning and mental health, has been documented. Caregiver overload was reduced and an improvement in the adoption of the role was observed, aspects that converge with the experience of the dyads and the caregivers in the support and backing provided by the model. CONCLUSION: The intervention was structured in five modules: case detection, complexity screening, comprehensive assessment with various instruments, individualized care and follow-up plan, and plan assessment. The nurse manager role is confirmed as that of a professional with the leadership capacity to articulate disciplines and actors, whilst also dealing with the day-to-day needs of people with complex health conditions. IMPACT: A comprehensive and integrated approach to patients with multiple diseases in a health insurance context marked by access barriers and fragmentation of health services. The study provides quantitative and qualitative evidence of the benefits of the case management model in Colombia for patients with multiple diseases and their family caregivers, particularly in terms of the psychosocial dimensions of health-related quality of life and dependence assessment. A significant impact on the caregiver role, as well as an improvement in perception and trust in the health system, was observed as a result of the overcoming of administrative barriers achieved by the nurse case manager. The findings are considered to be extremely useful for decision-makers and insurers in developing a case management model focused on comprehensive and individualized care plans, as well as for individuals with multiple diseases and their caregivers.

A Test for the Rapid Detection of the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus.

The cefazolin inoculum effect (CzIE) has been associated with therapeutic failures and mortality in invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections. A diagnostic test to detect the CzIE is not currently available. We developed a rapid (∼3 h) CzIE colorimetric test to detect staphylococcal-ß-lactamase (BlaZ) activity in supernatants after ampicillin induction. The test was validated using 689 bloodstream MSSA isolates recovered from Latin America and the United States. The cefazolin MIC determination at a high inoculum (107 CFU/ml) was used as a reference standard (cutoff ≥16 µg/ml). All isolates underwent genome sequencing. A total of 257 (37.3%) of MSSA isolates exhibited the CzIE by the reference standard method. The overall sensitivity and specificity of the colorimetric test was 82.5% and 88.9%, respectively. Sensitivity in MSSA isolates harboring type A BlaZ (the most efficient enzyme against cefazolin) was 92.7% with a specificity of 87.8%. The performance of the test was lower against type B and C enzymes (sensitivities of 53.3% and 72.3%, respectively). When the reference value was set to ≥32 µg/ml, the sensitivity for isolates carrying type A enzymes was 98.2%. Specificity was 100% for MSSA lacking blaZ The overall negative predictive value ranged from 81.4% to 95.6% in Latin American countries using published prevalence rates of the CzIE. MSSA isolates from the United States were genetically diverse, with no distinguishing genomic differences from Latin American MSSA, distributed among 18 sequence types. A novel test can readily identify most MSSA isolates exhibiting the CzIE, particularly those carrying type A BlaZ. In contrast to the MIC determination using high inoculum, the rapid test is inexpensive, feasible, and easy to perform. After minor validation steps, it could be incorporated into the routine clinical laboratory workflow.

Seeking an Adjustment from the Unnatural to the Supernatural: The Experience of Losing a Child from Cancer in Colombia.

AIMS: The death of a child with cancer can be devastating for his or her parents. This study sought to understand the way in which the process of parental grief develops after the death of a child with cancer. METHODS: The research used a grounded theory approach, in which 18 participants were enrolled including parents whose child died from cancer 5 months to 5 years before. In-depth interviews were conducted, which were analyzed using constant comparisons until theoretical saturation was reached. RESULTS: Fifteen subcategories were identified and grouped into three categories that explain what the grieving process represents to the parents over time (a) crossing a desert, (b) dying while alive, and (c) coming back to life. From the emerging relationships among the categories, the core category "seeking adjustment from the unnatural to the supernatural" arises. The results show that grief begins from the moment of diagnosis until long after the child's death. For parents, it entails understanding the disruption in the natural course of life, going through indescribable pain, and being spiritually reconnected with their child. CONCLUSIONS: These results enable nurses to design comprehensive interventions that meet the described needs of these parents.

LiaR-independent pathways to daptomycin resistance in Enterococcus faecalis reveal a multilayer defense against cell envelope antibiotics.

The lipopeptide antibiotic daptomycin (DAP) is a key drug against serious enterococcal infections, but the emergence of resistance in the clinical setting is a major concern. The LiaFSR system plays a prominent role in the development of DAP resistance (DAP-R) in enterococci, and blocking this stress response system has been proposed as a novel therapeutic strategy. In this work, we identify LiaR-independent pathways in Enterococcus faecalis that regulate cell membrane adaptation in response to antibiotics. We adapted E. faecalis OG1RF (a laboratory strain) and S613TM (a clinical strain) lacking liaR to increasing concentrations of DAP, leading to the development of DAP-R and elevated MICs to bacitracin and ceftriaxone. Whole genome sequencing identified changes in the YxdJK two-component regulatory system and a putative fatty acid kinase (dak) in both DAP-R strains. Deletion of the gene encoding the YxdJ response regulator in both the DAP-R mutant and wild-type OG1RF decreased MICs to DAP, even when a functional LiaFSR system was present. Mutations in dak were associated with slower growth, decreased membrane fluidity and alterations of cell morphology. These findings suggest that overlapping stress response pathways can provide protection against antimicrobial peptides in E. faecalis at a significant cost in bacterial fitness.

Novel Insights into the Classification of Staphylococcal ß-Lactamases in Relation to the Cefazolin Inoculum Effect.

Cefazolin has become a prominent therapy for methicillin-susceptible Staphylococcus aureus (MSSA) infections. However, an important concern is the cefazolin inoculum effect (CzIE), a phenomenon mediated by staphylococcal ß-lactamases. Four variants of staphylococcal ß-lactamases have been described based on serological methodologies and limited sequence information. Here, we sought to reassess the classification of staphylococcal ß-lactamases and their correlation with the CzIE. We included a large collection of 690 contemporary bloodstream MSSA isolates recovered from Latin America, a region with a high prevalence of the CzIE. We determined cefazolin MICs at standard and high inoculums by broth microdilution. Whole-genome sequencing was performed to classify the ß-lactamase in each isolate based on the predicted full sequence of BlaZ. We used the classical schemes for ß-lactamase classification and compared it to BlaZ allotypes found in unique sequences using the genomic information. Phylogenetic analyses were performed based on the BlaZ and core-genome sequences. The overall prevalence of the CzIE was 40%. Among 641 genomes, type C was the most predominant ß-lactamase (37%), followed by type A (33%). We found 29 allotypes and 43 different substitutions in BlaZ. A single allotype, designated BlaZ-2, showed a robust and statistically significant association with the CzIE. Two other allotypes (BlaZ-3 and BlaZ-5) were associated with a lack of the CzIE. Three amino acid substitutions (A9V, E112A, and G145E) showed statistically significant association with the CzIE (P = <0.01). CC30 was the predominant clone among isolates displaying the CzIE. Thus, we provide a novel approach to the classification of the staphylococcal ß-lactamases with the potential to more accurately identify MSSA strains exhibiting the CzIE.

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.

Detection of heterogeneous vancomycin intermediate resistance in MRSA isolates from Latin America.

BACKGROUND: Vancomycin is a common first-line option for MRSA infections. The heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) phenotype is associated with therapeutic failure. However, hVISA isolates are usually reported as vancomycin susceptible by routine susceptibility testing procedures. OBJECTIVES: To detect and characterize the hVISA phenotype in MRSA isolates causing infections in nine Latin American countries. METHODS: We evaluated a total of 1189 vancomycin-susceptible MRSA isolates recovered during 2006-08 and 2011-14. After an initial screening of hVISA using glycopeptide-supplemented agar strategies, the detection of hVISA was performed by Etest (GRD) and Macro-method (MET). Isolates deemed to be hVISA were subjected to population analysis profile/AUC (PAP/AUC) and WGS for further characterization. Finally, we interrogated alterations in predicted proteins associated with the development of the VISA phenotype in both hVISA and vancomycin-susceptible S. aureus (VSSA) genomes. RESULTS: A total of 39 MRSA isolates (3.3%) were classified as hVISA (1.4% and 5.6% in MRSA recovered from 2006-08 and 2011-14, respectively). Most of the hVISA strains (95%) belonged to clonal complex (CC) 5. Only 6/39 hVISA isolates were categorized as hVISA by PAP/AUC, with 6 other isolates close (0.87-0.89) to the cut-off (0.9). The majority of the 39 hVISA isolates exhibited the Leu-14→Ile (90%) and VraT Glu-156→Gly (90%) amino acid substitutions in WalK. Additionally, we identified 10 substitutions present only in hVISA isolates, involving WalK, VraS, RpoB and RpoC proteins. CONCLUSIONS: The hVISA phenotype exhibits low frequency in Latin America. Amino acid substitutions in proteins involved in cell envelope homeostasis and RNA synthesis were commonly identified. Our results suggest that Etest-based methods are an important alternative for the detection of hVISA clinical isolates.

Disrupting Membrane Adaptation Restores In Vivo Efficacy of Antibiotics Against Multidrug-Resistant Enterococci and Potentiates Killing by Human Neutrophils.

Daptomycin resistance in enterococci is often mediated by the LiaFSR system, which orchestrates the cell membrane stress response. Activation of LiaFSR through the response regulator LiaR generates major changes in cell membrane function and architecture (membrane adaptive response), permitting the organism to survive the antibiotic attack. Here, using a laboratory strain of Enterococcus faecalis, we developed a novel Caenorhabditis elegans model of daptomycin therapy and showed that disrupting LiaR-mediated cell membrane adaptation restores the in vivo activity of daptomycin. The LiaR effect was also seen in a clinical strain of daptomycin-resistant Enterococcus faecium, using a murine model of peritonitis. Furthermore, alteration of the cell membrane response increased the ability of human polymorphonuclear neutrophils to readily clear both E. faecalis and multidrug-resistant E. faecium. Our results provide proof of concept that targeting the cell membrane adaptive response restores the in vivo activity of antibiotics, prevents resistance, and enhances the ability of the innate immune system to kill infecting bacteria.

Linezolid- and Vancomycin-resistant Enterococcus faecium in Solid Organ Transplant Recipients: Infection Control and Antimicrobial Stewardship Using Whole Genome Sequencing.

BACKGROUND: Vancomycin-resistant enterococci are an important cause of healthcare-associated infections and are inherently resistant to many commonly used antibiotics. Linezolid is the only drug currently approved by the US Food and Drug Administration to treat vancomycin-resistant enterococci; however, resistance to this antibiotic appears to be increasing. Although outbreaks of linezolid- and vancomycin-resistant Enterococcus faecium (LR-VRE) in solid organ transplant recipients remain uncommon, they represent a major challenge for infection control and hospital epidemiology. METHODS: We describe a cluster of 4 LR-VRE infections among a group of liver and multivisceral transplant recipients in a single intensive care unit. Failure of treatment with linezolid in 2 cases led to a review of standard clinical laboratory methods for susceptibility determination. Testing by alternative methods including whole genome sequencing (WGS) and a comprehensive outbreak investigation including sampling of staff members and surfaces was performed. RESULTS: Review of laboratory testing methods revealed a limitation in the VITEK 2 system with regard to reporting resistance to linezolid. Linezolid resistance in all cases was confirmed by E-test method. The use of WGS identified a resistant subpopulation with the G2376C mutation in the 23S ribosomal RNA. Sampling of staff members' dominant hands as well as sampling of surfaces in the unit identified no contaminated sources for transmission. CONCLUSIONS: This cluster of LR-VRE in transplant recipients highlights the possible shortcomings of standard microbiology laboratory methods and underscores the importance of WGS to identify resistance mechanisms that can inform patient care, as well as infection control and antibiotic stewardship measures.

Mutations in cdsA and pgsA Correlate with Daptomycin Resistance in Streptococcus mitis and S. oralis.

We investigated the ability of several recent clinical viridans group streptococci (VGS) bloodstream isolates (Streptococcus mitis/S. oralis subgroup) from daptomycin (DAP)-naive patients to develop DAP resistance in vitro All strains rapidly developed high-level and stable DAP resistance. Substitutions in two enzymes involved in the cardiolipin biosynthesis pathway were identified, i.e., CdsA (phosphatidate cytidylyltransferase) and PgsA (CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase). These mutations were associated with complete disappearance of phosphatidylglycerol and cardiolipin from cell membranes. DAP interactions with the cell membrane differed in isolates with PgsA versus CdsA substitutions.

Effectiveness of a case management model for the comprehensive provision of health services to multi-pathological people.

AIM: To determine the effectiveness of a case management model for approaching multi-pathological people in a health promoting entity of the contributory healthcare scheme in Bogotá, Colombia between 2018 - . DESIGN: Mixed methods research. METHOD: The study contemplates two components: a quantitative component using a quasi-experimental analytical design before and after longitudinal intervention to determine the effectiveness of the case management model and a qualitative descriptive design to understand the experience of the participants about the model. The Administrative Department of Science, Technology and Innovation of Colombia (Colciencias) funded this project by means of call 777-November 2017, under the financing agreement No. 848-December 2017. DISCUSSION: Addressing problems deriving from the structure of the Colombian healthcare system is crucial for implementing case management models. Furthermore, the effectiveness of such models may be affected by power relations and market failures, but the proved potential of a model may represent a generalized benefit for the Colombian health system. IMPACT: In Colombia, considering complications and management of chronic non-communicable diseases as isolated cases is considered as the highest cost events in healthcare provision, since an average of 12.8 million pesos is invested in each patient. This has led to rethink the management in these patients by means of a comprehensive model that guarantees the effectiveness of healthcare delivery, in the framework of a healthcare system heavily affected by payment capacity, where the market has a strong predominance, such as the case of Colombia. TRIAL REGISTRATION NUMBER: RPCEC00000293.

Clonal Emergence of Invasive Multidrug-Resistant Staphylococcus epidermidis Deconvoluted via a Combination of Whole-Genome Sequencing and Microbiome Analyses.

Background: Pathobionts, bacteria that are typically human commensals but can cause disease, contribute significantly to antimicrobial resistance. Staphylococcus epidermidis is a prototypical pathobiont as it is a ubiquitous human commensal but also a leading cause of healthcare-associated bacteremia. We sought to determine the etiology of a recent increase in invasive S. epidermidis isolates resistant to linezolid. Methods: Whole-genome sequencing (WGS) was performed on 176 S. epidermidis bloodstream isolates collected at the MD Anderson Cancer Center in Houston, Texas, between 2013 and 2016. Molecular relationships were assessed via complementary phylogenomic approaches. Abundance of the linezolid resistance determinant cfr was determined in stool samples via reverse-transcription quantitative polymerase chain reaction. Results: Thirty-nine of the 176 strains were linezolid resistant (22%). Thirty-one of the 39 linezolid-resistant S. epidermidis infections were caused by a particular clone resistant to multiple antimicrobials that spread among leukemia patients and carried cfr on a 49-kb plasmid (herein called pMB151a). The 6 kb of pMB151a surrounding the cfr gene was nearly 100% identical to a cfr-containing plasmid isolated from livestock-associated staphylococci in China. Analysis of serial stool samples from leukemia patients revealed progressive staphylococcal domination of the intestinal microflora and an increase in cfr abundance following linezolid use. Conclusions: The combination of linezolid use plus transmission of a multidrug-resistant clone drove expansion of invasive, linezolid-resistant S. epidermidis. Our results lend support to the notion that a combination of antibiotic stewardship plus infection control measures may help to control the spread of a multidrug-resistant pathobiont.

Influence of Inoculum Effect on the Efficacy of Daptomycin Monotherapy and in Combination with ß-Lactams against Daptomycin-Susceptible Enterococcus faecium Harboring LiaSR Substitutions.

Enterococcus faecium isolates that harbor LiaFSR substitutions but are phenotypically susceptible to daptomycin (DAP) by current breakpoints are problematic, since predisposition to resistance may lead to therapeutic failure. Using a simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) model, we investigated DAP regimens (6, 8, and 10 mg/kg of body weight/day) as monotherapy and in combination with ampicillin (AMP), ceftaroline (CPT), or ertapenem (ERT) against E. faecium HOU503, a DAP-susceptible strain that harbors common LiaS and LiaR substitutions found in clinical isolates (T120S and W73C, respectively). Of interest, the efficacy of DAP monotherapy, at any dose regimen, was dependent on the size of the inoculum. At an inoculum of ∼109 CFU/g, DAP doses of 6 to 8 mg/kg/day were not effective and led to significant regrowth with emergence of resistant derivatives. In contrast, at an inoculum of ∼107 CFU/g, marked reductions in bacterial counts were observed with DAP at 6 mg/kg/day, with no resistance. The inoculum effect was confirmed in a rat model using humanized DAP exposures. Combinations of DAP with AMP, CPT, or ERT demonstrated enhanced eradication and reduced potential for resistance, allowing de-escalation of the DAP dose. Persistence of the LiaRS substitutions was identified in DAP-resistant isolates recovered from the SEV model and in DAP-resistant derivatives of an initially DAP-susceptible clinical isolate of E. faecium (HOU668) harboring LiaSR substitutions that was recovered from a patient with a recurrent bloodstream infection. Our results provide novel data for the use of DAP monotherapy and combinations for recalcitrant E. faecium infections and pave the way for testing these approaches in humans.

Runt-Related Transcription Factor 2 Induction During Differentiation of Wharton's Jelly Mesenchymal Stem Cells to Osteoblasts Is Regulated by Jumonji AT-Rich Interactive Domain 1B Histone Demethylase.

Novel bone regeneration approaches aim to obtain immature osteoblasts from somatic stem cells. Umbilical cord Wharton's jelly mesenchymal stem cells (WJ-MSCs) are an ideal source for cell therapy. Hence, the study of mechanisms involved in WJ-MSC osteoblastic differentiation is crucial to exploit their developmental capacity. Here, we have assessed epigenetic control of the Runt-related transcription factor 2 (RUNX2) osteogenic master regulator gene in WJ-MSC. We present evidence indicating that modulation of RUNX2 expression through preventing Jumonji AT-rich interactive domain 1B (JARID1B) histone demethylase activity is relevant to enhance WJ-MSC osteoblastic potential. Hence, JARID1B loss of function in WJ-MSC results in increased RUNX2/p57 expression. Our data highlight JARID1B activity as a novel target to modulate WJ-MSC osteoblastic differentiation with potential applications in bone tissue engineering. Stem Cells 2017;35:2430-2441.

An Analysis of the Epidemic of Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae: Convergence of Two Evolutionary Mechanisms Creates the "Perfect Storm".

Background: Carbapenem resistance is a critical healthcare challenge worldwide. Particularly concerning is the widespread dissemination of Klebsiella pneumoniae carbapenemase (KPC). Klebsiella pneumoniae harboring blaKPC (KPC-Kpn) is endemic in many areas including the United States, where the epidemic was primarily mediated by the clonal dissemination of Kpn ST258. We postulated that the spread of blaKPC in other regions occurs by different and more complex mechanisms. To test this, we investigated the evolution and dynamics of spread of KPC-Kpn in Colombia, where KPC became rapidly endemic after emerging in 2005. Methods: We sequenced the genomes of 133 clinical isolates recovered from 24 tertiary care hospitals located in 10 cities throughout Colombia, between 2002 (before the emergence of KPC-Kpn) and 2014. Phylogenetic reconstructions and evolutionary mapping were performed to determine temporal and genetic associations between the isolates. Results: Our results indicate that the start of the epidemic was driven by horizontal dissemination of mobile genetic elements carrying blaKPC-2, followed by the introduction and subsequent spread of clonal group 258 (CG258) isolates containing blaKPC-3. Conclusions: The combination of 2 evolutionary mechanisms of KPC-Kpn within a challenged health system of a developing country created the "perfect storm" for sustained endemicity of these multidrug-resistant organisms in Colombia.