Exploring avibactam and relebactam inhibition of Klebsiella pneumoniae carbapenemase D179N variant: role of the Ω loop-held deacylation water.

Klebsiella pneumoniae carbapenemase-2 (KPC-2) presents a clinical threat as this ß-lactamase confers resistance to carbapenems. Recent variants of KPC-2 in clinical isolates contribute to concerning resistance phenotypes. Klebsiella pneumoniae expressing KPC-2 D179Y acquired resistance to the ceftazidime/avibactam combination affecting both the ß-lactam and the ß-lactamase inhibitor yet has lowered minimum inhibitory concentrations for all other ß-lactams tested. Furthermore, Klebsiella pneumoniae expressing the KPC-2 D179N variant also manifested resistance to ceftazidime/avibactam yet retained its ability to confer resistance to carbapenems although significantly reduced. This structural study focuses on the inhibition of KPC-2 D179N by avibactam and relebactam and expands our previous analysis that examined ceftazidime resistance conferred by D179N and D179Y variants. Crystal structures of KPC-2 D179N soaked with avibactam and co-crystallized with relebactam were determined. The complex with avibactam reveals avibactam making several hydrogen bonds, including with the deacylation water held in place by Ω loop. These results could explain why the KPC-2 D179Y variant, which has a disordered Ω loop, has a decreased affinity for avibactam. The relebactam KPC-2 D179N complex revealed a new orientation of the diazabicyclooctane (DBO) intermediate with the scaffold piperidine ring rotated ~150° from the standard DBO orientation. The density shows relebactam to be desulfated and present as an imine-hydrolysis intermediate not previously observed. The tetrahedral imine moiety of relebactam interacts with the deacylation water. The rotated relebactam orientation and deacylation water interaction could potentially contribute to KPC-mediated DBO fragmentation. These results elucidate important differences that could aid in the design of novel ß-lactamase inhibitors.

Acinetobacter quorum sensing contributes to inflammation-induced inhibition of orthopaedic implant osseointegration.

Implant infection impairs osseointegration of orthopaedic implants by inducing inflammation. Acinetobacter spp. are increasingly prevalent multi-drug resistant bacteria that can cause osteomyelitis. Acinetobacter spp. can also cause inflammation and thereby inhibit osseointegration in mice. The purpose of the present study was to investigate the role of quorum sensing in this context. Therefore, wild-type bacteria were compared with an isogenic abaI mutant defective in quorum sensing in a murine osseointegration model. The abaI quorum- sensing mutant affected significantly less osseointegration and interleukin (IL) 1ß levels, without detectably altering other pro-inflammatory cytokines. Wild-type bacteria had fewer effects on IL1 receptor (IL1R)-/- mice. These results indicated that quorum sensing in Acinetobacter spp. contributed to IL1ß induction and the resultant inhibition of osseointegration in mice. Moreover, targeting the Gram-negative acyl-homoserine lactone quorum sensing may be particularly effective for patients with Acinetobacter spp. infections.

Structural Characterization of the D179N and D179Y Variants of KPC-2 ß-Lactamase: Ω-Loop Destabilization as a Mechanism of Resistance to Ceftazidime-Avibactam.

Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) present a global clinical threat, as these ß-lactamases confer resistance to carbapenems and oxyimino-cephalosporins. Recent clinically identified KPC variants with substitutions at Ambler position D179, located in the Ω loop, are resistant to the ß-lactam/ß-lactamase inhibitor combination ceftazidime-avibactam, but susceptible to meropenem-vaborbactam. To gain insights into ceftazidime-avibactam resistance conferred by D179N/Y variants of KPC-2, crystal structures of these variants were determined. The D179N KPC-2 structure revealed that the change of the carboxyl to an amide moiety at position 179 disrupted the salt bridge with R164 present in wild-type KPC-2. Additional interactions were disrupted in the Ω loop, causing a decrease in the melting temperature. Shifts originating from N179 were also transmitted toward the active site, including ∼1-Å shifts of the deacylation water and interacting residue N170. The structure of the D179Y KPC-2 ß-lactamase revealed more drastic changes, as this variant exhibited disorder of the Ω loop, with other flanking regions also being disordered. We postulate that the KPC-2 variants can accommodate ceftazidime because the Ω loop is displaced in D179Y or can be more readily displaced in D179N KPC-2. To understand why the ß-lactamase inhibitor vaborbactam is less affected by the D179 variants than avibactam, we determined the crystal structure of D179N KPC-2 in complex with vaborbactam, which revealed wild-type KPC-2-like vaborbactam-active site interactions. Overall, the structural results regarding KPC-2 D179 variants revealed various degrees of destabilization of the Ω loop that contribute to ceftazidime-avibactam resistance, possible substrate-assisted catalysis of ceftazidime, and meropenem and meropenem-vaborbactam susceptibility.

Clinical and biochemical markers in CIPN: A reappraisal.

The increased survival of cancer patients has raised growing public health concern on associated long-term consequences of antineoplastic treatment. Chemotherapy-induced peripheral neuropathy (CIPN) is a primarily sensory polyneuropathy, which may be accompanied by pain, autonomic disturbances, and motor deficit. About 70% of treated cancer patients might develop CIPN during or after the completion of chemotherapy, and in most of them such complication persists after six months from the treatment. The definition of the potential risk of development and resolution of CIPN according to a clinical and biochemical profile would be certainly fundamental to tailor chemotherapy regimen and dosage on individual susceptibility. In recent years, patient-reported and clinician-related tools along with quality of life instruments have been featured as primary outcomes in clinical setting and randomized trials. New studies on metabolomics markers are further pursuing accurate and easily accessible indicators of peripheral nerve damage. The aim of this review is to outline the strengths and pitfalls of current knowledge on CIPN, and to provide a framework for future potential developments of standardized protocols involving clinical and biochemical markers for CIPN assessment and monitoring.

Blood molecular biomarkers for chemotherapy-induced peripheral neuropathy: From preclinical models to clinical practice.

Chemotherapy-induced peripheral neuropathy (CIPN) has long been recognized as a clinically significant issue in patients treated with antineoplastic drugs. This common long-term toxic side-effect which negatively impacts the outcome of the disease can lead to disability and have detrimental effects on patients' quality of life. Since axonal injury is a prominent feature of CIPN, responsible for several sensory symptoms, including pain, sensory loss and hypersensitivity to mechanical and/or cold stimuli in the hands and feet, neurophysiological assessments remain the gold standard for clinical diagnosis of CIPN. Given the large impact of CIPN on cancer patients, there is increasing emphasis on biomarkers of adverse outcomes in safety assessment and translational research, to prevent permanent neuroaxonal damage. Since the results on reliable blood molecular markers for axonal degeneration are still controversial, here we provide a brief overview of blood molecular biomarkers used for assessing and/or predicting CIPN in preclinical and clinical settings.

Systemic involvement in adult-onset leukoencephalopathy with intracranial calcifications and cysts (Labrune syndrome) with a novel mutation of the SNORD118 gene.

BACKGROUND AND PURPOSE: Although Labrune syndrome is a well-known disorder characterized by a typical neuroradiological triad, namely leukoencephalopathy, intracranial calcifications and cysts, there are no reports of systemic involvement in this disorder. This paper attempts to describe a peculiar clinical manifestation related to a novel mutation in the SNORD118 gene. METHODS: Clinical examination, brain and total-body imaging, and neurophysiological and ophthalmological investigations were performed. Amplification of the SNORD118 gene and Sanger sequencing were integrated to investigate potential causative mutations. RESULTS: A 69-year-old woman, with a long history of episodes of vertigo and gait imbalance, was referred to our hospital for progressive cognitive and motor deterioration. Computed tomography and magnetic resonance imaging disclosed diffuse bilateral leukoencephalopathy in periventricular and deep white matter, widespread calcifications and numerous cysts in the brain, liver, pancreas and kidneys. The genetic analysis revealed two biallelic variants in the SNORD118 gene, one of which is novel (n.60G>C). CONCLUSIONS: This is the first report of adult-onset Labrune syndrome with an unusual systemic involvement presenting a novel mutation in the SNORD118 gene.

Epidemiology and genotypic characterisation of dissemination patterns of uropathogenic Escherichia coli in a community.

To characterise the dissemination patterns of uropathogenic Escherichia coli (UPEC) in a community, we conducted a study utilising molecular and fundamental descriptive epidemiology. The subjects, consisted of women having community-acquired acute urinary tract infection (UTI), were enrolled in the study from 2011 to 2012. UPEC isolates were subjected to antibacterial-susceptibility testing, O serogrouping, phylotyping, multilocus-sequence typing with phylogenetic-tree analysis and pulsed-field-gel electrophoresis (PFGE). From the 209 unique positive urinary samples 166 UPEC were isolated, of which 129 were fully susceptible to the tested antibiotics. Of the 53 sequence types (STs), the four most prevalent STs (ST95, ST131, ST73 and ST357) accounted for 60% of all UPEC strains. Antimicrobial resistance was less frequently observed for ST95 and ST73 than for the others. A majority of rare STs and a few common STs constituted the diversity pattern within the population structure, which was composed of the two phylogenetically distinct clades. Eleven genetically closely related groups were determined by PFGE, which accounted for 42 of the 166 UPEC isolates, without overt geo-temporal clustering. Our results indicate that a few major lineages of UPEC, selected by unidentified factors, are disseminated in this community and contribute to a large fraction of acute UTIs.

Dual ß-Lactam Combinations Highly Active against Mycobacterium abscessus Complex In Vitro.

As a consequence of a growing population of immunocompromised individuals, including transplant recipients and cystic fibrosis patients, there has been a dramatic increase in chronic infections caused by Mycobacterium abscessus complex (MABC) strains that are usually recalcitrant to effective antibiotic therapy. The recent rise of macrolide resistance in MABC has further complicated this clinical dilemma, dramatizing the need for novel agents. The repurposing of current antibiotics is one rapid path from discovery to patient care. In this study, we have discovered that dual ß-lactams, and specifically the combination of ceftazidime with either ceftaroline or imipenem, are synergistic and have clinically relevant activities, with MIC50s of 0.25 (ceftaroline with 100 µg/ml ceftazidime) and 0.5 µg/ml (imipenem with 100 µg/ml ceftazidime) against clinical MABC isolates. Similar synergy was observed in time-kill studies against the M. abscessus ATCC 19977 strain using clinically achievable concentrations of either imipenem (4 µg/ml) or ceftaroline (2 µg/ml), as the addition of ceftazidime at concentrations of ≥50 µg/ml showed a persistent bactericidal effect over 5 days. Treatment of THP-1 human macrophages infected with three different M. abscessus clinical isolates supported the in vitro findings, as the combination of 100 µg/ml ceftazidime and 0.125 µg/ml ceftaroline or 100 µg/ml ceftazidime and 0.25 µg/ml imipenem dramatically reduced the CFU counts to near baseline levels of infection. This study's finding that there is synergy between certain ß-lactam combinations against M. abscessus infection provides optimism toward identifying an optimum dual ß-lactam treatment regimen.IMPORTANCE The emergence of chronic MABC infections among immunocompromised populations and their inherent and acquired resistance to effective antibiotic therapy have created clinical challenges in advancing patients for transplant surgery and treating those with disease. There is an urgent need for new treatment regimens, and the repurposing of existing antibiotics provides a rapid strategy to advance a laboratory finding to patient care. Our recent discoveries that dual ß-lactams, specifically the combination of ceftazidime with ceftaroline or ceftazidime with imipenem, have significant in vitro MIC values and kill curve activities and are effective against infected THP-1 human macrophages provide optimism for a dual ß-lactam treatment strategy against MABC infections. The unexpected synergistic activities reported in this study create a new path of discovery to repurpose the large family of ß-lactam drugs.

Predictors of outcome in patients with severe sepsis or septic shock due to extended-spectrum ß-lactamase-producing Enterobacteriaceae.

PURPOSE: There are few data in the literature regarding sepsis or septic shock due to extended-spectrum ß-lactamases (ESBL)-producing Enterobacteriaceae (E). The aim of this study was to assess predictors of outcome in septic patients with bloodstream infection (BSI) caused by ESBL-E. METHODS: Patients with severe sepsis or septic shock and BSI due to ESBL-E were selected from the INCREMENT database. The primary endpoint of the study was the evaluation of predictors of outcome after 30 days from development of severe sepsis or septic shock due to ESBL-E infection. Three cohorts were created for analysis: global, empirical-therapy and targeted-therapy cohorts. RESULTS: 367 septic patients were analysed. Overall mortality was 43.9% at 30 days. Escherichia coli (62.4%) and Klebsiella pneumoniae (27.2%) were the most frequent isolates. ß-lactam/ß-lactamase inhibitor (BLBLI) combinations were the most empirically used drug (43.6%), followed by carbapenems (29.4%). Empirical therapy was active in vitro in 249 (67.8%) patients, and escalation of antibiotic therapy was reported in 287 (78.2%) patients. Cox regression analysis showed that age, Charlson Comorbidity Index, McCabe classification, Pitt bacteremia score, abdominal source of infection and escalation of antibiotic therapy were independently associated with 30-day mortality. No differences in survival were reported in patients treated with BLBLI combinations or carbapenems in empirical or definitive therapy. CONCLUSIONS: BSI due to ESBL-E in patients who developed severe sepsis or septic shock was associated with high 30-day mortality. Comorbidities, severity scores, source of infection and antibiotic therapy escalation were important determinants of unfavorable outcome.

New agents for the treatment of infections with Gram-negative bacteria: restoring the miracle or false dawn?

BACKGROUND: Antibiotic resistance in Gram-negative resistance has developed without a commensurate response in the successful development of antibiotic agents, though recent progress has been made. AIMS: This review aims to provide a summary of the existing evidence on efficacy, spectrum of activity and the development of resistance of new agents that have been licensed or have completed advanced clinical trials and that possess activity against resistant Gram-negative organisms. SOURCES: A review of the published literature via MEDLINE database was performed. Relevant clinical trials were identified with the aid of the clinicaltrials.gov registry. Further data were ascertained from review of abstracts from recent international meetings and pharmaceutical companies. CONTENT: Data on the mechanism of action, microbiological spectrum, clinical efficacy and development of resistance are reported for new agents that have activity against Gram-negative organisms. This includes the ß-lactam/ß-lactamase inhibitor combinations ceftazidime/avibactam, ceftolozane/tazobactam, imipenem/cilastatin/relebactam, meropenem/vaborbactam and aztreonam/avibactam; cefiderocol, a siderophore cephalosporin; plazomicin and eravacycline. IMPLICATIONS: The development of new agents with activity against multidrug-resistant Gram-negative pathogens has provided important therapeutic options for clinicians. Polymyxins appear to have been supplanted by new agents as first-line therapy for Klebsiella pneumoniae carbapenemase producers. Cefiderocol and ceftazidime/avibactam/aztreonam are promising options for metallo-ß-lactamase producers, and cefiderocol and ceftolozane/tazobactam for multiply resistant Pseudomonas aeruginosa, but definitive data showing clinical efficacy is as yet lacking. Reports of the development of resistance early after the release and use of new agents is of concern. Orally administered options and agents active effective against Acinetobacter baumannii are under-represented in clinical development.

In Vitro Discordance with In Vivo Activity: Humanized Exposures of Ceftazidime-Avibactam, Aztreonam, and Tigecycline Alone and in Combination against New Delhi Metallo-ß-Lactamase-Producing Klebsiella pneumoniae in a Murine Lung Infection Model.

The management of infections with New Delhi metallo-beta-lactamase-1 (NDM)-producing bacteria remains clinically challenging given the multidrug resistant (MDR) phenotype associated with these bacteria. Despite resistance in vitro, ceftazidime-avibactam previously demonstrated in vivo activity against NDM-positive Enterobacteriaceae Herein, we observed in vitro synergy with ceftazidime-avibactam and aztreonam against an MDR Klebsiella pneumoniae harboring NDM. In vivo, humanized doses of ceftazidime-avibactam monotherapy resulted in >2 log10 CFU bacterial reduction; therefore, no in vivo synergy was observed.

Successful treatment of a disseminated infection with extensively drug-resistant Klebsiella pneumoniae in a liver transplant recipient with a fosfomycin-based multidrug regimen.

Donor-derived infections with multidrug-resistant gram-negative bacteria are associated with poor outcomes, in part because of limited treatment options. Here, we describe a case of donor-derived, disseminated infection with colistin-resistant, carbapenemase-producing Klebsiella pneumoniae in a liver transplant recipient that was cured with addition of intravenous fosfomycin to a multidrug regimen, in conjunction with aggressive surgical source control. Intravenous fosfomycin represents a promising adjunctive agent for use in treatment of extensively drug-resistant infections in immunocompromised hosts.

Validation of the UPDRS section IV for detection of motor fluctuations in Parkinson's disease.

INTRODUCTION: The UPDRS-IV represents the most common screening tool to assess motor fluctuations in patients with PD despite the lack of a clinimetric validation. OBJECTIVES: We evaluated sensitivity and specificity of UPDRS-IV using a 12-h waking-day motor assessment as the gold standard. METHODS: We consecutively enrolled PD patients who underwent a 12-h waking-day motor assessment in the study. Patients were clinically evaluated every 2 h for 12 h using the UPDRS-III. Motor scores were reported as a line graph and six blinded raters classified patients as having or not having motor fluctuations. The UPDRS-IV was used in order to assess the presence of predictable and unpredictable motor fluctuations according to items 36-38. RESULTS: Sixty two PD patients were enrolled in the study. According to the raters' evaluations, 39 (62.9%) were classified as having motor fluctuations, while according to the UPDRS-IV 47 (75.8%) presented a motor fluctuation giving a sensitivity of 87.2% (95%CI 72.6-95.7) and a specificity of 43.5% (95%CI 23.2-65.5). CONCLUSION: Our study results confirm the high level of sensitivity with a lower level of specificity of UPDRS-IV to screen motor fluctuations in PD patients.

Spectrum of excess mortality due to carbapenem-resistant Klebsiella pneumoniae infections.

Patients infected or colonized with carbapenem-resistant Klebsiella pneumoniae (CRKp) are often chronically and acutely ill, which results in substantial mortality unrelated to infection. Therefore, estimating excess mortality due to CRKp infections is challenging. The Consortium on Resistance against Carbapenems in K. pneumoniae (CRACKLE) is a prospective multicenter study. Here, patients in CRACKLE were evaluated at the time of their first CRKp bloodstream infection (BSI), pneumonia or urinary tract infection (UTI). A control cohort of patients with CRKp urinary colonization without CRKp infection was constructed. Excess hospital mortality was defined as mortality in cases after subtracting mortality in controls. In addition, the adjusted hazard ratios (aHR) for time-to-hospital-mortality at 30 days associated with infection compared with colonization were calculated in Cox proportional hazard models. In the study period, 260 patients with CRKp infections were included in the BSI (90 patients), pneumonia (49 patients) and UTI (121 patients) groups, who were compared with 223 controls. All-cause hospital mortality in controls was 12%. Excess hospital mortality was 27% in both patients with BSI and those with pneumonia. Excess hospital mortality was not observed in patients with UTI. In multivariable analyses, BSI and pneumonia compared with controls were associated with aHR of 2.59 (95% CI 1.52-4.50, p <0.001) and 3.44 (95% CI 1.80-6.48, p <0.001), respectively. In conclusion, in patients with CRKp infection, pneumonia is associated with the highest excess hospital mortality. Patients with BSI have slightly lower excess hospital mortality rates, whereas excess hospital mortality was not observed in hospitalized patients with UTI.

Analysis of the Structure and Function of FOX-4 Cephamycinase.

Class C ß-lactamases poorly hydrolyze cephamycins (e.g., cefoxitin, cefotetan, and moxalactam). In the past 2 decades, a new family of plasmid-based AmpC ß-lactamases conferring resistance to cefoxitin, the FOX family, has grown to include nine unique members descended from the Aeromonas caviae chromosomal AmpC. To understand the basis for the unique cephamycinase activity in the FOX family, we determined the first X-ray crystal structures of FOX-4, apo enzyme and the acyl-enzyme with its namesake compound, cefoxitin, using the Y150F deacylation-deficient variant. Notably, recombinant expression of N-terminally tagged FOX-4 also yielded an inactive adenylylated enzyme form not previously observed in ß-lactamases. The posttranslational modification (PTM), which occurs on the active site Ser64, would not seem to provide a selective advantage, yet might present an opportunity for the design of novel antibacterial drugs. Substantial ligand-induced changes in the enzyme are seen in the acyl-enzyme complex, particularly the R2 loop and helix H10 (P289 to N297), with movement of F293 by 10.3 Å. Taken together, this study provides the first picture of this highly proficient class C cephamycinase, uncovers a novel PTM, and suggests a possible cephamycin resistance mechanism involving repositioning of the substrate due to the presence of S153P, N289P, and N346I substitutions in the ligand binding pocket.

Fulminant endocarditis and disseminated infection caused by carbapenem-resistant Acinetobacter baumannii in a renal-pancreas transplant recipient.

Acinetobacter baumannii is an important cause of healthcare-associated infections, and is particularly problematic among patients who undergo organ transplantation. We describe a case of fulminant sepsis caused by carbapenem-resistant A. baumannii harboring the blaOXA-23 carbapenemase gene and belonging to international clone II. This isolate led to the death of a patient 6 days after simultaneous kidney-pancreas transplantation. Autopsy findings revealed acute mitral valve endocarditis, myocarditis, splenic and renal emboli, peritonitis, and pneumonia. This case highlights the severe nature of certain A. baumannii infections and the vulnerability of transplanted patients to the increasingly intractable "high-risk" clones of multidrug-resistant organisms.

Tigecycline therapy for carbapenem-resistant Klebsiella pneumoniae (CRKP) bacteriuria leads to tigecycline resistance.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an increasing global threat. Here, we describe the prevalence and impact of tigecycline use in a cohort of patients with CRKP bacteriuria nested within a multicentre, prospective study. In the 21-month study period, 260 unique patients were included. Tigecycline was given to 80 (31%) patients. The use of tigecycline during the index hospitalization was significantly associated with the subsequent development of tigecycline resistance in the same patient (OR, 6.13; 95% CI, 1.15-48.65; p 0.03). In conclusion, the use of tigecycline with CRKP bacteriuria is common, and is associated with the subsequent development of tigecycline resistance.

Multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates: a zone of high heterogeneity (HHZ) as a tool for epidemiological studies.

Comparison of genome-wide, high-resolution restriction maps of Klebsiella pneumoniae clinical isolates, including an NDM-1 producer, and in silico-generated restriction maps of sequenced genomes revealed a highly heterogeneous region we designated the 'high heterogeneity zone' (HHZ). The HHZ consists of several regions, including a 'hot spot' prone to insertions and other rearrangements. The HHZ is a characteristic genomic area that can be used in the identification and tracking of outbreak-causing strains.

Taking stock of infections and antibiotic resistance in the elderly and long-term care facilities: A survey of existing and upcoming challenges.

Treating elderly patients with infections represents one of the greatest challenges to health-care providers. Older adults are the largest growing sector of the population and suffer excessively from infectious diseases such as pneumonia, urinary tract infections (UTIs), and skin and soft-tissue infections. Often because of disabilities, the elderly require treatment of infectious diseases in long-term care facilities (LTCFs). As a result of antibiotic use, LTCFs have become "reservoirs of resistance" and multi-drug resistant (MDR) pathogens are frequently recovered. Clinicians also need to be aware of the impairment of immune function and other emerging chronic infections (HIV, HCV) that are now present in the elderly. Despite vigilance regarding this issue, delays in diagnosis and initiation of therapy are common. This article reviews the changing landscape of infections in the elderly and the challenge these syndromes present in the context of an increasing older population that requires dedicated resources.

Streptococcus anginosus (Streptococcus milleri Group) Pyomyositis in a 50-Year-Old Man with Acquired Immunodeficiency Syndrome: Case Report and Review of Literature.

We describe the first reported case of bacterial pyomyositis of the right thigh caused by Streptococcus anginosus (S. milleri group) in an HIV-infected patient. The clinical presentation was complicated by multiple ring-enhancing lesions detected on magnetic resonance imaging of the brain. Evaluation for central nervous system toxoplasmosis,syphilis, and cryptococcal infection was negative. Aggressive antibiotic therapy directed against S. anginosus and surgical debridement were limb salvaging. Clinicians should considerS. anginosus as a causative pathogen in HIV-associated pyomyositis, particularly in complex presentations. Prompt surgical drainage may minimize complications due to S. anginosus, a pathogen associated with significant sequelae due to its invasive nature.