Mutant prevention concentration of colistin alone and in combination with rifampicin for multidrug-resistant Acinetobacter baumannii.

Colistin-susceptible isolates of Acinetobacter baumannii often contain subpopulations that are resistant to colistin. Monotherapy with colistin can lead to selective growth of these subpopulations and emergence of colistin-resistant strains. Our objectives were to explore the susceptibility pattern of colistin-resistant subpopulations and investigate if combining colistin with a second antibiotic could prevent their selective growth. Four colistin-susceptible clinical isolates of A. baumannii and one reference isolate were used. The mutant prevention concentration (MPC) of colistin, i.e. the concentration required to block growth of all single-step-mutant subpopulations, was determined by plating an inoculum of 109 CFU on Mueller Hinton agar (MHA)-plates containing 2-fold dilutions of colistin (0.125-128 mg/L). Susceptibility testing of colistin-resistant subpopulations, obtained in the MPC assay, was performed with Etest. The MPC of colistin, in combination with rifampicin, was determined by plating an inoculum of 109 CFU on MHA-plates containing colistin (0.125-128 mg/L) and fixed concentrations of rifampicin (1.1 mg/L or 4.4 mg/L). The colistin-resistant subpopulations demonstrated increased susceptibility to a number of agents compared to their main populations. These subpopulations were even susceptible to agents that normally are inactive against gram-negative bacteria and all had rifampicin MICs of < 0.002 mg/L. The combination of colistin and rifampicin completely inhibited the growth of all colistin-resistant subpopulations and significantly lowered the MPC of colistin for A. baumannii. Combining colistin with rifampicin could be a way to prevent selective growth of colistin-resistant subpopulations of A. baumannii and possibly the emergence of colistin-resistant strains.

Heterogeneous glycopeptide intermediate Staphylococcus epidermidis isolated from prosthetic joint infections.

Methicillin-resistant Staphylococcus epidermidis (MRSE) poses a major problem in prosthetic joint infections (PJIs). Vancomycin is often considered the drug of choice in the empirical treatment of staphylococcal PJIs. As recent decades have seen reports of heterogeneous glycopeptide intermediate S. aureus (hGISA), our aim was to examine the prevalence of heterogeneous glycopeptide intermediate S. epidermidis (hGISE) in PJIs. S. epidermidis isolates (n = 122) from 119 patients in three Swedish counties between 1993 and 2012 were included. All were isolated from perioperative tissue samples from revision surgery in clinically verified PJIs. Antimicrobial susceptibility testing against staphylococcal antibiotics was performed. The macromethod Etest (MME) and glycopeptide resistance detection (GRD) Etest were used to detect hGISE. Standard minimal inhibitory concentration (MIC) determination revealed no vancomycin-resistant isolates, while teicoplanin resistance was detected in 14 out of 122 isolates (11.5%). hGISE was found in 95 out of 122 isolates (77.9%), 64 out of 67 of isolates with teicoplanin MIC >2 mg/L (95.5%) and 31 out of 55 of isolates with teicoplanin MIC ≤2 mg/L (56.4%). Thus, the presence of hGISE cannot be ruled out by teicoplanin MIC ≤2 mg/L alone. Multidrug resistance was detected in 86 out of 95 hGISE isolates (90.5%) and in 16 out of 27 isolates (59.3%), where hGISE could not be detected. In conclusion, hGISE detected by MME or GRD was common in this material. However, hGISE is difficult to detect with standard laboratory diagnostic routines. Glycopeptide treatment may not be sufficient in many of these PJIs, even if standard MIC classifies the isolated S. epidermidis as susceptible.

ADP-ribosylation factor 1 and its activation of phospholipase D are important for the assembly of very low density lipoproteins.

The role of ADP-ribosylation factor 1 (ARF-1) in the assembly of very low density lipoproteins (VLDL) was investigated by expressing dominant-negative mutants in McA-RH7777 cells. Transient expression of ARF-1(T31N), a GDP-restrictive mutant, significantly inhibited apolipoprotein B-100 (apoB-100) VLDL production without influencing the biosynthesis of apoB-100 low density lipoproteins or total apoB production (indicating that it inhibited the second step of VLDL assembly) and without altering total protein production or biosynthesis of transferrin, phosphatidylcholine, or triglycerides. These effects were confirmed in stable inducible transfectants. In contrast, expression of an ARF-1 mutant lacking the N-terminal 17 amino acids, which has no myristoylation site and cannot interact with the microsomal membrane, did not affect VLDL assembly. Thus, active ARF-1 is needed for the second step of the process. To further explore these observations, we developed a cell-free system based on the postnuclear supernatant isolated from McA-RH7777 cells. In this system, 10-15% of the apoB-100 pool was converted to VLDL in a time- and temperature-dependent way. The assembly process was highly dependent on a heat-stable factor in the d > 1.21 g/ml infranatant of fetal calf serum; this factor was not present in low density lipoproteins or VLDL. Brefeldin A inhibited VLDL assembly in this system, as did a synthetic peptide (corresponding to N-terminal amino acids 2-17 of ARF-1) that displaces ARF-1 from the membrane. Thus, active ARF-1 is also needed for cell-free assembly of VLDL. Guanosine 5'-3-O-(thio)triphosphate also inhibited VLDL assembly in this system, indicating that the process requires ongoing hydrolysis of GTP. 1-Butanol, which inhibits the formation of phosphatidic acid (PA) and instead gives rise to phosphatidylbutanol, inhibited VLDL assembly, whereas 2-butanol, which does not inhibit PA formation, failed to do so. Thus, phospholipase D (PLD)-catalyzed formation of PA from phosphatidylcholine is essential for VLDL assembly. In support of this conclusion, exogenous PLD prevented brefeldin A from inhibiting the assembly process. Our results indicate that ARF-1 participates in the second step of VLDL assembly through a process that involves activation of PLD and production of PA.

Assembly of very low density lipoprotein: a two-step process of apolipoprotein B core lipidation.

The liver plays a primary role in lipid metabolism. Important functions include the synthesis and incorporation of hydrophobic lipids, triacylglycerols and cholesteryl esters into the core of water-miscible particles called lipoproteins and the secretion of these particles into the circulation for transport to distant tissues. In this article, we present a brief overview of one aspect of the assembly process of very low density lipoproteins, namely, possible mechanisms for combining core lipids with apolipoprotein B. This is a complex process in which apolipoprotein B interacts with core lipids to form very low density lipoproteins by a two-step process that can be dissociated biochemically.

The Brief Medication Questionnaire: a tool for screening patient adherence and barriers to adherence.

Self-report tools for monitoring adherence can be useful in identifying patients who need assistance with their medications, assessing patient concerns, and evaluating new programs. The aim of this study is to test the validity of the Brief Medication Questionnaire (BMQ), a new self-report tool for screening adherence and barriers to adherence. The tool includes a 5-item Regimen Screen that asks patients how they took each medication in the past week, a 2-item Belief Screen that asks about drug effects and bothersome features, and a 2-item Recall Screen about potential difficulties remembering. Validity was assessed in 20 patients using the Medication Events Monitoring System (MEMS). Results varied by type of non-adherence, with the Regimen and Belief Screens having 80-100% sensitivity for "repeat" non-adherence and the Recall Screen having 90% sensitivity for "sporadic" non-adherence. The BMQ appears more sensitive than existing tools and may be useful in identifying and diagnosing adherence problems.

Musculoskeletal pain and analgesic therapy in a very old population.

The aim of this study was to examine the prevalence of musculoskeletal pain and use of analgesic drugs in a population of very old people. The investigation is based on data from the Kungsholmen project. Of the 2638 inhabitants aged 75 or more in a parish of central Stockholm 1800 were examined. Of the subjects. 60% reported trouble with pain at musculoskeletal locations, while only 40% of the pain reporting subjects used analgesics, one third of which were non-prescription minor analgesics. Prescription analgesics (non-steroidal antiinflammatory drugs or centrally acting drugs) were used by one fourth of the pain reporting subjects, and taken on a regular basis in only half of the cases. Potent opioids were used by less than 1% of the population. There was no increase in pain with increasing age, but an increase in use of minor analgesics with age >85. Women more often reported pain and had a higher consumption of analgesics. Light opioids were more often used by subjects with multifocal pain. Subjects living in sheltered accommodation used more analgesics, particularly light opioids, than did those living in their own homes or in institutions. Subjects with low or intermediate educational status more often reported pain and used light opioids to a larger extent than did the highly educated. These results indicate that musculoskeletal pain is relatively common among the very old, but does not seem to be a severe problem for the majority, considering the low proportion of subjects using prescription analgesics regularly. However, the very low use of potent opioids indicates that some of the elderly suffering from cancer and other severe pain causing diseases might be undertreated.

Resident characteristics and organizational factors influencing the quality of drug use in Swedish nursing homes.

Appropriateness of drug use is an important indicator of the quality of care in nursing homes. In this study, we analyzed the influence of resident characteristics and selected organizational factors on the appropriateness of psychotropic drug use in 33 Swedish nursing homes. Specific criteria based on published guidelines and recommendations were developed to measure appropriateness. Residents diagnosed with a psychiatric disorder and younger residents had more deviations from the criteria; however, resident mix did not explain variations in appropriateness of drug use at the facility level. Facilities with better nurse staffing and drug intervention teams had fewer deviations from the criteria, but only 15-20% of the variation in drug prescribing was explained by these predictors.

Physician and staff assessments of drug interventions and outcomes in Swedish nursing homes.

OBJECTIVE: To describe the type and frequency of drug-related problems discussed in regular team meetings conducted in 15 Swedish nursing homes and report physician and staff assessments of these interventions and residents' outcomes. DATA SOURCES AND METHODS: The data were collected within the context of a controlled trial with the primary aim of exploring the effects of regular team interventions on drug prescribing practices in Swedish nursing homes. In 15 experimental nursing homes, the residents' drug therapy was discussed regularly by a team consisting of a pharmacist, a physician, nurses, undernurses (similar to licensed practical nurses), and nurse's aides. The pharmacist documented problems, made changes, and observed outcomes. Following the intervention period, a questionnaire was sent to the medical staff that contained items regarding perceived outcomes, the intervention's impact on knowledge of drug therapy in the elderly, and attitudes toward the pharmacist's role. RESULTS: Unclear indication and problematic choice of drugs were the most common drug-related problems discussed. In 19% of the situations, therapy changes were reported to have had a beneficial effect on the residents' clinical status; in 47% of the situations, staff reported no observable outcome from changes, suggesting that the changes had been appropriate. Finally, medical staff claimed in the follow-up survey that their knowledge about drug therapy had increased; they expressed an overall positive attitude toward this interactive collaboration. CONCLUSIONS: Regular intervention conducted by a multidisciplinary team incorporating a pharmacist can effectively improve prescribing practices, increase staff knowledge about appropriate drug therapy in the elderly, and result in improved quality of care for nursing home residents.

The impact of regular multidisciplinary team interventions on psychotropic prescribing in Swedish nursing homes.

OBJECTIVE: To evaluate the impact of regular multidisciplinary team interventions on the quantity and quality of psychotropic drug prescribing in Swedish nursing homes. DESIGN: A randomized controlled trial. SETTING: A sample of 33 nursing homes: 15 experimental homes and 18 control homes representing 5% of all Swedish nursing homes. PARTICIPANTS: The sample consisted of 1854 long-term care residents with an average age of 83 years. Seventy percent of the residents were women, and 42% had a documented diagnosis of dementia. An additional 5% had a psychotic disorder, and 7% had a diagnosis of depression. INTERVENTION: Experimental homes participated in an outreach program that was designed to influence drug use through improved teamwork among physicians, pharmacists, nurses, and nurses' assistants. Multidisciplinary team meetings were held on a regular basis throughout the 12-month study period. MEASUREMENTS: Lists of each resident's prescriptions were collected 1 month before and 1 month after the 12-month intervention. Measures included the proportion of residents with any psychotropic drug, polymedicine, and therapeutic duplication and proportion of residents with nonrecommended and acceptable drugs in each psychotropic drug class, as defined by current Swedish guidelines. RESULTS: Baseline results show extensive psychotropic drug prescribing, with the most commonly prescribed drugs being hypnotics (40%), anxiolytics (40%), and antipsychotics (38%). After 12 months of team meetings in the experimental homes, there was a significant decrease in the prescribing of psychotics (-19%), benzodiazepine hypnotics (-37%), and antidepressants (-59%). Orders for more acceptable antidepressants also increased in the experimental homes. In the control homes there was increased use of acceptable antidepressants, but there were no significant reductions in other drug classes. CONCLUSIONS: There is excessive prescription of psychotropic drugs in Swedish nursing homes. Improved teamwork among caregivers can improve prescribing as defined by clinical guidelines.

Drug use in Swedish nursing homes.

OBJECTIVE: To describe the overall drug use in Swedish nursing homes and to comment on the impact of regular multidisciplinary team interventions on the quantity of inappropriate medications. DESIGN AND SETTING: This randomised, controlled trial was carried out in a sample of 33 nursing homes, 15 experimental homes and 18 control homes, representing 5% of all Swedish nursing homes. PARTICIPANTS: The sample consisted of 1854 nursing home residents with an average age of 83 years. 70% of the residents were female and 42% had a documented diagnosis of dementia. An additional 5% had a psychotic disorder and 7% had a diagnosis of depression. MEASUREMENTS: Lists of each resident's prescriptions were collected 1 month before and 1 month after the 12-month intervention. Measures included the proportion of residents receiving any particular drug. The number of drugs classified by guidelines as inappropriate was also measured before and after the intervention period. RESULTS: The nursing home residents were prescribed on average 7.7 medications, the majority on a regular basis. The most frequently prescribed group of drugs was laxatives, followed by psychotropic agents and cardiovascular drugs. Some groups of drugs decreased in the intervention homes. CONCLUSION: Extensive and somewhat inappropriate drug use in Swedish nursing homes is a significant and serious problem. Careful monitoring is necessary to ensure well tolerated drug therapy in this frail population. Neither written information from the Medical Products Agency, nor the campaign in the pharmacies alone had an impact on drug use in Swedish nursing homes. Multidisciplinary team discussions contributed to improved drug use, but more research is needed to understand the mechanisms behind this finding.

Drug use by demented and non-demented elderly people.

AIM: to determine the use of drugs by demented and non-demented elderly people in a population, by dementia status and type, age, sex and accommodation type. METHOD: data were obtained from the Kungsholmen project, a longitudinal community study of people over 75 in Stockholm, Sweden. RESULTS: 85% used at least one medicinal drug, and of these 12% were demented. Mean numbers of drugs used were 2.8 for demented and 3.2 for non-demented people. 45% of demented people and 38% of non-demented people used psychotropic agents. Psychotropic use was higher in women and increased with institutionalization. Antipsychotic agents were used more by demented (22%) than by non-demented (3.5%) people: this was largely explained by differences in accommodation type. The odds ratio (OR) for use of antipsychotics by those in institutions compared with those living in their own homes was 9.32. Opioids were commonly prescribed for demented people. The proportions taking opioids in those using analgesics were 42% in demented and 23% in non-demented people (OR 2.07). Laxatives were used by 18% of the demented people in institutions compared with 39% of non-demented people in institutions. CONCLUSION: being in an institution had a stronger association with the use of certain drugs (e.g. psychotropics) than did dementia status. Demented people, especially those in institutions, used a large number of antipsychotics and opioids, but fewer laxatives and minor analgesics. Prescribers and institutional staff should be aware of these factors so they can optimize patient treatment.

: prevalence and association with drug use.

In a cross-sectional study, we have investigated the prevalence of self-reported symptoms and their association with medicinal drug use in elderly people. Data from the Kungsholmen Project were used, a population-based study of elderly people aged 75 years and over in Stockholm, Sweden. The study sample comprised 1800 persons. Information on the occurrence of 22 different symptoms and the actual drug use was obtained at interviews with the participants. The relation of symptoms to age, gender and housing, and their association with drug use was analysed using logistic regression. The most commonly reported symptoms were pain and tiredness. In general, symptoms were more common in women and at higher ages. Many of the associations between symptoms and drug use reflected established treatments. However, some were suggestive of inappropriate treatment or dosage; for example, the association between tiredness and the use of anxiolytics and hypnotics-sedatives.

Use of cardiovascular drugs in an older Swedish population.

OBJECTIVE: To describe the use of cardiovascular drugs in an older population with respect to age, sex, housing type, and creatinine clearance. DESIGN: A cross-sectional survey. PARTICIPANTS: All residents of a district of Stockholm (Kungsholmen), Sweden, aged 75 and older, living in institutions or at home. MEASUREMENTS: Cardiovascular drug use, serum creatinine, electrolytes, height, weight, and symptoms. RESULTS: A total of 43 cardiovascular (CV) drugs were used. The most common drugs were digoxin (used by 18.2%), furosemide (16.4%), and glyceryl trinitrate (12.4%). Drugs with an antihypertensive effect accounted for 61% of all CV drugs. CV drug use increased with age for cardiac glycosides and diuretics, but decreased with age for calcium antagonists and beta-blockers. Drug doses tended to be less than the recommended daily dose except for a few drugs, e.g., furosemide. There was a trend toward decreasing dose with increasing age, but this was not significant. Diuretics were the only CV drugs used more often in women. People living in institutional care used the least amount of CV drugs. The dose of drugs taken did not appear to be related to estimated creatinine clearance. Comparisons between drug use and complaint of symptoms showed a strong correlation between the use of cardiac glycosides and anorexia, calcium antagonists and constipation, and nitrates and vertigo. There were weaker correlations with cardiac glycosides and visual disturbances and with potassium sparing diuretics and a high potassium. CONCLUSIONS: CV drugs are used commonly in older people. We suggest that the symptoms correlating with cardiac glycoside use may be signs of unrecognized toxicity, and this may relate to our finding that drug use is often not tailored to renal function as measured by creatinine clearance.

The use of medicines with anticholinergic effects in older people: a population study in an urban area of Sweden.

OBJECTIVE: To investigate the use of medicines with anticholinergic properties among older people in an urban population in Sweden. DESIGN: A cross-sectional survey. SETTING: Ordinary homes, sheltered accommodations, nursing homes, and geriatric departments. PARTICIPANTS: All residents aged 75 and older in a district of Stockholm, Sweden. MEASUREMENTS: Structured interviews with older persons, their relatives and/or health care personnel; prescription forms; medical records. RESULTS: The overall use of medicines with anticholinergic effects was comparatively low. Doses of these medicines were also generally low. Concurrent use of several such medicines was uncommon. The most prevalent therapeutic/pharmacological group was neuroleptics. In contrast, antidepressants were used by few older people. The prevalence of medicines with anticholinergic effects was highest at institutions, where neuroleptics were frequent and use of low-potency neuroleptics was not uncommon. CONCLUSION: Our results indicate that the risk of anticholinergic side effects may be quite low in the present population as a whole. However, there may be grounds for revising the therapy in institutions, where the use of neuroleptics was shown to be high and low-potency neuroleptics, known to have a higher incidence of anticholinergic side effects, were not avoided.

International pharmacy.

Several worldwide initiatives involve pharmacy education and practice; however, the International Pharmaceutical Federation is the only worldwide pharmacy organization currently in existence. This is the first report of the good pharmacy practice initiative published in the United States. We hope that it will stimulate interest in international pharmacy.

Glycine codon discrimination and the nucleotide in position 32 of the anticodon loop.

Using an in vitro protein-synthesizing system that allowed us to monitor separately the reading of each glycine codon, we have previously shown, that in constructs based on glycine tRNA1 from Escherichia coli the nature of the nucleotide in position 32 determines the ability of the anticodon UCC to discriminate between the glycine codons. Thus, with a U in position 32 the anticodon UCC discriminated according to the wobble rules, but with a C in this position it had lost its ability to discriminate. In the present paper we show that the same is true also for constructs based on mycoplasma glycine tRNA. When C32 in the wild type was changed to U32, the anticodon UCC discriminated between the glycine codons, while in wild type mycoplasma glycine tRNA it did not. Furthermore, when U32 was changed to C32 in glycine tRNA1(CCC), the anticodon CCC loses its ability to discriminate. We therefore conclude that the nature of the nucleotide in position 32 determines the discriminatory ability of both anticodons UCC and CCC in the glycine tRNA1 structural background, and that the same is true for the anticodon UCC in the mycoplasma glycine tRNA background.

Undiscriminating codon reading with adenosine in the wobble position.

To investigate the reading properties of adenosine in the wobble position we have used site-directed mutagenesis of the Escherichia coli glycine tRNA1(CCC) gene to substitute the nucleotide A in the wobble position of the corresponding tRNA. The effect of this change on the ability of the tRNA to discriminate between the nucleotides in the third position of the glycine codons has been investigated. We have compared the ability of the mutant glycine tRNA1(UCC) and glycine tRNA1(ACC) as well as the mycoplasma glycine tRNA(UCC) to read the glycine codons. The results showed that glycine tRNA1(ACC) unlike glycine tRNA1(UCC) did not fully discriminate between the glycine codons. These experiments were carried out using a new in vitro protein synthesizing system that allows us to monitor the reading of all four glycine codons. In the present paper we give a detailed description of this new in vitro system.

The nucleotide in position 32 of the tRNA anticodon loop determines ability of anticodon UCC to discriminate among glycine codons.

We have investigated the influence of structures in the tRNA anticodon loop and stem on the ability of the anticodon to discriminate among codons. We had previously shown that anticodon UCC, when placed in the structural context of tRNA(Gly1) from Escherichia coli, discriminated efficiently between the glycine codons, as required by the wobble rules. Thus, this anticodon read GGA and GGG but did not read GGU and GGC, whereas in mycoplasma tRNA(Gly), the same anticodon did not discriminate among the glycine codons. We have now determined the reading properties of three constructions based on tRNA(Gly1) containing the anticodon UCC in different structural contexts. In one of these constructs, tRNA(Gly1-ASL), the anticodon loop and stem are the same as in mycoplasma tRNA(Gly). The second construct, tRNA(Gly1-AS), has an anticodon stem identical with the mycoplasma tRNA(Gly), whereas in the last construct, tRNA(Gly1-C32), the only difference from tRNA(Gly1)(UCC) is that the uridine in position 32 of the anticodon loop has been replaced by cytidine. These constructs were tested for ability to read glycine codons in an in vitro protein-synthesizing system that allowed us to monitor separately the reading of each codon. We found that the anticodon UCC, when present in tRNA(Gly1-AS), discriminated among the glycine codons, whereas in the constructs tRNA(Gly1-ASL) and tRNA(Gly1-C32), the same anticodon had lost its ability to discriminate--i.e., it behaved as in mycoplasma tRNA(Gly). These results strongly suggest that nt 32 of the anticodon loop of tRNA(Gly1)(UCC) decisively influences the reading properties of the anticodon UCC.

Codon reading properties of an unmodified transfer RNA.

We have previously shown that the Mycoplasma mycoides glycine tRNA (anticodon UCC) effectively reads the codons GGU and GGC in violation of the classic codon reading rules. We have attempted to elucidate what structural elements in this tRNA molecule confer this translational property and in the course of this investigation T7 RNA polymerase transcription of the corresponding gene was used to produce a tRNA devoid of modified nucleosides. Using an in vitro translation system the ability of this tRNA to read the 4 glycine codons (GGU, GGC, and GGG) was tested and it was shown to be as efficient as its normal, fully modified counterpart in the reading of all four codons. This result demonstrates that a tRNA devoid of modified nucleosides is able to efficiently sustain protein synthesis in vitro and, furthermore, that the normal modification pattern of the Mycoplasma glycine tRNA is not essential for the ability of this tRNA to read the glycine codons GGU and GGC effectively.