Mechanism of action and initial evaluation of a membrane active all-D-enantiomer antimicrobial peptidomimetic.

Development of therapy against infections caused by antibiotic-resistant pathogens is a major unmet need in contemporary medicine. In previous work, our group chemically modified an antimicrobial peptidomimetic motif for targeted applications against cancer and obesity. Here, we show that the modified motif per se is resistant to proteolytic degradation and is a candidate antiinfective agent. We also show that the susceptibility of microorganisms to the drug is independent of bacterial growth phase. Moreover, this peptidomimetic selectively interferes with the integrity and function of the microbial surface lipid bilayer, data indicative that bacterial death results from membrane disruption followed by dissipation of membrane potential. Finally, we demonstrate two potential translational applications: use against biofilms and synergy with antibiotics in use. In summary, we introduce the mechanism of action and the initial evaluation of a prototype drug and a platform for the development of D-enantiomer antimicrobial peptidomimetics that target bacterial membranes of certain gram-negative problem pathogens with promising translational applications.

FKS mutant Candida glabrata: risk factors and outcomes in patients with candidemia.

BACKGROUND: Echinocandins are recommended for Candia glabrata candidemia. Mutations in the FKS1 and FKS2 genes are associated with echinocandin resistance. Few studies have assessed risk factors for FKS mutant isolates and outcomes in patients receiving micafungin treatment. METHODS: Patients with C. glabrata bloodstream infection admitted to a large, tertiary care hospital between 2009 and 2012 were included in this study. For each isolate, FKS1 and FKS2 genes were sequenced to identify mutations. Risk factors for FKS mutations and treatment outcomes in patients receiving an echinocandin were assessed using multivariate logistic regression. RESULTS: Seventy-two patients were included in the study of which 13 (18%) had an FKS mutant isolate. The only significant predictor for FKS mutations was prior echinocandin exposure (odds ratio [OR], 19.9; 95% confidence interval [CI], 4.7-84.7; P ≤ .01). Treatment failure occurred in 17 (30%) of 57 patients who received an echinocandin and was more common in patients with FKS mutants (6 of 10; 60%) compared with non-FKS mutants (11 of 47; 23%). Underlying gastrointestinal disorder (OR, 4.7; 95% CI, 1.1-20.9; P = .04) and prior echinocandin exposure (OR, 8.3; 95% CI, 1.7-40.4; P ≤ .01) were independent predictors of echinocandin treatment failure. Treatment response and echinocandin minimum inhibitory concentrations varied among specific FKS mutations. CONCLUSIONS: FKS mutations were identified in 18% of 72 patients with C. glabrata candidemia. Common risk factors for FKS mutant isolates included previous echinocandin exposure, which also influenced response rates.

Clinical practice patterns in hospitalized patients at risk for invasive candidiasis: role of antifungal stewardship programs in an era of rapid diagnostics.

BACKGROUND: Rapid diagnostic tests for Candida are becoming available that may supplement traditional microbiological identification. OBJECTIVE: Assess clinical practice patterns in patients with or at risk of candidiasis who may benefit from the use of rapid diagnostic tests. METHODS: This was a prospective cohort study of patients with candidemia or receiving systemic antifungals conducted at a university-affiliated tertiary care hospital. Time to initiation of therapy, Candida species, time to identification, and indications for antifungal use were assessed. RESULTS: A total of 162 patients with candidemia aged 58 ± 17 years were identified. Average time to yeast identification yeast was 2.2 ± 1.3 days and varied by Candida species (range = 0.6-7.9 days). Average time for patient to start antifungal therapy was 3.5 ± 2.1 days. In Monte Carlo simulations, average time to initiation of antifungal therapy was 0.6 ± 0.2 days for T2Candida, 2.6 ± 1.3 days for PNA-FISH (fluorescence in situ hybridization using peptide nucleic acid probes), and 2.5 ± 1.4 days for MALDI-TOF (matrix-assisted laser desorption/ionization time of flight). Use of T2Candida on the day of the blood culture collection resulted in 3136 to 6078 fewer doses of echinocandins annually per 5000 patients. CONCLUSION: Many interventions are possible for antifungal stewardship programs to improve care of patients at risk for systemic candidiasis, including rapid identification of yeast species and limiting unnecessary antifungal agents. Technology enabling rapid diagnosis of Candida will be paramount to appropriate, cost-effective treatment of patients with or at risk for candidiasis.

An evaluation of multiple phenotypic screening methods for Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae may display MICs to carbapenems within susceptible or intermediate ranges, prompting confirmatory testing. Four phenotypic methods to detect KPC producers were evaluated against a collection of clinical Enterobacteriaceae isolates. Meropenem-phenylboronic acid double disk synergy testing demonstrated the best performance with 100% sensitivity and specificity.

Detection of invasive Bartonella infections with next-generation sequencing of microbial cell-free DNA.

We report 9 patients with invasive Bartonella infections, including 5 with endocarditis, who were diagnosed with microbial cell-free DNA next-generation sequencing and Bartonella serology studies. Diagnosis with plasma mcfDNA NGS enabled a faster clinical and laboratory diagnosis in 8 patients. Prompt diagnosis impacted antibiotic management in all 9 patients.

The impact of a blood-culture diagnostic stewardship intervention on utilization rates and antimicrobial stewardship.

Blood-culture overutilization is associated with increased cost and excessive antimicrobial use. We implemented an intervention in the adult intensive care unit (ICU), combining education based on the DISTRIBUTE algorithm and restriction to infectious diseases and ICU providers. Our intervention led to reduced blood-culture utilization without affecting safety metrics.

Impact of prior inappropriate fluconazole dosing on isolation of fluconazole-nonsusceptible Candida species in hospitalized patients with candidemia.

Prior use of fluconazole is a modifiable risk factor for the isolation of fluconazole-nonsusceptible Candida species. Optimization of the use of fluconazole by appropriate dose or duration may be able to minimize the risk of resistance. The objective of this study was to evaluate the effects of prior fluconazole therapy, including the dose and duration, on fluconazole susceptibility among Candida species isolated from hospitalized patients with candidemia. A retrospective cohort study of hospitalized patients with a first occurrence of nosocomial candidemia, from 2006 to 2009, was carried out. The relationships between the initial dose and duration of prior fluconazole therapy and the isolation of fluconazole-nonsusceptible Candida species were assessed. An initial fluconazole dose greater than 2 mg/kg and less than 6 mg/kg of body weight was considered suboptimal. A total of 177 patients were identified, of whom 133 patients aged 61 ± 16 years (56% male, 51% Caucasian, 51% with an APACHE II score of ≥ 15) had candidemia more than 2 days after the hospital admission day. Nine of 107 (8%) patients with fluconazole-susceptible Candida species and 9 of 26 (35%) patients with fluconazole-nonsusceptible Candida species had prior fluconazole exposure (risk ratio [RR], 3.03; 95% confidence interval [95% CI], 1.57 to 5.86; P, 0.0022). Preexposure with an initial dose of fluconazole greater than 2 mg/kg and less than 6 mg/kg occurred in 3 of 9 (33%) and 8 of 9 (89%) patients with fluconazole-susceptible and fluconazole-nonsusceptible Candida species, respectively (P, 0.0498). We conclude that patients with candidemia due to fluconazole-nonsusceptible Candida species were more likely to have received prior fluconazole therapy. Suboptimal initial dosing of prior fluconazole therapy was associated with candidemia with fluconazole-nonsusceptible Candida species.

An integrated approach to evaluate different tetracycline derivatives for formulary decisions.

PURPOSE: Stenotrophomonas maltophilia has emerged as a critical opportunistic pathogen associated with significant morbidity and mortality. Tetracycline derivatives have been recognized as alternative treatment options, but they have varied pharmacokinetic properties. An integrated approach to different tetracycline derivatives for formulary decisions is reported. METHODS: The minimum inhibitory concentration (MIC) data from clonally diverse bloodstream S. maltophilia isolates were examined, along with the pharmacokinetic profiles of 4 tetracycline derivatives, to predict achievable pharmacodynamic exposures with standard intravenous dosing regimens. Antimicrobial therapy was assessed using the ratio of daily drug acquisition cost relative to the ratio of the free-drug area under the time-concentration curve (fAUC) to minimum inhibitory concentration (MIC) for 90% of isolates (fAUC/MIC90). RESULTS: In our analysis, minocycline had the greatest fAUC/MIC90. Doxycycline was the most financially preferred agent, as calculated using 2020 average wholesale price for base-case estimates of drug acquisition cost. CONCLUSION: An integrated evaluation for antimicrobial formulary decision-making addressed local susceptibility data, pharmacokinetics, pharmacodynamics, dosing regimens, and drug acquisition costs. This comprehensive method is more objective than the conventional approach and warrants validation.

MIC profiling of ceftazidime/avibactam against two carbapenemase-producing Klebsiella pneumoniae isolates.

OBJECTIVES: The aim of this study was to correlate the results of a modified susceptibility testing method with outcomes of ceftazidime/avibactam (CAZ/AVI) therapy. METHODS: Two bloodstream K. pneumoniae isolates (CAZ/AVI-susceptible) from an abdominal source were recovered from two unrelated patients. Both patients were treated with CAZ/AVI but had discordant outcomes: KP118 (eradication within 24 h) and KP286 (persistent bacteraemia for over 30 days). Carbapenemase production in the two isolates was confirmed by Carba NP test. The CAZ minimum inhibitory concentration (MIC) was determined with escalating AVI concentrations (0-16 mg/L). The concentration-response was characterised by the sigmoid inhibitory maximum effect model. The best-fit parameter values were used to predict %T > MICi associated with CAZ/AVI exposures expected in peritoneal fluid after standard dosing (2.5 g every 8 h). These CAZ/AVI exposures were simulated in a hollow-fibre infection model (HFIM), and the bacterial responses were correlated with observed clinical outcomes. RESULTS: The AVI-dependent reduction in CAZ MIC was well characterised in both bacterial isolates (r2 ≥ 0.98). In the HFIM, sustained suppression of KP118 (T > MICi = 100%) was observed over 5 days, but not with KP286 (T > MICi < 100%). These observations are consistent with the clinical course of the patients. CONCLUSIONS: The discordant patient outcomes could be potentially explained by MIC profiling of CAZ/AVI. This method appears to be more robust than conventional susceptibility testing in predicting positive clinical outcome of CAZ/AVI therapy, and the clinical utility of this approach should be further investigated.

Prevalence of extended-spectrum beta-lactamase and carbapenemase-producing bloodstream isolates of Klebsiella pneumoniae in a tertiary care hospital.

To improve prescribing of empiric therapy, the local molecular epidemiology of extended-spectrum beta-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPCs) in bloodstream isolates of K. pneumoniae were evaluated. Isolates resistant to third generation cephalosporins were screened phenotypically for ESBLs and carbapenemases, and subsequently confirmed by PCR for the presence of ESBL (blaTEM, blaSHV and blaCTX-M) and carbapenemase (blaKPC, blaVIM, blaNDM and blaOXA-48) genes. Hydrolytic activity (functional gene expression) was quantified using a nitrocefin degradation assay and correlated to ceftazidime or meropenem MIC. Clonality was assessed by repetitive element-based PCR. Beta-lactamases were functionally expressed in 13 isolates (15.5%); 7 (53.8%) harboured blaCTX-M-15 and 6 (46.2%) carried the blaKPC-2 gene. Correlation of hydrolytic activity to MIC yielded a coefficient of 98% for isolates expressing ESBLs alone and 56% for carbapenemase producers. Four unique ESBL-expressing clones and five carbapenem-resistant clones were identified. All 13 resistant isolates were susceptible to ceftazidime/avibactam (MIC ≤ 8/4 mg/L).

Prevalence and predictors of spontaneous bacterial peritonitis due to ceftriaxone-resistant organisms at a large tertiary centre in the USA.

OBJECTIVES: The epidemiology of spontaneous bacterial peritonitis (SBP) due to ceftriaxone-resistant organisms has not been well studied in the USA. The primary objective of this study was to assess the prevalence and predictors of ceftriaxone-resistant SBP at a large US tertiary-care centre. METHODS: This 1:1:4 case-case-control study included 141 adults with liver cirrhosis admitted from November 2011 to March 2016. Case group 1 were patients with SBP with a ceftriaxone-resistant organism (n=21). Case group 2 were patients with SBP with a ceftriaxone-susceptible organism (n=26). The control group were patients without SBP (n=94). Multiple logistic regression analysis was used to identify predictors of ceftriaxone-resistant SBP. RESULTS: Fifty isolates were identified from 47 patients with culture-positive SBP (case groups 1 and 2). Of these 50 isolates, 32 (64%) were Gram-negatives [mostly Enterobacteriaceae (91%)], 15 (30%) were Gram-positives and 3 (6%) were Candida spp. The prevalence of ceftriaxone resistance in patients with culture-positive SBP was 45% (21/47). The most common ceftriaxone-resistant organisms were ESBL-producing Enterobacteriaceae (45%). Independent predictors of ceftriaxone-resistant SBP included duration of ß-lactam therapy in the past 90days (aOR=1.07, 95% CI 1.01-1.13) and recent invasive gastrointestinal procedure (aOR=12.47, 95% CI 2.74-56.67). CONCLUSIONS: The prevalence of ceftriaxone-resistant SBP was significant at a US tertiary centre. Local epidemiological data and identification of risk factors associated with ceftriaxone-resistant SBP, e.g. increased usage of previous ß-lactam therapy and invasive gastrointestinal procedure, may help clinicians identify patients requiring alternative empirical antibiotics.

Mycobacterium bovis BCG vaccination modulates TNF-alpha production after pulmonary challenge with virulent Mycobacterium tuberculosis in guinea pigs.

Tumor necrosis factor-alpha (TNF-alpha) plays critical and opposing roles in the pathogenesis of tuberculosis (TB). We examined the effects of Mycobacterium bovis BCG vaccination on TNF-alpha production in three distinct guinea pig leukocyte populations before and after pulmonary infection with M. tuberculosis H37Rv. Following BCG vaccination alone, and following challenge, bronchoalveolar lavage cells (BALC), resident peritoneal cells (PC), and splenocytes (SPC) were stimulated with purified protein derivative (PPD). Before virulent challenge, BCG vaccination clearly enhanced the ability of BALC, PC and SPC to produce TNF-alpha in response to PPD stimulation ex vivo. Following challenge, the TNF-alpha production of all three leukocyte populations from BCG-vaccinated animals remained relatively constant at pre-challenged levels. In sharp contrast, 5 weeks post-challenge, all three leukocyte populations from unvaccinated animals produced very high amounts of TNF-alpha in response to PPD. Three weeks post-challenge, SPC from one of the unvaccinated animals produced higher levels of TNF-alpha but the others produced lower levels of TNF-alpha than BCG-vaccinated animals. As expected, BCG vaccination reduced the levels of virulent mycobacteria in both the lungs and spleens. Thus, BCG vaccination allows guinea pigs to modulate TNF-alpha levels in conjunction with a reduction in bacillary loads in their tissues.

Cross-reaction of anti-human CD monoclonal antibodies on guinea pig cells: a summary of the guinea pig section of the HLDA8 animal homologues data.

A panel of 377 commercially available monoclonal antibodies (mAbs) specific for a total of 144 CD antigens was submitted to the animal homologue section of the Eighth International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies in a range of vertebrate species. Each of the mAbs in this study was screened for positive reactivity with guinea pig splenocytes by flow cytometry. In the first phase of this study 36 of the total 367 mAbs (9.81%) cross-reacted with splenocyte surface molecules. The majority (26 of 36) of these cross-reactive mAbs were analysed further to confirm appropriate cell subset expression by two-color immunofluorescence. Our results indicate that 15 anti-human CD9, CD10, CD14, CD20 (two clones), CD22, CD25, CD29 (two clones), CD32, CD47 (two clones), CD49d, CD49e, and CD86 mAbs exhibit clear cross-reactivity with guinea pig splenocytes. These mAb can potentially be added to the limited repertoire of reagents available for studies in this model system. This data clearly indicates that mouse anti-human CD mAb guinea pig cross-reactions have been defined and that an aim of this HLDA8 section has been fulfilled, i.e., to identify mAbs which recognize conserved, species-independent CD epitopes. These results will contribute to the availability of mAbs and tools in veterinary medicine and immunology.

Disseminated coccidioidomycosis with multifocal musculoskeletal disease involvement.

We report a rare case of disseminated coccidioidomycosis with multifocal musculoskeletal involvement. The patient presented to the emergency department with left shoulder pain and swelling. Magnetic resonance imaging of the left shoulder revealed enhancing soft tissue masses, bony lesions, and fluid collections in and around the glenohumeral joint with involvement of the proximal humerus, glenoid, and rotator cuff musculature. Multiple additional areas of involvement were subsequently discovered. Fungal cultures confirmed coccidioidomycosis infection at all surgical sites with superimposed polymicrobial bacterial infection in the left shoulder.

Activity of minocycline against Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae clinical isolates, with comparison to doxycycline and tigecycline.

We examined the in vitro activity of minocycline against 103 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae isolates and found approximately half had susceptible (26%) or intermediate (26%) MICs. For a subset of 35 isolates, susceptibility was highest to tigecycline (71% FDA vs. 20% EUCAST) followed by minocycline (14%) and then doxycycline (6%).

Evaluating the role of tumor necrosis factor-alpha in experimental pulmonary tuberculosis in the guinea pig.

Tumor necrosis factor-alpha (TNF-alpha) is suggested to play multiple roles in immune and pathologic responses in tuberculosis. In this study, we have developed a system for the expression of recombinant guinea pig TNF-alpha (rgpTNF-alpha). Using rgpTNF-alpha along with neutralizing anti-rgpTNF-alpha antiserum, we tested the effect of modulating the levels of TNF-alpha on antigen-specific T cell proliferation in splenocytes. By neutralizing TNF-alpha in the supernatant of PPD-pulsed splenocytes with anti-rgpTNF-alpha, we observed hyperproliferation. Conversely, the addition of rgpTNF-alpha resulted in a significant suppression of PPD-induced lymphoproliferation. In addition, when unvaccinated and BCG-vaccinated guinea pigs were treated with polyclonal rgpTNF-alpha antiserum throughout the first 3 weeks following low-dose, pulmonary infection with Mycobacterium tuberculosis H37Rv, we observed splenomegaly in BCG-vaccinated guinea pigs. We also detected higher levels of splenic granuloma organization in the non-vaccinated group as well as a significant number of plasma cells associated with granulomata from the BCG-vaccinated group. These results suggest that modulating the availability of TNF-alpha in BCG-vaccinated guinea pigs can lead to immuno-dysregulation and, perhaps, the inappropriate enhancement of humoral immunity. Conversely, abrogating TNF-alpha activity in the context of a hyperinflammatory response in non-vaccinated guinea pigs may, in fact, rescue them from immunopathological consequences of overproducing TNF-alpha.

Outcomes and Risk Factors for Mortality among Patients Treated with Carbapenems for Klebsiella spp. Bacteremia.

BACKGROUND: Extensive dissemination of carbapenemase-producing Enterobacteriaceae has led to increased resistance among Klebsiella species. Carbapenems are used as a last resort against resistant pathogens, but carbapenemase production can lead to therapy failure. Identification of risk factors for mortality and assessment of current susceptibility breakpoints are valuable for improving patient outcomes. AIM: The objective of this study was to evaluate outcomes and risk factors for mortality among patients treated with carbapenems for Klebsiella spp. bacteremia. METHODS: Patients hospitalized between 2006 and 2012 with blood cultures positive for Klebsiella spp. who received ≥ 48 hours of carbapenem treatment within 72 hours of positive culture were included in this retrospective study. Patient data were retrieved from electronic medical records. Multivariate logistic regression was used to identify risk factors for 30-day hospital mortality. RESULTS: One hundred seven patients were included. The mean patient age was 61.5 years and the median APACHE II score was 13 ± 6.2. Overall, 30-day hospital mortality was 9.3%. After adjusting for confounding variables, 30-day mortality was associated with baseline APACHE II score (OR, 1.17; 95% CI, 1.01-1.35; P = 0.03), length of stay prior to index culture (OR, 1.03; 95% CI, 1.00-1.06; P = 0.04), and carbapenem non-susceptible (imipenem or meropenem MIC > 1 mg/L) infection (OR, 9.08; 95% CI, 1.17-70.51; P = 0.04). CONCLUSIONS: Baseline severity of illness and length of stay prior to culture were associated with 30-day mortality and should be considered when treating patients with Klebsiella bacteremia. These data support the change in carbapenem breakpoints for Klebsiella species.