Policy driven changes in animal research practices: mapping researchers' attitudes towards animal-free innovations using the Netherlands as an example.

BACKGROUND: Reducing the number of animals used in experiments has become a priority for the governments of many countries. For these reductions to occur, animal-free alternatives must be made more available and, crucially, must be embraced by researchers. METHODS: We conducted an international online survey for academics in the field of animal science (N = 367) to explore researchers' attitudes towards the implementation of animal-free innovations. Through this survey, we address three key questions. The first question is whether scientists who use animals in their research consider governmental goals for animal-free innovations achievable and whether they would support such goals. Secondly, responders were asked to rank the importance of ten roadblocks that could hamper the implementation of animal-free innovations. Finally, responders were asked whether they would migrate (either themselves or their research) if increased animal research regulations in their country of residence restricted their research. RESULTS: While nearly half (40%) of the responders support governmental goals, the majority (71%) of researchers did not consider such goals achievable in their field within the near future. In terms of roadblocks for implementation of animal-free methods, ~ 80% of the responders considered 'reliability' as important, making it the most highly ranked roadblock. However, all other roadblocks were reported by most responders as somewhat important, suggesting that they must also be considered when addressing animal-free innovations. Importantly, a majority reported that they would consider migration to another country in response to a restrictive animal research policy. Thus, governments must consider the risk of researchers migrating to other institutes, states or countries, leading to a 'brain-drain' if policies are too strict or suitable animal-free alternatives are not available. CONCLUSION: Our findings suggest that development and implementation of animal-free innovations are hampered by multiple factors. We outline three pillars concerning education, governmental influence and data sharing, the implementation of which may help to overcome these roadblocks to animal-free innovations.

No increase in frequency of antifungal resistance among yeasts isolated from normally sterile sites in patients at Foothills Hospital from 1993 to 1996.

OBJECTIVE: To determine the prevalence of resistance to antifungal drugs among yeasts isolated from sterile sites from patients in one hospital and the relationship of resistance to antifungal use, and to assess whether resistance was increasing. METHOD: Susceptibility testing performed by National Committee for Clinical Laboratory Standards (NCCLS) (Villanova, Pennsylvania) microdilution method and by E test. Antifungal use was determined by selected chart review and from pharmacy data. SPECIMENS AND SETTING: Tertiary care adult hospital with neonatal intensive care. POPULATION STUDIED: Distinct yeast isolates from sterile site specimens collected during the years 1993 to 1996. RESULTS: A total of 132 yeast isolates were studied, of which 78 (59%) were Candida albicans. The proportion of C albicans remained steady over the four-year period, and there was no trend to increased resistance among C albicans. The number of isolates of all species with fluconazole microdilution minimum inhibitory concentration (MIC) greater than 8 mg/L in each of the four years were one of 32 in 1996, three of 26 in 1994, six of 33 in 1995, and one of 41 in 1996. A single isolate had an itraconazole microdilution MIC greater than 0.5 mg/L in each year. Prior use of antifungal therapy was rare in this patient population. CONCLUSIONS: The increase in resistance to antifungal drugs reported by some centres did not occur in this institution over the course of the study. This experience may reflect differences in infection control practices and in patterns of use of antifungal agents. The NCCLS method was found to be superior to the E test as a routine method for testing susceptibility of yeasts.

Serum lipids in early pregnancy and risk of pre-eclampsia.

OBJECTIVE: To determine whether first and late second trimester serum total and high density lipoprotein cholesterol are associated with blood pressure, uterine artery pulsatility index and pregnancy outcome. DESIGN: A prospective cohort study. Data were analysed using multiple linear and logistic regression analysis. PARTICIPANTS: Three hundred and ninety-three pregnant women requesting chorionic villus sampling because of advanced maternal age (36 years and older). MAIN OUTCOME MEASURES: Serum total cholesterol and high density lipoprotein cholesterol were measured by an automated enzymatic method. Uterine artery flow velocity waveforms were recorded using continuous Doppler ultrasound. Pregnancy outcome was assessed by questionnaire. RESULTS: First trimester serum total cholesterol was significantly associated with the risk of pre-eclampsia, with the adjusted relative risk exceeding 5 for women with serum total cholesterol levels above 6.0 mmol/1 when compared with women with a cholesterol level below 5.0 mmol/1. First trimester serum total cholesterol also showed a significant relationship with diastolic blood pressure (coefficient of linear regression = 0.02 (mmol/1)/mm Hg, 95% CI = 0.01 to 0.03), and the change in both diastolic and systolic blood pressure from the first to the late second trimester was associated with linear changes in serum total cholesterol and high density lipoprotein cholesterol. CONCLUSIONS: These data suggest a relation between serum lipids in early pregnancy and the development of pre-eclampsia.

Early uterine artery Doppler velocimetry and the outcome of pregnancy in women aged 35 years and older.

The objective of this paper was to determine whether first- and second-trimester uterine artery Doppler velocimetry are associated with pregnancy complications in women of advanced maternal age. A prospective cohort study of 352 women aged 35 years and older was studied. The pulsatility index (PI) values at 12-13 weeks of gestation were significantly associated with development of hypertensive disorders, a small-for-gestational-age infant and gestational diabetes, with a relative risk exceeding 4, 2 and 8, respectively for women with PI values in the highest quartile (> 1.67) of the PI distribution when compared with the lowest quartile of the PI distribution (< 1.24). At 23-27 weeks' gestation, uterine artery PI values were found to be associated with preterm delivery with a gestational age-adjusted risk of 10.6 for women with PI values in the highest quartile of PI (> 1.24) when compared with PI values in the lowest quartile of the PI distribution (< 1.09). No associations existed between uterine artery PI, antepartum hemorrhage and Cesarean section rate. The risk estimates for any of the outcome parameters were not affected by maternal age. Results indicate that hemodynamic changes detectable in the uterine artery as early as the first trimester of pregnancy are associated with an increased risk of hypertensive disorders, a small-for-gestational-age infant and gestational diabetes. A similar association exists in the late second trimester of pregnancy, with an increased risk of preterm delivery.

Calcium metabolism, calcium supplementation and hypertensive disorders of pregnancy.

In recent years growing attention has been directed towards the possible role of calcium in the development of pregnancy-induced hypertension and preeclampsia. Several studies describe calcium metabolism in normal and hypertensive pregnancy, but so far, they have shown discrepant and inconsistent results. Intracellular free calcium, which plays an important role in vascular smooth muscle contraction, has been claimed as a pathogenic factor in hypertensive disorders of pregnancy. Although there is discordance in the data, a possible role of intracellular calcium in the development of hypertensive disorders of pregnancy cannot be excluded. Observational studies in pregnant women suggest an inverse association between calcium intake and the incidence of hypertensive disorders of pregnancy. Despite large methodological differences, the results from the calcium supplementation trials support this finding. Although it is rather difficult to isolate the effect of calcium intake from the intake of other mineral elements, results from calcium supplementation trials are supportive for calcium being the most important. Proposed mechanisms by which calcium supplementation may lower blood pressure involve changes in parathyroid hormone (PTH) level, the renin-angiotensin system and calcium as a modifier of vascular agent regulation, but none of these have yet been elucidated. At present, circumstantial evidence suggest a positive role for calcium in the prevention of hypertensive disorders of pregnancy, but definite evidence is lacking and further research is warranted.

The predictive value of uterine artery flow velocity waveforms in miscarriage in older women.

OBJECTIVE: To determine whether uterine artery blood flow velocity measurements can predict miscarriage in older women. DESIGN: Prospective study. SUBJECTS: Three hundred and ninety-three women aged 35 years and older in the first trimester of pregnancy. MAIN OUTCOME MEASURES: Miscarriage, genetic abortion, pulsatility index (PI), maternal age, gestational age at intake. RESULTS: Twenty women miscarried; 10 pregnancies were terminated because of chromosomal anomalies. Maternal age and gestational age at intake were significantly associated with miscarriage rate (P = 0.01 and P = 0.001, respectively). Uterine artery PI values declined significantly during the first trimester (P = 0.001). However, no association was found between uterine artery PI values and miscarriage rate. PI, maternal age and gestational age at intake were not essentially different between women who miscarried before or after chorionic villus sampling. No association was found between PI, maternal age, and gestational age at intake and genetic abortion rate. CONCLUSIONS: Uterine artery blood flow velocity waveforms, as expressed by the pulsatility index, bear no relation to miscarriage.

DNA probe technology for rapid detection of Haemophilus influenzae in clinical specimens.

In a previous study, we reported that a 5-kilobase Haemophilus influenzae DNA fragment involved in penicillin-binding protein expression could be used as a probe for specific detection of H. influenzae strains (F. Malouin and L. E. Bryan, Mol. Cell. Probes 1:221-232, 1987). Here, we report the ability of this probe to detect H. influenzae in clinical specimens. In a bacterial dot experiment, there was strong hybridization of the 32P-labeled probe to nonencapsulated and serotype a through f H. influenzae strains. The detection of H. influenzae in body fluids was then evaluated by using pooled human serum, urine, cerebrospinal fluid, and sputum as dilution media for H. influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, and Escherichia coli cells. At 65 degrees C, the probe hybridized to H. influenzae and H. aegyptius (greater than or equal to 10(5) cells) in all fluids. There was no hybridization with the E. coli negative control, and H. parainfluenzae hybridized when greater than or equal to 10(7) cells were used. Experiments performed at 73 and 80 degrees C permitted elimination of H. parainfluenzae hybridization. The detection of H. influenzae in 232 sputa from patients with respiratory tract infections was very specific (96 to 97%) and sensitive (74 to 100%) when the total time of the procedure was sufficient (6 to 24 h) and when the experiments were performed at 80 degrees C. In addition, the probe detected three of three and four of four H. influenzae-infected cerebrospinal fluids and blood cultures, respectively, and did not react with pneumococcus- or streptococcus-infected cerebrospinal fluids. Finally, by using a small-scale procedure, the probe rapidly detected H. influenzae in cerebrospinal fluid and sputum specimens (4 and 8 h, respectively). These results imply prompt diagnosis of H. influenzae infections caused by nonencapsulated and serotype a through f strains.

Mechanism of aminoglycoside antibiotic resistance in anaerobic bacteria: Clostridium perfringens and Bacteroides fragilis.

Cell-free amino acid incorporation using ribosomes from strains of either Clostridium perfringens or Bacteroides fragilis was shown to be susceptible to inhibition by streptomycin and gentamicin. Ribosomes bound dihydrostreptomycin as effectively as ribosomes from Escherichia coli. No inactivation of streptomycin or gentamicin was detected by cell extracts of either anaerobic bacterial species. B. fragilis, grown without added hemin, menadione, and fumarate, and C. perfringens did not show any time-dependent accumulation of dihydrostreptomycin or gentamicin at concentrations tested. Decreased resistance to aminoglycosides and time-dependent uptake of dihydrostreptomycin at 500 mug/ml was observed with B. fragilis grown with hemin, menadione, and fumarate. With the last additions, cytochrome b was detected by cytochrome spectra of B. fragilis. These results demonstrate that anaerobic bacteria unable to carry out oxygen- or nitrate-dependent electron transport are resistant to streptomycin and gentamicin because of failure to transport aminoglycosides. The induction of fumarate-dependent electron transport in B. fragilis is associated with some aminoglycoside transport that is of poor efficiency relative to bacteria with electron transport to oxygen or nitrate.

Effects of membrane-energy mutations and cations on streptomycin and gentamicin accumulation by bacteria: a model for entry of streptomycin and gentamicin in susceptible and resistant bacteria.

Several mutants of Escherichia coli affecting aerobic energy generation and energization of the bacterial membrane have been examined for their effect on streptomycin and gentamicin accumulation and susceptibility. A heme-deficient mutant (K207) and two mutants (CJ-8 [colicin K insensitive] and NR-70) associated with defective aerobic active transport were associated with decreased transport of streptomycin and gentamicin and increased resistance to those antibiotics. These mutants also exhibited increased resistance to several other aminoglycoside antibiotics, but not the aminocyclitol spectinomycin. The same observations were made with a ubiquinone-deficient mutant, but a strA derivative of this mutant was shown additionally to be saturable for streptomycin accumulation at a concentration four or more times lower than that required for saturation of the parent. A mutant uncoupled for adenosine 5'-triphosphate synthesis from electron transport and membrane Mg-adenosine 5'-triphosphatase deficient was hypersensitive to those aminoglycosides tested and spectinomycin, and showed enhanced transport of streptomycin and gentamicin. A variety of compounds structurally related to streptomycin were examined at high concentrations for inhibition of streptomycin uptake in a strA mutant of E. coli K-12 SA 1306, but no evidence for competition was detected, suggesting the absence of a common transport carrier. Four different divalent cations were shown to inhibit streptomycin and gentamicin accumulation in E. coli K-12 SA 1306. Divalent cations were shown to inhibit uptake of these two drugs in two bacterial species with distinct cell wall structures, Pseudomonas aeruginosa and Staphylococcus aureus, and to inhibit streptomycin uptake in spheroplasts of streptomycin-susceptible and -resistant E. coli. However, calcium had almost no inhibitory effect on streptomycin uptake by the ubiquinone-deficient mutant E. coli AN66. These and previous findings have been used to formulate a model for aminoglycoside entry into bacteria using a low-affinity membranous complex involved in membrane energization that includes respiratory quinones, which probably act to bind and transport aminoglycosides across the cell membrane. This phase of transport is associated with the lowest accumulation rate (termed energy-dependent phase I) that is rate limiting for susceptibility. It is further proposed that subsequent association of the membrane-bound aminoglycoside with higher-affinity binding sites on membrane-associated ribosomes carrying out a normal ribosomal cycle and protein synthesis results in a more rapid transport rate (termed energy-dependent phase II). The increased rate could result from a state of membrane energization analogous to that causing enhanced aminoglycoside transport rates seen in the uncoupled mutant, AN120. How this model explains the mechanism by which enzymatically modified aminoglycosides render cells resistant to unmodified aminoglycosides is also discussed.

Gentamicin resistance in clinical-isolates of Pseudomonas aeruginosa associated with diminished gentamicin accumulation and no detectable enzymatic modification.

Three strains of Pseudomonas aeruginosa resistant to gentamicin obtained as representative gentamicin-resistant clinical isolates from the University of Alberta Hospital (UAH) in Edmonton, Canada were characterized to determine their mechanism of resistance. All strains showed wide aminoglycoside resistance (tobramycin, sisomicin, amikacin, streptomycin, kanamycin, SCH 20569) but contained no evidence of gentamicin-acetylating, adenylating or phosphorylating activity. Gentamicin inhibited amino-acid incorporation in cell-free systems equally well with either ribosomes or soluble cell fractions obtained from either resistant or sensitive strains. Plasmid DNA was detected in two strains but resistance could not be transferred by conjugation to either P. aeruginosa or Escherichia coli recipients. The resistant strains showed a marked reduction in energy-dependent accumulation of gentamicin compared to a sensitive strain. These strains which are common at UAH are most likely resistant due to a failure of gentamicin to be transported across the cytoplasmic membrane to ribosomal sites until relatively high external gentamicin concentrations.

Streptomycin accumulation in susceptible and resistant strains of Escherichia coli and Pseudomonas aeruginosa.

Streptomycin accumulation by susceptible strains of Escherichia coli and Pseudomonas aeruginosa has been shown to be prevented or inhibited by inhibitors of electron transport, sulfhydryl groups and protein synthesis, and agents that uncouple oxidative phosphorylation. Streptomycin is recovered from cells in an unchanged form and is intracellularly concentrated above extracellular concentrations. Accumulation kinetics are multiphasic; an initial phase which cannot be prevented by the above inhibitors is unable to cause inhibition of cell growth or loss of cell viability. Prevention of further phases of uptake does prevent these events. Inhibitor-susceptible accumulation is time dependent and begins almost immediately upon exposure of cells to streptomycin. Streptomycin accumulation remains energy dependent even when cells are losing acid-soluble [(3)H]adenine, presumably through loss of permeability control. These results demonstrate that streptomycin accumulation necessary for inhibition of cell growth or cell death requires energy and is not a process of diffusion or secondary to membrane leakage. Streptomycin accumulation in ribosomally resistant mutants of E. coli and P. aeruginosa is similar in that both energy-independent and energy-dependent accumulation can be demonstrated. The total energy-dependent accumulation is, however, significantly lower than that in streptomycin-susceptible cells due to the absence of an additional energy-dependent phase of accumulation, which seems dependent on ribosomal binding of streptomycin. Ribosomally resistant strains can be shown to concentrate streptomycin accumulated by the energy-dependent process above the external concentration in nutrient broth but not in Trypticase soy broth. The energy-dependent accumulation can be saturated in the Str(r) strain of E. coli in nutrient broth, implying limited accumulation sites.

Gentamicin accumulation by sensitive strains of Escherichia coli and Pseudomonas aeruginosa.

Gentamicin accumulation with time shows multiphasic kinetics in strains of Escherichia coli and Pseudomonas aeruginosa. All but first phase accumulation may be prevented or reduced by inhibitors of electron transport, by a sulfhydryl poison, by agents which uncouple electron transport and oxidative phosphorylation and by an inhibitor of protein synthesis. The phases of accumulation which are sensitive to these inhibitors are required for loss of cell viability. Gentamicin can be extracted from cells in an unchanged form as judged by paper chromatography and is concentrated 4 to 250 times over extracellular concentrations within the bacterial cell. Gentamicin accumulation has been shown to occur before there is any evidence or release of acid-soluble 3H-adenine from cells. These data demonstrate that productive gentamicin accumulation capable of causing cell death is by active transport.

Gentamicin resistance in Pseudomonas aeruginosa: R-factor-mediated resistance.

By disk diffusion antimicrobial susceptibility testing, 11% of 313 consecutive strains of Pseudomonas aeruginosa, examined during July to October 1973, were resistant to gentamicin (minimal inhibitory concentration 12.5 to >100 mug/ml), and a further 31% were moderately resistant (6.25 to 12.5 mug/ml) to gentamicin at the University of Alberta Hospital in Edmonton, Canada. Of 45 gentamicin-resistant strains from that hospital, none possessed R-factors or gentamicin-inactivating enzymes. Eight of 13 strains obtained from three American sources, which contained gentamicin-acetylating (12 strains) or -adenylating (1 strain) activity, conjugally transferred both gentamicin resistance and antibiotic-inactivating activity. P. aeruginosa recipients were much more effective for detection of transferable gentamicin resistance than Escherichia coli recipients, although not all P. aeruginosa were equally as effective as recipients. One strain, POW 151, transferred resistance to both carbenicillin and gentamicin as well as to several other antibiotics. R-factors detected belonged to P-2 and P-3 (Com 6, C) incompatibility groups. Expression of gentamicin resistance due to acetylation of gentamicin was subject to marked phenotypic lag, especially in recipient strain P. aeruginosa 280. This was shown to result in the failure to detect gentamicin resistance transfer if the concentration of gentamicin in selection media was too high (>2.5 mug/ml for strain 280). Some but not all recipients were changed in pyocine type upon acquisition of R-factors.

Characteristics of R931 and other Pseudomonas aeruginosa R factors.

R factors were detected in 3.3% of 233 hospital isolates of Pseudomonas aeruginosa using P. aeruginosa recipients in conjugations. Transferred markers included streptomycin, tetracycline, and sulfonamide resistance. Gentamicin resistance was transferred from two strains previously shown to acetylate gentamicin. A group of R factors exemplified by R931 were characterized by failure to transfer to Escherichia coli recipients. Such R factors formed a single compatibility group when examined in a P. aeruginosa recipient. Other P. aeruginosa R factors, including RP4, showed stable coexistence with the R931 group. It is proposed that RP4 and similar R factors be members of the P-1 compatibility group and that R931, R3108, R209, and R130 be members of a group termed P-2. The buoyant densities of all R factors examined were similar, about 1.716 to 1.719 g/cm(3). The content of R-factor deoxyribonucleic acid (DNA) relative to the total DNA varied among the different R factors, ranging from about 18 +/- 2% in log-phase cells of 931 (R931) to undetectable for 679 (R679). However, R679, which transferred from strain 679 at extremely low and irregular frequencies to an E. coli host, was shown to represent about 4% R-factor DNA in that host. The relative DNA content of R931 appeared to decline in the stationary growth phase of 931 (R931) or 280 (R931). R931 covalently closed circular DNA was isolated by ethidium bromide-CsCl gradient centrifugation and examined by electron microscopy. Two major molecular distributions existed, having contour lengths of 0.5 and 12.4 mum. The molecular weights were estimated to be 10(6) and 25 x 10(6). Both molecules were under relaxed replication control. R factor R931 exists as a naturally occurring high-frequency transfer system in P. aeruginosa strains 931 and 1310. However, in strain 280 it acts as if subject to fertility repression. Other members of the P-2 compatibility group also are high-frequency transfer systems in the natural host and in strain 1310. RP4 is restricted from recipient strain 1310. Some additional recipient effects were noted in that strains 1310 or 280 sometimes differed in recipient effectiveness with a given donor. Agglutination reactions with absorbed antiserum were able to distinguish between two members of the same R-factor compatibility group, R931 and R3108.

Transferable drug resistance in Pseudomonas aeruginosa.

Three strains of Pseudomonas aeruginosa were demonstrated to transfer double-drug resistance by conjugation to a P. aeruginosa recipient at frequencies of 10(-4) to 10(-2) per recipient cell. Two of the three strains also transferred to Escherichia coli at frequencies which were 10(3)- to 10(5)-fold lower, but the third strain could not be demonstrated to do so. The latter strain, however, conferred maleness on the Pseudomonas recipient. The transfer of streptomycin resistance was associated with the acquisition of streptomycin phosphorylase by both P. aeruginosa and E. coli recipients. Maximal broth mating frequencies were obtained with nonagitated cultures less than 1 mm in depth. A pyocine selection system based on donor sensitivity and recipient resistance is described and appears to have future value as a generalized selective device for use after matings.

Mechanisms and spectrum of streptomycin resistance in a natural population of Pseudomonas aeruginosa.

A survey of 200 strains of Pseudomonas aeruginosa isolated from clinical specimens was made in an attempt to correlate the spectrum of their streptomycin resistance and the mechanism of resistance. The strains can be classified into three groups, according to their level of resistance to streptomycin: susceptible, low-level resistant, and high-level resistant strains. The mechanism of resistance of high-level resistant strains is either an R factor-mediated inactivation of streptomycin by phosphorylation or streptomycin-resistant ribosomes. However, such high-level resistant strains comprised less than 10% of the total strains isolated; the majority of the strains resistant to streptomycin were of low-level resistance. The latter are associated with a diminished uptake of streptomycin, and no evidence of streptomycin inactivation, resistant ribosomes, or R factors could be detected. The most probable explanation of low-level resistance is reduced permeability to streptomycin. Modification of the growth medium used in uptake studies simultaneously affected strongly both streptomycin incorporation and the minimal inhibitory concentration of streptomycin.