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.

Fusobacteria behaving badly: Masquerading strains of strictly anaerobic Escherichiacoli misidentified due to the deletion of the hemB gene.

Three strictly anaerobic strains of Escherichia coli were misidentified as Fusobacterium mortiferum, due to a deletion of the hemB gene which is involved in anaerobic respiration. An unusual antimicrobial susceptibility pattern sparked the further diagnostic strategies that eventually identified these strains as true anaerobic E. coli This phenomenon is more common than appreciated and can have an impact on clinical practice including persistent and relapsing infections.

Fusobacteriumnucleatum Adheres to Clostridioides difficile via the RadD Adhesin to Enhance Biofilm Formation in Intestinal Mucus.

BACKGROUND & AIMS: Although Clostridioides difficile infection (CDI) is known to involve the disruption of the gut microbiota, little is understood regarding how mucus-associated microbes interact with C difficile. We hypothesized that select mucus-associated bacteria would promote C difficile colonization and biofilm formation. METHODS: To create a model of the human intestinal mucus layer and gut microbiota, we used bioreactors inoculated with healthy human feces, treated with clindamycin and infected with C difficile with the addition of human MUC2-coated coverslips. RESULTS: C difficile was found to colonize and form biofilms on MUC2-coated coverslips, and 16S rRNA sequencing showed a unique biofilm profile with substantial cocolonization with Fusobacterium species. Consistent with our bioreactor data, publicly available data sets and patient stool samples showed that a subset of patients with C difficile infection harbored high levels of Fusobacterium species. We observed colocalization of C difficile and F nucleatum in an aggregation assay using adult patients and stool of pediatric patients with inflammatory bowel disease and in tissue sections of patients with CDI. C difficile strains were found to coaggregate with F nucleatum subspecies in vitro; an effect that was inhibited by blocking or mutating the adhesin RadD on Fusobacterium and removal of flagella on C difficile. Aggregation was shown to be unique between F nucleatum and C difficile, because other gut commensals did not aggregate with C difficile. Addition of F nucleatum also enhanced C difficile biofilm formation and extracellular polysaccharide production. CONCLUSIONS: Collectively, these data show a unique interaction of between pathogenic C difficile and F nucleatum in the intestinal mucus layer.

The metabolic profile of Bifidobacterium dentium reflects its status as a human gut commensal.

BACKGROUND: Bifidobacteria are commensal microbes of the mammalian gastrointestinal tract. In this study, we aimed to identify the intestinal colonization mechanisms and key metabolic pathways implemented by Bifidobacterium dentium. RESULTS: B. dentium displayed acid resistance, with high viability over a pH range from 4 to 7; findings that correlated to the expression of Na+/H+ antiporters within the B. dentium genome. B. dentium was found to adhere to human MUC2+ mucus and harbor mucin-binding proteins. Using microbial phenotyping microarrays and fully-defined media, we demonstrated that in the absence of glucose, B. dentium could metabolize a variety of nutrient sources. Many of these nutrient sources were plant-based, suggesting that B. dentium can consume dietary substances. In contrast to other bifidobacteria, B. dentium was largely unable to grow on compounds found in human mucus; a finding that was supported by its glycosyl hydrolase (GH) profile. Of the proteins identified in B. dentium by proteomic analysis, a large cohort of proteins were associated with diverse metabolic pathways, indicating metabolic plasticity which supports colonization of the dynamic gastrointestinal environment. CONCLUSIONS: Taken together, we conclude that B. dentium is well adapted for commensalism in the gastrointestinal tract.

Visualization of fidaxomicin association with the exosporium layer of Clostridioides difficile spores.

BACKGROUND: Fidaxomicin has novel pharmacologic effects on C. difficile spore formation including outgrowth inhibition and persistent spore attachment. However, the mechanism of fidaxomicin attachment on spores has not undergone rigorous microscopic studies. MATERIALS & METHODS: Fidaxomicin attachment to C. difficile spores of three distinct ribotypes and C. difficile mutant spores with inactivation of exosporium or spore-coat protein-coding genes were visualized using confocal microscopy with a fidaxomicin-bodipy compound (green fluorescence). The pharmacologic effect of the fidaxomicin-bodipy compound was determined. Confocal microscopy experiments included direct effect on C. difficile wild-type and mutant spores, effect of exosporium removal, and direct attachment to a comparator spore forming organism, Bacillus subtilis. RESULTS: The fidaxomicin-bodipy compound MIC was 1 mg/L compared to 0.06 mg/L for unlabeled fidaxomicin, a 16-fold increase. Using confocal microscopy, the intracellular localization of fidaxomicin into vegetative C. difficile cells was observed consistent with its RNA polymerase mechanism of action and inhibited spore outgrowth. The fidaxomicin-bodipy compound was visualized outside of the core of C. difficile spores with no co-localization with the membrane staining dye FM4-64. Exosporium removal reduced fidaxomicin-bodipy association with C. difficile spores. Reduced fidaxomicin-bodipy was observed in C. difficile mutant spores for the spore surface proteins CdeC and CotE. CONCLUSION: This study visualized a direct attachment of fidaxomicin to C. difficile spores that was diminished with mutants of specific exosporium and spore coat proteins. These data provide advanced insight regarding the anti-spore properties of fidaxomicin.

Systems biology evaluation of refractory Clostridioides difficile infection including multiple failures of fecal microbiota transplantation.

BACKGROUND: Fecal microbiota transplantation (FMT) aims to cure Clostridioides difficile infection (CDI) through reestablishing a healthy microbiome and restoring colonization resistance. Although often effective after one infusion, patients with continued microbiome disruptions may require multiple FMTs. In this N-of-1 study, we use a systems biology approach to evaluate CDI in a patient receiving chronic suppressive antibiotics with four failed FMTs over two years. METHODS: Seven stool samples were obtained between 2016-18 while the patient underwent five FMTs. Stool samples were cultured for C. difficile and underwent microbial characterization and functional gene analysis using shotgun metagenomics. C. difficile isolates were characterized through ribotyping, whole genome sequencing, metabolic pathway analysis, and minimum inhibitory concentration (MIC) determinations. RESULTS: Growing ten strains from each sample, the index and first four recurrent cultures were single strain ribotype F078-126, the fifth was a mixed culture of ribotypes F002 and F054, and the final culture was ribotype F002. One single nucleotide polymorphism (SNP) variant was identified in the RNA polymerase (RNAP) ß-subunit RpoB in the final isolated F078-126 strain when compared to previous F078-126 isolates. This SNV was associated with metabolic shifts but phenotypic differences in fidaxomicin MIC were not observed. Microbiome differences were observed over time during vancomycin therapy and after failed FMTs. CONCLUSION: This study highlights the importance of antimicrobial stewardship in patients receiving FMT. Continued antibiotics play a destructive role on a transplanted microbiome and applies selection pressure for resistance to the few antibiotics available to treat CDI.

Human intestinal enteroids as a model of Clostridioides difficile-induced enteritis.

Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton. C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.NEW & NOTEWORTHY In this article, we developed a novel model of Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear C. difficile-conditioned media suitable for live-cell imaging.

Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates.

OBJECTIVES: To describe, for the first time (to the best of our knowledge), the genetic mechanisms of vancomycin resistance in clinical isolates of Clostridioides difficile ribotype 027. METHODS: Clinical isolates and laboratory mutants were analysed: genomically to identify resistance mutations; by transcriptional analysis of vanGCd, the vancomycin resistance operon encoding lipid II d-alanine-d-serine that is less bound by vancomycin than native lipid II d-alanine-d-alanine; by imaging of vancomycin binding to cell walls; and for changes in vancomycin bactericidal activity and autolysis. RESULTS: Vancomycin-resistant laboratory mutants and clinical isolates acquired mutations to the vanSR two-component system that regulates vanGCd. The substitutions impaired VanSR's function, resulting in constitutive transcription of vanGCd. Resistance was reversed by silencing vanG, encoding d-alanine-d-serine ligase in the vanGCd operon. In resistant cells, vancomycin was less bound to the cell wall septum, the site where vancomycin interacts with lipid II. Vancomycin's bactericidal activity was reduced against clinical isolates and laboratory mutants (64 and ≥1024 mg/L, respectively) compared with WT strains (4 mg/L). Truncation of the potassium transporter TrkA occurred in laboratory mutants, which were refractory to autolysis, accounting for their survival in high drug concentrations. CONCLUSIONS: Ribotype 027 evolved first-step resistance to vancomycin by constitutively expressing vanGCd, which is otherwise silent. Experimental evolutions and bactericidal assays show that ribotype 027 can acquire mutations to drastically enhance its tolerance to vancomycin. Thus, further epidemiological studies are warranted to examine the extent to which vancomycin resistance impacts clinical outcomes and the potential for these strains to evolve higher-level resistance, which would be devastating.

Clostridioides (Formerly Clostridium) difficile Infection During Hospitalization Increases the Likelihood of Nonhome Patient Discharge.

BACKGROUND: Clostridioides (formerly Clostridium) difficile infection (CDI) is associated with significant morbidity and mortality, including frequent hospitalizations. However, the impact of CDI after hospital discharge is poorly understood. The purpose of this study was to assess patient discharge disposition and understand CDI-related risk factors for nonhome discharge. METHODS: Using a nationally representative database of Veterans Health Administration (VHA) patients (2003-2014) and a validation database from hospitalized non-VHA patients in Houston, Texas, admission and discharge disposition was obtained for patients with CDI and matched controls. Incidence of and clinical/microbiologic risk factors for nonhome discharge were assessed using these databases. RESULTS: A total of 15173 VHA patients with CDI and 48599 non-CDI control patients originally admitted from the community were included. Significantly more patients with CDI were discharged to a nonhome location compared with controls (18% vs 8%; P < .0001), most commonly hospice/death (12%) or nursing home/long-term care facility (6%). Results were confirmed using a propensity-matched analysis and a validation cohort of 1941 hospitalized patients with CDI in Houston, Texas. Age, comorbidities, severe CDI, and ribotypes F027, F001, and F053-163 were associated with a nonhome discharge (P < .05 for all). CONCLUSIONS: Hospitalized patients with CDI frequently required a higher level of medical care residence at discharge compared with non-CDI patients. Risk factors for discharge to a higher level of care included CDI disease severity and variables associated with recurrent CDI.

Nitric oxide production by glomerular podocytes.

Nitric Oxide (NO), a potent vasodilator and vital signaling molecule, has been shown to contribute to the regulation of glomerular ultrafiltration. However, whether changes in NO occur in podocytes during the pathogenesis of salt-sensitive hypertension has not yet been thoroughly examined. We showed here that podocytes produce NO, and further hypothesized that hypertensive animals would exhibit reduced NO production in these cells in response to various paracrine factors, which might contribute to the damage of glomeruli filtration barrier and development of proteinuria. To test this, we isolated glomeruli from the kidneys of Dahl salt-sensitive (SS) rats fed a low salt (LS; 0.4% NaCl) or high salt (HS; 4% NaCl, 3 weeks) diets and loaded podocytes with either a combination of NO and Ca2+ fluorophores (DAF-FM and Fura Red, respectively) or DAF-FM alone. Changes in fluorescence were observed with confocal microscopy in response to adenosine triphosphate (ATP), angiotensin II (Ang II), and hydrogen peroxide (H2O2). Application of Ang II resulted in activation of both NO and intracellular calcium ([Ca2+]i) transients. In contrast, ATP promoted [Ca2+]i transients, but did not have any effects on NO production. SS rats fed a HS diet for 3 weeks demonstrated impaired NO production: the response to Ang II or H2O2 in podocytes of glomeruli isolated from SS rats fed a HS diet was significantly reduced compared to rats fed a LS diet. Therefore, glomerular podocytes from hypertensive rats showed a diminished NO release in response to Ang II or oxidative stress, suggesting that podocytic NO signaling is dysfunctional in this condition and likely contributes to the development of kidney injury.

Intravital imaging of the kidney in a rat model of salt-sensitive hypertension.

Hypertension is one of the most prevalent diseases worldwide and a major risk factor for renal failure and cardiovascular disease. The role of albuminuria, a common feature of hypertension and robust predictor of cardiorenal disorders, remains incompletely understood. The goal of this study was to investigate the mechanisms leading to albuminuria in the kidney of a rat model of hypertension, the Dahl salt-sensitive (SS) rat. To determine the relative contributions of the glomerulus and proximal tubule (PT) to albuminuria, we applied intravital two-photon-based imaging to investigate the complex renal physiological changes that occur during salt-induced hypertension. Following a high-salt diet, SS rats exhibited elevated blood pressure, increased glomerular sieving of albumin (GSCalb = 0.0686), relative permeability to albumin (+Δ16%), and impaired volume hemodynamics (-Δ14%). Serum albumin but not serum globulins or creatinine concentration was decreased (-0.54 g/dl), which was concomitant with increased filtration of albumin (3.7 vs. 0.8 g/day normal diet). Pathologically, hypertensive animals had significant tubular damage, as indicated by increased prevalence of granular casts, expansion and necrosis of PT epithelial cells (+Δ2.20 score/image), progressive augmentation of red blood cell velocity (+Δ269 µm/s) and micro vessel diameter (+Δ4.3 µm), and increased vascular injury (+Δ0.61 leakage/image). Therefore, development of salt-induced hypertension can be triggered by fast and progressive pathogenic remodeling of PT epithelia, which can be associated with changes in albumin handling. Collectively, these results indicate that both the glomerulus and the PT contribute to albuminuria, and dual treatment of glomerular filtration and albumin reabsorption may represent an effective treatment of salt-sensitive hypertension.

Novel antibiotics in development to treat Clostridium difficile infection.

PURPOSE OF REVIEW: Clostridium difficile infections (CDI) remain a challenge to treat clinically due primarily to limited number of antibiotics available and unacceptably high recurrence rates. Because of this, there has been significant demand for creating innovative therapeutics, which has resulted in the development of several novel antibiotics. RECENT FINDINGS: This review updates seven different antibiotics that are currently in development to treat CDI including fidaxomicin, surotomycin, ridinilazole, ramoplanin, cadazolid, LFF571, and CRS3123. Available preclinical and clinical data are compared between these antibiotics. SUMMARY: Many of these new antibiotics display almost ideal properties for antibiotics directed against CDI. Despite these properties, not all clinical development of these compounds has been successful. These studies have provided key insights into the pathogenesis of CDI and will continue to inform future drug development. Successful phase III clinical trials should result in several new and novel antibiotics to treat CDI.

Mutation of Plekha7 attenuates salt-sensitive hypertension in the rat.

PLEKHA7 (pleckstrin homology domain containing family A member 7) has been found in multiple studies as a candidate gene for human hypertension, yet functional data supporting this association are lacking. We investigated the contribution of this gene to the pathogenesis of salt-sensitive hypertension by mutating Plekha7 in the Dahl salt-sensitive (SS/JrHsdMcwi) rat using zinc-finger nuclease technology. After four weeks on an 8% NaCl diet, homozygous mutant rats had lower mean arterial (149 ± 9 mmHg vs. 178 ± 7 mmHg; P < 0.05) and systolic (180 ± 7 mmHg vs. 213 ± 8 mmHg; P < 0.05) blood pressure compared with WT littermates. Albumin and protein excretion rates were also significantly lower in mutant rats, demonstrating a renoprotective effect of the mutation. Total peripheral resistance and perivascular fibrosis in the heart and kidney were significantly reduced in Plekha7 mutant animals, suggesting a potential role of the vasculature in the attenuation of hypertension. Indeed, both flow-mediated dilation and endothelium-dependent vasodilation in response to acetylcholine were improved in isolated mesenteric resistance arteries of Plekha7 mutant rats compared with WT. These vascular improvements were correlated with changes in intracellular calcium handling, resulting in increased nitric oxide bioavailability in mutant vessels. Collectively, these data provide the first functional evidence that Plekha7 may contribute to blood pressure regulation and cardiovascular function through its effects on the vasculature.

Evaluating the Effects of Surotomycin Treatment on Clostridium difficile Toxin A and B Production, Immune Response, and Morphological Changes.

Surotomycin is a cyclic lipopeptide in development for Clostridium difficile-associated diarrhea. This study aimed to assess the impact of surotomycin exposure on C. difficile toxin A and B concentrations and the associated changes in immune response in comparison to vancomycin and metronidazole. Time-kill curve assays were performed using strain R20291 (BI/NAP1/027) at supra-MICs (4× and 40×) and sub-MICs (0.5×) of surotomycin and comparators. Following treatment, CFU counts, toxin A and B concentrations, and cellular morphological changes using scanning electron microscopy were examined. Inflammatory response was determined by measuring interleukin-8 (IL-8) concentrations from polarized Caco-2 cells exposed to antibiotic-treated C. difficile growth media. Supra-MICs (4× and 40×) of surotomycin resulted in a reduction of vegetative cells over 72 h (4-log difference, P < 0.01) compared to controls. These results correlated with decreases of 77% and 68% in toxin A and B production at 48 h, respectively (P < 0.005, each), which resulted in a significant reduction in IL-8 concentration compared to controls. Similar results were observed with comparator antibiotics. Bacterial cell morphology showed that the cell wall was broken apart by surotomycin treatment at supra-MICs while sub-MIC studies showed a "deflated" phenotype plus a rippling effect. These results suggest that surotomycin has potent killing effects on C. difficile that results in reduced toxin production and attenuates the immune response similar to comparator antibiotics. The morphological data also confirm observations that surotomycin is a membrane-active antibiotic.

The NRF2 knockout rat: a new animal model to study endothelial dysfunction, oxidant stress, and microvascular rarefaction.

Nuclear factor (erythroid-derived 2)-like-2 (NRF2) is a master antioxidant and cell protective transcription factor that upregulates antioxidant defenses. In this study we developed a strain of Nrf2 null mutant rats to evaluate the role of reduced NRF2-regulated antioxidant defenses in contributing to endothelial dysfunction and impaired angiogenic responses during salt-induced ANG II suppression. Nrf2(-/-) mutant rats were developed using transcription activator-like effector nuclease technology in the Sprague-Dawley genetic background, and exhibited a 41-bp deletion that included the start codon for Nrf2 and an absence of immunohistochemically detectable NRF2 protein. Expression of mRNA for the NRF2-regulated indicator enzymes heme oxygenase-1, catalase, superoxide dismutase 1, superoxide dismutase 2, and glutathione reductase was significantly lower in livers of Nrf2(-/-) mutant rats fed high salt (HS; 4% NaCl) for 2 wk compared with wild-type controls. Endothelium-dependent dilation to acetylcholine was similar in isolated middle cerebral arteries (MCA) of Nrf2(-/-) mutant rats and wild-type littermates fed low-salt (0.4% NaCl) diet, and was eliminated by short-term (3 days) HS diet in both strains. Low-dose ANG II infusion (100 ng/kg sc) reversed salt-induced endothelial dysfunction in MCA and prevented microvessel rarefaction in wild-type rats fed HS diet, but not in Nrf2(-/-) mutant rats. The results of this study indicate that suppression of NRF2 antioxidant defenses plays an essential role in the development of salt-induced oxidant stress, endothelial dysfunction, and microvessel rarefaction in normotensive rats and emphasize the potential therapeutic benefits of directly upregulating NRF2-mediated antioxidant defenses to ameliorate vascular oxidant stress in humans.

Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.

OBJECTIVES: Ridinilazole (SMT19969) is a narrow-spectrum, non-absorbable antimicrobial with activity against Clostridium difficile undergoing clinical trials. The purpose of this study was to assess the pharmacological activity of ridinilazole and assess the effects on cell morphology. METHODS: Antibiotic killing curves were performed using the epidemic C. difficile ribotype 027 strain, R20291, using supra-MIC (4× and 40×) and sub-MIC (0.125×, 0.25× and 0.5×) concentrations of ridinilazole. Following exposure, C. difficile cells were collected for cfu counts, toxin A and B production, and morphological changes using scanning electron and fluorescence microscopy. Human intestinal cells (Caco-2) were co-incubated with ridinilazole-treated C. difficile growth medium to determine the effects on host inflammatory response (IL-8). RESULTS: Treatment at supra-MIC concentrations (4× and 40× MIC) of ridinilazole resulted in a significant reduction in vegetative cells over 72 h (4 log difference, P < 0.01) compared with controls without inducing spore formation. These results correlated with a 75% decrease in toxin A production (P < 0.05) and a 96% decrease in toxin B production (P < 0.05). At sub-MIC levels (0.5× MIC), toxin A production was reduced by 91% (P < 0.01) and toxin B production was reduced by 100% (P < 0.001), which resulted in a 74% reduction in IL-8 release compared with controls (P < 0.05). Sub-MIC (0.5×)-treated cells formed filamentous structures ∼10-fold longer than control cells. Following fluorescence labelling, the cell septum was not forming in sub-MIC-treated cells, yet the DNA was dividing. CONCLUSIONS: Ridinilazole had robust killing effects on C. difficile that significantly reduced toxin production and attenuated the inflammatory response. Ridinilazole also elicited significant cell division effects suggesting a potential mechanism of action.

A novel method for imaging the pharmacological effects of antibiotic treatment on Clostridium difficile.

Clostridium difficile is a significant cause of nosocomial-acquired infection that results in severe diarrhea and can lead to mortality. Treatment options for C. difficile infection (CDI) are limited, however, new antibiotics are being developed. Current methods for determining efficacy of experimental antibiotics on C. difficile involve antibiotic killing rates and do not give insight into the drug's pharmacologic effects. Considering this, we hypothesized that by using scanning electron microscopy (SEM) in tandem to drug killing curves, we would be able to determine efficacy and visualize the phenotypic response to drug treatment. To test this hypothesis, supraMIC kill curves were conducted using vancomycin, metronidazole, fidaxomicin, and ridinilazole. Following collection, cells were either plated or imaged using a scanning electron microscope (SEM). Consistent with previous reports, we found that the tested antibiotics had significant bactericidal activity at supraMIC concentrations. By SEM imaging and using a semi-automatic pipeline for image analysis, we were able to determine that vancomycin and to a lesser extent fidaxomicin and ridinilazole significantly affected the cell wall, whereas metronidazole, fidaxomicin, and ridinilazole had significant effects on cell length suggesting a metabolic effect. While the phenotypic response to drug treatment has not been documented previously in this manner, the results observed are consistent with the drug's mechanism of action. These techniques demonstrate the versatility and reliability of imaging and measurements that could be applied to other experimental compounds. We believe the strategies laid out here are vital for characterizing new antibiotics in development for treating CDI.

Innate inflammatory response and immunopharmacologic activity of micafungin, caspofungin, and voriconazole against wild-type and FKS mutant Candida glabrata isolates.

The direct or indirect interactions that antifungals have with the host immune response may play a significant role in defining their activity in vivo. However, the impact that acquired antifungal resistance has on the immunopharmacologic activity of antifungals is not well described. We assessed the immunopharmacologic activity of caspofungin, micafungin, and voriconazole among isolates of Candida glabrata with or without FKS-mediated echinocandin resistance. Clinical bloodstream isolates of C. glabrata from patients who did (n = 5) or did not (n = 3) develop persistent candidemia and who did (n = 2) or did not (n = 11) harbor FKS gene mutations were included. A cell-based assay was used to compare differences in macrophage activation among isolates when grown in the presence or absence of subinhibitory concentrations of caspofungin, micafungin, or voriconazole. In the absence of antifungals, macrophage activation was significantly lower for index C. glabrata isolates obtained from persistent candidemia patients than for those from nonpersistent patients (33% versus 79% increase over negative controls, respectively; P < 0.01). Growth of isolates possessing wild-type FKS genes in subinhibitory concentrations of micafungin or caspofungin, but not voriconazole, significantly increased macrophage inflammatory responses compared to untreated controls (1.25- to 2.75-fold increase, P < 0.01). For isolates harboring the FKS2 hot spot 1 (HS1) S663P mutation, however, a significant increase was observed only with micafungin treatment (1.75-fold increase versus negative control, P < 0.01). Macrophage activation correlated with the level of unmasking of ß-glucan in the cell wall. The diminished macrophage inflammatory response to isolates that caused persistent candidemia and differential immunopharmacologic activity of echinocandins among FKS mutants suggest that certain strains of C. glabrata may have a higher propensity for immunoevasion and development of antifungal resistance during treatment.

Comparison of Risk Stratification Approaches to Identify Patients with Clostridioides difficile Infection at Risk for Multidrug-Resistant Organism Gut Microbiota Colonization.

INTRODUCTION: Multidrug-resistant organisms (MDRO) commonly colonize the gut microbiota of patients with Clostridioides difficile infection (CDI). This increases the likelihood of systemic infections with these MDROs. To help guide MDRO screening and/or empiric antibiotic therapy, we derived and compared predictive indices for MDRO gut colonization in patients with CDI. METHODS: This was a multicenter, retrospective cohort study of adult patients with CDI from July 2017 to April 2018. Stool samples were screened for MDRO via growth and speciation on selective antibiotic media and confirmed using resistance gene polymerase chain reaction. A regression-based risk score for MDRO colonization was constructed. Predictive performance via area under the receiver operating characteristic curve (aROC) of this index was compared with two other simplified risk stratification approaches: (1) prior healthcare exposure and/or high-CDI risk antibiotics; (2) number of prior high-CDI risk antibiotics. RESULTS: 50 (20.8%) of 240 included patients had MDRO colonization; 35 (14.6%) VRE, 18 (7.5%) MRSA, 2 (0.8%) CRE. Prior fluoroquinolone (aOR 2.404, 95% CI 1.095-5.279) and prior vancomycin (1.996, 95% CI 1.014-3.932) were independently associated with MDRO colonization while prior clindamycin (aOR 3.257, 95% CI 0.842-12.597) and healthcare exposure (aOR 2.138, 95% CI 0.964-4.740) were retained as explanatory variables. The regression-based risk score significantly predicted MDRO colonization (aROC 0.679, 95% CI 0.595-0.763), but was not significantly more predictive than prior healthcare exposure + prior antibiotics (aROC 0.646, 95% CI 0.565-0.727) or number of prior antibiotic exposures (aROC 0.642, 95% CI 0.554-0.730); P > 0.05 for both comparisons. CONCLUSION: A simplified approach using prior healthcare exposure and receipt of prior antibiotics known to increase CDI risk identified patients at risk for MDRO gut microbiome colonization as effectively as individual patient/antibiotic risk modeling.

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

Objectives: To characterize a bla CMY variant associated with ceftazidime-avibactam (CZA) resistance from a serially collected Escherichia coli isolate. Methods: A patient with an intra-abdominal infection due to recurrent E. coli was treated with CZA. On day 48 of CZA therapy, E. coli with a CZA MIC of >256 mg/L was identified from abdominal drainage. Illumina WGS was performed on all isolates to identify potential resistance mechanisms. Site-directed mutants of CMY ß-lactamase were constructed to identify amino acid residues responsible for CZA resistance. Results: WGS revealed that all three isolates were E. coli ST410. The CZA-resistant strain uniquely acquired a novel CMY ß-lactamase gene, herein called bla CMY-185 , harbored on an IncIγ-type 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 CZA resistance with an MIC of 32 mg/L. Single CMY-2 mutants did not confer reduced CZA susceptibility. However, double and triple mutants containing N346Y previously associated with CZA resistance in other AmpC enzymes, conferred CZA 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 the steric hindrance between the side chain of Y346 and the sulfate group of avibactam. Conclusion: We identified CZA 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 CZA resistance.