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.

Differential in vitro susceptibility to ampicillin/ceftriaxone combination therapy among Enterococcus faecalis infective endocarditis clinical isolates.

OBJECTIVES: To investigate the genomic diversity and ß-lactam susceptibilities of Enterococcus faecalis collected from patients with infective endocarditis (IE). METHODS: We collected 60 contemporary E. faecalis isolates from definite or probable IE cases identified between 2018 and 2021 at the University of Pittsburgh Medical Center. We used whole-genome sequencing to study bacterial genomic diversity and employed antibiotic checkerboard assays and a one-compartment pharmacokinetic-pharmacodynamic (PK/PD) model to investigate bacterial susceptibility to ampicillin and ceftriaxone both alone and in combination. RESULTS: Genetically diverse E. faecalis were collected, however, isolates belonging to two STs, ST6 and ST179, were collected from 21/60 (35%) IE patients. All ST6 isolates encoded a previously described mutation upstream of penicillin-binding protein 4 (pbp4) that is associated with pbp4 overexpression. ST6 isolates had higher ceftriaxone MICs and higher fractional inhibitory concentration index values for ampicillin and ceftriaxone (AC) compared to other isolates, suggesting diminished in vitro AC synergy against this lineage. Introduction of the pbp4 upstream mutation found among ST6 isolates caused increased ceftriaxone resistance in a laboratory E. faecalis isolate. PK/PD testing showed that a representative ST6 isolate exhibited attenuated efficacy of AC combination therapy at humanized antibiotic exposures. CONCLUSIONS: We find evidence for diminished in vitro AC activity among a subset of E. faecalis IE isolates with increased pbp4 expression. These findings suggest that alternate antibiotic combinations against diverse contemporary E. faecalis IE isolates should be evaluated.

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.

LiaX is a surrogate marker for cell envelope stress and daptomycin non-susceptibility in Enterococcus faecium.

Daptomycin (DAP) is often used as a first-line therapy to treat vancomycin-resistant Enterococcus faecium infections, but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP minimum inhibitory concentrations (MICs) have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system, and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface-exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium with an activated LiaFSR system. Unlike E. faecalis, E. faecium LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX enzyme-linked immunosorbent assay (ELISA). We then assessed 86 clinical E. faecium bloodstream isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-resistant clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-susceptible isolates by standard MIC determination also had elevated LiaX ELISAs compared to a well-characterized DAP-susceptible strain. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many E. faecium isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

Co-Occurrence of Gram-Negative Rods in Patients with Hematologic Malignancy and Sinopulmonary Mucormycosis.

Both Mucorales and Gram-negative rods (GNRs) commonly infect patients with hematological malignancies (HM); however, their co-occurrence is understudied. Therefore, we retrospectively reviewed the records of 63 patients with HM and proven or probable sinopulmonary mucormycosis at MD Anderson Cancer Center (Houston, Texas) from 2000-2020. Seventeen out of sixty-three reviewed patients (27.0%) had sinopulmonary co-occurrence of GNRs (most commonly Pseudomonas aeruginosa and Stenotrophomonas maltophilia) within 30 days of a positive Mucorales culture or histology demonstrating Mucorales species. Eight of seventeen co-isolations of Mucorales and GNRs were found in same-day samples. All 15 patients with GNR co-occurrence and reported antimicrobial data had received anti-Pseudomonal agents within 14 days prior to diagnosis of mucormycosis and 5/15 (33.3%) had received anti-Stenotrophomonal agents. Demographic and clinical characteristics of patients with and without GNR co-occurrence were comparable. Forty-two-day all-cause mortality was high (34.9%) and comparable in patients with (41.2%) and without (32.6%) GNR detection (p = 0.53). In summary, over a quarter of heavily immunosuppressed patients with sinopulmonary mucormycosis harbored GNRs in their respiratory tract. Although no impact on survival outcomes was seen in a background of high mortality in our relatively underpowered study, pathogenesis studies are needed to understand the mutualistic interplay of GNR and Mucorales and their influence on host responses.

The emergence of cefiderocol resistance in Pseudomonas aeruginosa from a heteroresistant isolate during prolonged therapy.

Cefiderocol is a siderophore cephalosporin designed to target multi-drug-resistant Gram-negative bacteria. Previously, the emergence of cefiderocol non-susceptibility has been associated with mutations in the chromosomal cephalosporinase (PDC) along with mutations in the PirA and PiuA/D TonB-dependent receptor pathways. Here, we report a clinical case of cefiderocol-resistant P. aeruginosa that emerged in a patient during treatment. This resistance was associated with mutations not previously reported, suggesting potential novel pathways to cefiderocol 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.

High-level ceftazidime/avibactam resistance in Escherichia coli conferred by the novel plasmid-mediated ß-lactamase CMY-185 variant.

OBJECTIVES: To characterize a blaCMY variant associated with ceftazidime/avibactam resistance from a serially collected Escherichia coli isolate. METHODS: A patient with an intra-abdominal infection due to recurrent E. coli was treated with ceftazidime/avibactam. On Day 48 of ceftazidime/avibactam therapy, E. coli with a ceftazidime/avibactam MIC of >256 mg/L was identified from abdominal drainage. Illumina and Oxford Nanopore Technologies WGS was performed on serial isolates to identify potential resistance mechanisms. Site-directed mutants of CMY ß-lactamase were constructed to identify amino acid residues responsible for ceftazidime/avibactam resistance. RESULTS: WGS revealed that all three isolates were E. coli ST410. The ceftazidime/avibactam-resistant strain uniquely acquired a novel CMY ß-lactamase gene, herein called blaCMY-185, harboured on an IncI-γ/K1 conjugative plasmid. The CMY-185 enzyme possessed four amino acid substitutions relative to CMY-2, including A114E, Q120K, V211S and N346Y, and conferred high-level ceftazidime/avibactam resistance with an MIC of 32 mg/L. Single CMY-2 mutants did not confer reduced ceftazidime/avibactam susceptibility. However, double and triple mutants containing N346Y previously associated with ceftazidime/avibactam resistance in other AmpC enzymes, conferred ceftazidime/avibactam MICs ranging between 4 and 32 mg/L as well as reduced susceptibility to the newly developed cephalosporin, cefiderocol. Molecular modelling suggested that the N346Y substitution confers the reduction of avibactam inhibition due to steric hindrance between the side chain of Y346 and the sulphate group of avibactam. CONCLUSIONS: We identified ceftazidime/avibactam resistance in E. coli associated with a novel CMY variant. Unlike other AmpC enzymes, CMY-185 appears to require an additional substitution on top of N346Y to confer ceftazidime/avibactam resistance.

Molecular Basis of Cell Membrane Adaptation in Daptomycin-Resistant Enterococcus faecalis.

Daptomycin is a last-resort lipopeptide antibiotic that disrupts cell membrane (CM) and peptidoglycan homeostasis. Enterococcus faecalis has developed a sophisticated mechanism to avoid daptomycin killing by re-distributing CM anionic phospholipids away from the septum. The CM changes are orchestrated by a three-component regulatory system, designated LiaFSR, with a possible contribution of cardiolipin synthase (Cls). However, the mechanism by which LiaFSR controls the CM response and the role of Cls are unknown. Here, we show that cardiolipin synthase activity is essential for anionic phospholipid redistribution and daptomycin resistance since deletion of the two genes ( cls1 and cls2 ) encoding Cls abolished CM remodeling. We identified LiaY, a transmembrane protein regulated by LiaFSR, as an important mediator of CM remodeling required for re-distribution of anionic phospholipid microdomains via interactions with Cls1. Together, our insights provide a mechanistic framework on the enterococcal response to cell envelope antibiotics that could be exploited therapeutically.

LiaX is a surrogate marker for cell-envelope stress and daptomycin non-susceptibility in Enterococcus faecium.

Daptomycin (DAP) is often used as a first line therapy to treat vancomycin-resistant Enterococcus faecium (VR Efm ) infections but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP MICs have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ . In Enterococcus faecalis , LiaX is surface exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis , LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium . Here, we found that liaX is essential in E. faecium ( Efm ) with an activated LiaFSR system. Unlike E. faecalis , Efm LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX ELISA. We then assessed 86 clinical E. faecium BSI isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-R clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-S isolates by standard MIC determination had elevated LiaX ELISAs above the established cut-off. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many Efm isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

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.

The evolution of motivational interviewing.

This review traces the development of motivational interviewing (MI) from its happenstance beginnings and the first description published in this journal in 1983, to its continuing evolution as a method that is now in widespread practice in many professions, nations and languages. The efficacy of MI has been documented in hundreds of controlled clinical trials, and extensive process research sheds light on why and how it works. Developing proficiency in MI is facilitated by feedback and coaching based on observed practice after initial training. The author reflects on parallels between MI core processes and the characteristics found in 70 years of psychotherapy research to distinguish more effective therapists. This suggests that MI offers an evidence-based therapeutic style for delivering other treatments more effectively. The most common use of MI now is indeed in combination with other treatment methods such as cognitive behaviour therapies.

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.

A Case of Successful Treatment of Recurrent Urinary Tract Infection by Extended-Spectrum ß-Lactamase Producing Klebsiella pneumoniae Using Oral Lyophilized Fecal Microbiota Transplant.

Recurrent urinary tract infections (UTIs) are a challenging clinical entity that can be frustrating for patient and physician alike. Repeated rounds of antibiotics can select for multidrug-resistant organisms, further complicating care. We describe the successful use of fecal microbiota transplantation (FMT) for the treatment of recurrent extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae UTIs in a patient with an ileal conduit and urostomy. In the 18 months after FMT, the patient had not experienced new infections with ESBL-producing organisms. The urine and stool microbiomes of the patient were tracked before and post-FMT using 16s RNA sequencing with measurement of α-diversity. Sequencing of the recipient microbiota did not mirror the donor stool taxa at either site, but an increase in the relative proportion of the genus Bacteroides as compared with Prevotella was noted in the stool post-transplant. FMTs may be a promising treatment option for recurrent multidrug-resistant infections.

Bacterial cell membranes and their role in daptomycin resistance: A review.

Lipids play a major role in bacterial cells. Foremost, lipids are the primary constituents of the cell membrane bilayer, providing structure and separating the cell from the surrounding environment. This makes the lipid bilayer a prime target for antimicrobial peptides and membrane-acting antibiotics such as daptomycin. In response, bacteria have evolved mechanisms by which the membrane can be adapted to resist attack by these antimicrobial compounds. In this review, we focus on the membrane phospholipid changes associated with daptomycin resistance in enterococci, Staphylococcus aureus, and the Viridans group streptococci.

Human Dectin-1 deficiency impairs macrophage-mediated defense against phaeohyphomycosis.

Subcutaneous phaeohyphomycosis typically affects immunocompetent individuals following traumatic inoculation. Severe or disseminated infection can occur in CARD9 deficiency or after transplantation, but the mechanisms protecting against phaeohyphomycosis remain unclear. We evaluated a patient with progressive, refractory Corynespora cassiicola phaeohyphomycosis and found that he carried biallelic deleterious mutations in CLEC7A encoding the CARD9-coupled, ß-glucan-binding receptor, Dectin-1. The patient's PBMCs failed to produce TNF-α and IL-1ß in response to ß-glucan and/or C. cassiicola. To confirm the cellular and molecular requirements for immunity against C. cassiicola, we developed a mouse model of this infection. Mouse macrophages required Dectin-1 and CARD9 for IL-1ß and TNF-α production, which enhanced fungal killing in an interdependent manner. Deficiency of either Dectin-1 or CARD9 was associated with more severe fungal disease, recapitulating the human observation. Because these data implicated impaired Dectin-1 responses in susceptibility to phaeohyphomycosis, we evaluated 17 additional unrelated patients with severe forms of the infection. We found that 12 out of 17 carried deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular C-terminal domain and impaired Dectin-1-dependent cytokine production. Thus, we show that Dectin-1 and CARD9 promote protective TNF-α- and IL-1ß-mediated macrophage defense against C. cassiicola. More broadly, we demonstrate that human Dectin-1 deficiency may contribute to susceptibility to severe phaeohyphomycosis by certain dematiaceous fungi.

Antimicrobial Susceptibility Testing for Enterococci.

Enterococci are major, recalcitrant nosocomial pathogens with a wide repertoire of intrinsic and acquired resistance determinants and the potential of developing resistance to all clinically available antimicrobials. As such, multidrug-resistant enterococci are considered a serious public health threat. Due to limited treatment options and rapid emergence of resistance to all novel agents, the clinical microbiology laboratory plays a critical role in deploying accurate, reproducible, and feasible antimicrobial susceptibility testing methods to guide appropriate treatment of patients with deep-seated enterococcal infections. In this review, we provide an overview of the advantages and disadvantages of existing manual and automated methods that test susceptibility of Enterococcus faecium and Enterococcus faecalis to ß-lactams, aminoglycosides, vancomycin, lipoglycopeptides, oxazolidinones, novel tetracycline-derivatives, and daptomycin. We also identify unique problems and gaps with the performance and clinical utility of antimicrobial susceptibility testing for enterococci, provide recommendations for clinical laboratories to circumvent select problems, and address potential future innovations that can bridge major gaps in susceptibility testing.