In Vitro Selection of Ceftazidime-Avibactam Resistance in Enterobacteriaceae with KPC-3 Carbapenemase

Ceftazidime-avibactam is active against most Enterobacteriaceae isolates with KPC carbapenemases. We investigated whether this activity could be compromised by mutation. Single-step and multistep selections were attempted using ceftazidime-avibactam (avibactam fixed at 1 or 4 μg/ml) versus two strains each of Enterobacter cloacae and Klebsiella pneumoniae, all with the KPC-3 enzyme. Mutant bla_KPC alleles were sequenced, and their parentage was confirmed by typing. Ceftazidime-avibactam selected mutants at up to 16× MIC, with frequencies of ca. 10⁻⁹. This contrasted with previous experience for ceftaroline-avibactam, where mutant frequencies under similar conditions were <10⁻⁹. The MICs of ceftazidime with 1 μg/ml avibactam for the ceftazidime-avibactam-selected mutants rose from 1 to 8 μg/ml to 16 to >256 μg/ml and those of ceftazidime with 4 μg/ml avibactam from 0.25 to 1 μg/ml to 4 to 128 μg/ml; ceftaroline-avibactam MICs rose less, typically from 0.5 to 1 μg/ml to 1 to 8 μg/ml. The MICs of carbapenems and cephalosporins except ceftazidime and piperacillin-tazobactam were reduced for many mutants. Sequencing of bla_KPC revealed point and insertion changes in 12/13 mutants investigated, representing all four parents; one mutant lacked bla_KPC changes and possibly had reduced permeability. Amino acid changes commonly involved Ω loop alterations or 1 to 6 amino acid insertions immediately C-terminal to this loop. The most frequent change, seen in four mutants from three strains, was Asp179Tyr, replacing a residue that ordinarily forms a salt bridge to stabilize the Ω loop. Since ceftaroline-avibactam was less affected than ceftazidime-avibactam, we postulate that these mutations increase ceftazidimase specificity rather than conferring avibactam resistance. The clinical relevance remains uncertain.     

Human Granulocytic Anaplasmosis in the United States from 2008 to 2012: A Summary of National Surveillance Data

Human granulocytic anaplasmosis is an acute, febrile illness transmitted by the ticks Ixodes scapularis and Ixodes pacificus in the United States. We present a summary of passive surveillance data for cases of anaplasmosis with onset during 2008–2012. The overall reported incidence rate (IR) was 6.3 cases per million person-years. Cases were reported from 38 states and from New York City, with the highest incidence in Minnesota (IR = 97), Wisconsin (IR = 79), and Rhode Island (IR = 51). Thirty-seven percent of cases were classified as confirmed, almost exclusively by polymerase chain reaction. The reported case fatality rate was 0.3% and the reported hospitalization rate was 31%. IRs, hospitalization rates, life-threatening complications, and case fatality rates increased with age group. The IR increased from 2008 to 2012 and the geographic range of reported cases of anaplasmosis appears to have increased since 2000–2007. Our findings are consistent with previous case series and recent reports of the expanding range of the tick vector I. scapularis.     

Burnout among Primary Care Providers and Staff: Evaluating the Association with Practice Adaptive Reserve and Individual Behaviors

BackgroundWorkplace burnout among healthcare professionals is a critical public health concern. Few studies have examined organizational and individual factors associated with burnout across healthcare professional groups.ObjectiveThe purpose of this study was to examine the association between practice adaptive reserve (PAR) and individual behavioural response to change and burnout among healthcare professionals in primary care.DesignThis cross-sectional study used survey data from 154 primary care practices participating in the EvidenceNOW Heart of Virginia Healthcare initiative.ParticipantsWe analysed data from 1279 healthcare professionals in Virginia. Our sample included physicians, advanced practice clinicians, clinical support staff and administrative staff.Main MeasuresWe used the PAR instrument to measure organizational capacity for change and the Change Diagnostic Index© (CDI) to measure individual behavioural response, which achieved a 76% response rate. Logistic regression analysis was used to estimate the effects of PAR and CDI on burnout.Key ResultsAs organizational capacity for change increased, burnout in healthcare professionals decreased by 51% (OR: 0.49; 95% CI, 0.33, 0.73). As healthcare professionals showed improved response toward change, burnout decreased by 84% (OR: 0.16; 95% CI, 0.11, 0.23). Analysis by healthcare professional type revealed a significant association between high organizational capacity for change, positive response to change and low burnout among administrative staff (OR: 2.92; 95% CI, 1.37, 6.24). Increased hours of work per week was associated with higher odds of burnout (OR: 1.07; 95% CI, 1.05, 1.10) across healthcare professional groups.ConclusionAs transformation efforts in primary care continue, it is critical to understand the influence of these initiatives on healthcare professionals’ well-being. Efforts to reduce burnout among healthcare professionals are needed at both a system and organizational level. Building organizational capacity for change, supporting providers and staff during major change and consideration of individual workload may reduce levels of burnout.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11606-020-06367-z.KEY WORDS: adaptive reserve, burnout, practice transformation, primary care, well-being     

In Vitro Activity of Ceftazidime-Avibactam against 338 Molecularly Characterized Gentamicin-Nonsusceptible Gram-Negative Clinical Isolates Obtained from Patients in Canadian Hospitals

The mechanism of aminoglycoside resistance among 338 gentamicin-nonsusceptible Gram-negative bacteria (207 Enterobacteriaceae and 131 Pseudomonas aeruginosa) was assessed, and the in vitro activity of ceftazidime-avibactam against these isolates was determined. Aminoglycoside-modifying enzymes were detected in 91.8% of Enterobacteriaceae and 13.7% of P. aeruginosa isolates. A single strain of Klebsiella pneumoniae harbored a 16S rRNA methylase (ArmA). The ceftazidime-avibactam MIC₉₀ values were 0.5 μg/ml (MIC, ≤8 μg/ml for 100% of isolates) and 16 μg/ml (MIC, ≤8 μg/ml for 87.8% of isolates) against gentamicin-nonsusceptible Enterobacteriaceae and P. aeruginosa isolates, respectively.     

Activity of Ceftazidime-Avibactam against Fluoroquinolone-Resistant Enterobacteriaceae and Pseudomonas aeruginosa

Ceftazidime-avibactam and comparator antibiotics were tested by the broth microdilution method against 200 Enterobacteriaceae and 25 Pseudomonas aeruginosa strains resistant to fluoroquinolones (including strains with the extended-spectrum β-lactamase [ESBL] phenotype and ceftazidime-resistant strains) collected from our institution. The MICs and mechanisms of resistance to fluoroquinolone were also studied. Ninety-nine percent of fluoroquinolone-resistant Enterobacteriaceae strains were inhibited at a ceftazidime-avibactam MIC of ≤4 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference). Ceftazidime-avibactam was very active against ESBL Escherichia coli (MIC₉₀ of 0.25 mg/liter), ESBL Klebsiella pneumoniae (MIC₉₀ of 0.5 mg/liter), ceftazidime-resistant AmpC-producing species (MIC₉₀ of 1 mg/liter), non-ESBL E. coli (MIC₉₀ of ≤0.125 mg/liter), non-ESBL K. pneumoniae (MIC₉₀ of 0.25 mg/liter), and ceftazidime-nonresistant AmpC-producing species (MIC₉₀ of ≤0.5 mg/liter). Ninety-six percent of fluoroquinolone-resistant P. aeruginosa strains were inhibited at a ceftazidime-avibactam MIC of ≤8 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference), with a MIC₉₀ of 8 mg/liter. Additionally, fluoroquinolone-resistant mutants from each species tested were obtained in vitro from two strains, one susceptible to ceftazidime and the other a β-lactamase producer with a high MIC against ceftazidime but susceptible to ceftazidime-avibactam. Thereby, the impact of fluoroquinolone resistance on the activity of ceftazidime-avibactam could be assessed. The MIC₉₀ values of ceftazidime-avibactam for the fluoroquinolone-resistant mutant strains of Enterobacteriaceae and P. aeruginosa were ≤4 mg/liter and ≤8 mg/liter, respectively. We conclude that the presence of fluoroquinolone resistance does not affect Enterobacteriaceae and P. aeruginosa susceptibility to ceftazidime-avibactam; that is, there is no cross-resistance.     

Preventable Major Cardiovascular Events Associated With Uncontrolled Glucose,Blood Pressure,and Lipids and Active Smoking in Adults With Diabetes With and Without Cardiovascular Disease: A Contemporary Analysis

OBJECTIVEThe objective of this study was to assess the incidence of major cardiovascular (CV) hospitalization events and all-cause deaths among adults with diabetes with or without CV disease (CVD) associated with inadequately controlled glycated hemoglobin (A1C), high LDL cholesterol (LDL-C), high blood pressure (BP), and current smoking.RESULTSMean (SD) age at baseline was 59 (14) years; 48% of subjects were female, 45% were white, and 31% had CVD. Mean follow-up was 59 months. Event rates per 100 person-years for adults with diabetes and CVD versus those without CVD were 6.0 vs. 1.7 for MI/ACS, 5.3 vs. 1.5 for stroke, 8.4 vs. 1.2 for HF, 18.1 vs. 40 for all CV events, and 23.5 vs. 5.0 for all-cause mortality. The percentages of CV events and deaths associated with inadequate risk factor control were 11% and 3%, respectively, for those with CVD and 34% and 7%, respectively, for those without CVD.CONCLUSIONSAdditional attention to traditional CV risk factors could yield further substantive reductions in CV events and mortality in adults with diabetes.     

Parkinson disease-associated mutation R1441H in LRRK2 prolongs the “active state” of its GTPase domain

Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (Roc_R1441H) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD) (1–5). LRRK2 is a large (2,527-aa) multidomain protein consisting of seven putative domains (2), including a Ras-like GTPase domain called Ras of complex proteins (Roc), followed by a domain called C-terminal of Roc (COR), which is then followed by a kinase domain (Kin). It remains unclear how perturbations of these activities result in disease; however, the most common mutation in LRRK2-associated PD, G2019S in the kinase domain, shows higher kinase activity than wild type; therefore, its overactivation might be associated with disease pathogenesis (6).The tandem Roc-COR-Kin arrangement suggests that their activities might be coupled such that the GTPase activity of Roc might modulate the kinase activity. Indeed, several studies have shown that GTP binding to the Roc domain regulates the activity of the Kin domain (7, 8). Moreover, a PD-associated mutation in the Roc domain (R1441C) has been shown to have higher kinase activity (9), thus suggesting that mutations in the Roc domain, also up-regulate kinase activity.Understanding the function of Roc and its mechanism of action is important for understanding the mechanism of PD pathogenesis and therapeutic development. However, because of the lack sufficient quantity of protein samples amendable for detailed investigations, the biochemical properties and enzymatic activities of the Roc domain of LRRK2 are poorly understood.Here, we describe a stably folded construct of human Roc domain that enabled us to investigate quantitatively its biochemical and enzymatic properties. The results revealed that a PD-causing mutation R1441H in the Roc domain renders it less active at hydrolyzing GTP, as well as having higher affinity for GTP, than its wild-type counterpart, thereby increasing the residence time of its GTP-bound “active state,” which is associated with PD pathogenesis (8).     

Cardiac specific ATP-sensitive K(+) channel (K(ATP)) overexpression results in embryonic lethality

Transgenic mice overexpressing SUR1 and gain of function Kir6.2[?N30, K185Q] K_ATP channel subunits, under cardiac α-myosin heavy chain (αMHC) promoter control, demonstrate arrhythmia susceptibility and premature death. Pregnant mice, crossed to carry double transgenic progeny, which harbor high levels of both overexpressed subunits, exhibit the most extreme phenotype and do not deliver any double transgenic pups. To explore the fetal lethality and embryonic phenotype that result from K_ATP overexpression, wild type (WT) and K_ATP overexpressing embryonic cardiomyocytes were isolated, cultured and voltage-clamped using whole cell and excised patch clamp techniques. Whole mount embryonic imaging, Hematoxylin and Eosin (H&;E) and α smooth muscle actin (αSMA) immunostaining were used to assess anatomy, histology and cardiac development in K_ATP overexpressing and WT embryos. Double transgenic embryos developed in utero heart failure and 100% embryonic lethality by 11.5 days post conception (dpc). K_ATP currents were detectable in both WT and K_ATP-overexpressing embryonic cardiomyocytes, starting at early stages of cardiac development (9.5 dpc). In contrast to adult cardiomyocytes, WT and K_ATP-overexpressing embryonic cardiomyocytes exhibit basal and spontaneous K_ATP current, implying that these channels may be open and active under physiological conditions. At 9.5 dpc, live double transgenic embryos demonstrated normal looping pattern, although all cardiac structures were collapsed, probably representing failed, non-contractile chambers. In conclusion, K_ATP channels are present and active in embryonic myocytes, and overexpression causes in utero heart failure and results in embryonic lethality. These results suggest that the K_ATP channel may have an important physiological role during early cardiac development.     

Occupational transmission of hepatitis C in healthcare workers and factors associated with seroconversion: UK surveillance data

The study aims were to describe a case series of occupationally acquired hepatitis C (HCV) infections in UK healthcare workers and examine factors associated with transmission using exposure data reported to the Health Protection Agency between July 1997 and December 2007. Fifteen reported cases of documented HCV seroconversion occurred after percutaneous exposure, the majority from hollow-bore needles used in the source patient's vein or artery and contaminated with blood or blood-stained fluid. The seroconversion rate was 2.2% (14/626). In multivariable analysis of healthcare workers with percutaneous exposure to blood or blood-stained fluid, we demonstrate that blood sampling procedures (odds ratio [OR], 5.75; 95% CI, 1.33-24.91; P = 0.01) and depth of injury (OR for deep vs superficial injury, 21.99; 95% CI, 2.02-239.61; P = 0.02) are independently associated with a greater risk of HCV seroconversion. This is the first UK study of occupationally acquired HCV in healthcare workers. It has reinforced our knowledge of risk factors for HCV transmission. Most of these exposures and transmissions were preventable. Healthcare employers should provide regular education on the risks of occupational exposure and prevention through standard infection control procedures. They should ensure the availability of effective prevention measures and facilitate prompt reporting and adequate follow-up of exposures.