The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E-coli strains in human urine

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract. The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. Deliberate colonization of UTI-susceptible individuals with E. coli 83972 has been used successfully as an alternative approach for the treatment of patients who are refractory to conventional therapy. Colonization with strain 83972 appears to prevent infection with UPEC strains in such patients despite the fact that this strain is unable to express the primary adhesins involved in UTI, viz. P and type 1 fimbriae. Here we investigated the growth characteristics of E. coli 83972 in human urine and show that it can outcompete a representative spectrum of UPEC strains for growth in urine. The unique ability of ABU E. coli 83972 to outcompete UPEC in urine was also demonstrated in a murine model of human UTI, confirming the selective advantage over UPEC in vivo. Comparison of global gene expression profiles of E. coli 83972 grown in lab medium and human urine revealed significant differences in expression levels in the two media; significant down-regulation of genes encoding virulence factors such as hemolysin, lipid A, and capsular pollysaccharides was observed in cells grown in urine. Clearly, divergent abilities of ABU E. coli and UPEC to exploit human urine as a niche for persistence and survival suggest that these key differences may be exploited for preventative and/or therapeutic approaches.

Measurement of total phospholipids in urine of patients treated with gentamicin

Aims The excretion of phospholipids in urine may be a marker of the early renal toxicity of the aminoglycoside antibiotics. Urinary phospholipids are formed in myeloid bodies which develop in the lysosomes of proximal tubules during treatment with the aminoglycosides, and overflow into the urine. Methods Published assays were modified in order to measure the total phospholipid concentrations in human urine. Phospholipids were extracted from freeze-dried urine samples, digested in concentrated sulphuric acid, and the inorganic phosphorus content determined by complexing with ammonium molybdate and measuring the absorbance at 820 nm. Ten septicaemic patients treated with gentamicin for 5-7 days had significantly higher urine phospholipid concentrations than 10 healthy untreated control subjects (P<0.0001). There was a negative Linear relationship between phospholipid excretion and creatinine clearance (r(2) = 0.71). Results In 34 patients with acute pyelonephritis, increased phospholipid concentrations were observed prior to treatment compared with healthy controls (P<0.001) and did not alter during treatment with gentamicin. However, the phospholipid concentrations decreased significantly after treatment was completed (P<0.03). Conclusions These studies suggest that urinary phospholipids may indicate early aminoglycoside toxicity but with poor specificity, as many of the infections being treated may themselves be associated with phospholipiduria.

The excitatory behavioral and antianalgesic pharmacology of normorphine-3-glucuronide after intracerebroventricular administration to rats

In the adult male Sprague-Dawley rat, a species commonly used to study tolerance to the antinociceptive effects of morphine, approximate to 10% of the morphine dose is metabolized to normorphine-3-glucuronide (NM3G). In contrast, NM3G is a relatively minor metabolite of morphine in human urine reportedly accounting for approximate to 1% of the morphine dose. To date, the pharmacology of NM3G has been poorly characterized. Therefore, our studies were designed to determine whether the intrinsic pharmacology of NM3G is similar to that of morphine-3-glucuronide (M3G), the major metabolite of morphine, which has been shown to be a potent central nervous system (CNS) excitant and to attenuate the intrinsic antinociceptive effects of morphine in rats. The CNS excitatory potency of NM3G was found to be approximately half that of M3G, inducing convulsions in rats at intracerebroventricular (i.c.v.) doses of greater than or equal to 16.8 nmol. When administered before morphine (70 nmol i.c.v.), NM3G (8.9 nmol i.c.v.) attenuated antinociception for up to 2 hr, but when administered after morphine, no significant attenuation of morphine antinociception was observed. Thus, after i.c.v. administration, NM3G like M3G, is a potent CNS excitant and antianalgesic in the rat. NM3G may therefore play a role in the development of tolerance to the antinociceptive effects of morphine in the rat as has been proposed previously for M3G.

Asymptomatic bacteriuria Escherichia coli strain 83972 carries mutations in the foc locus and is unable to express F1C fimbriae

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infection (UTI), very little is known about the mechanisms by which these strains colonize the urinary tract. Bacterial adhesion conferred by specific surface-associated adhesins is normally considered as a prerequisite for colonization of the urinary tract. The prototype ABU E coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. This study characterized the molecular status of one of the primary adhesion factors known to be associated with UTI, namely F1C fimbriae, encoded by the foc gene cluster. F1C fimbriae recognize receptors present in the human kidney and bladder. Expression of the foc genes was found to be up-regulated in human urine. It was also shown that although strain 83972 contains a seemingly intact foc gene cluster, F1C fimbriae are not expressed. Sequencing and genetic complementation revealed that the focD gene, encoding a component of the F1C transport and assembly system, was non-functional, explaining the inability of strain 83972 to express this adhesin. The data imply that E. coli 83972 has lost its ability to express this important colonization factor as a result of host-driven evolution. The ancestor of the strain seems to have been a pyelonephritis strain of phylogenetic group B2. Strain 83972 therefore represents an example of bacterial adaptation from pathogenicity to commensalism through virulence factor loss.

Determination of pindolol enantiomers in human plasma and urine by simple liquid-liquid extraction and high-performance liquid chromatography

A simple method for the measurement of pindolol enantiomers by HPLC is presented. Alkalinized serum or urine is extracted with ethyl acetate and the residue remaining after evaporation of the organic layer is then derivatised with (S)-(-)-alpha-methylbenzyl isocyanate. The diastereoisomers of derivatised pindolol and metoprolol (internal standard) are separated by high-performance liquid chromatography (HPLC) using a C-18 silica column and detected using fluorescence (excitation lambda: 215 nm, emission lambda: 320 nm). The assay displays reproducible linearity for pindolol enantiomers with a correlation coefficient of r(2) greater than or equal to 0.998 over the concentration range 8-100 ng ml(-1) for plasma and 0.1-2.5 mu g ml(-1) for urine. The coefficient of variation for accuracy and precision of the quality control samples for both plasma and urine are consistently

Detection and differentiation of human polyomaviruses JC and BK by LightCycler PCR

Human polyomaviruses JC and BK may cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy and hemorrhagic cystitis. Molecular detection by PCR is recognized as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, a real-time PCR assay using the LightCycler platform was evaluated and compared to an in-house PCR assay using a conventional detection method. A total of 122 urine specimens were tested, and human polyomavirus was detected in 49 specimens (40%) by both conventional PCR and LightCycler PCR. The remaining 73 specimens (60%) were found negative by both assays. For 46 of the 49 positive specimens, LightCycler PCR and conventional PCR identified the same polyomavirus type. These samples included 30 samples with JC virus (JCV), 14 samples with BK virus (BKV), and 2 samples in which both viruses were detected. In the remaining three samples, both JCV and BKV were detected by the conventional assay, but only JCV was detected by the LightCycler assay. The results of this study show that the LightCycler PCR assay displays sensitivity and specificity similar to those of a conventional PCR assay. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid detection and differentiation of JCV and BKV in the clinical laboratory.

Cadmium levels in the lung, liver, kidney cortex, and urine samples from Australians without occupational exposure to metals

The authors undertook this study to assess levels of cadmium exposure in the general population. Samples of lung, liver, and kidney were obtained from 61 cadavers (43 males, 18 females; 2-89 yr of age, mean age = 38.5 yr) who died from accidental causes and who were subject to postmortem examinations at the John Tonge Centre for Forensic Sciences, Queensland Health Scientific Services, Brisbane, Australia, in 1997 and 1998. Samples of bladder urine were also obtained from 22 cadavers. Tissue and urine samples were analyzed for cadmium, zinc, and copper with inductively coupled plasm (ICP) mass spectrometry. The overall mean values for cadmium in the lung, liver, and kidney cortex samples were 0.13, 0.95, and 15.45 mug/gm wet tissue weight. The average renal cadmium level in subjects with high lung-cadmium levels (n = 13) was 6 mug/gm wet tissue weight higher than that of similarly aged subjects who had medium lung-cadmium levels (n = 30). In females, the average level of cadmium in the liver was 74% greater than in males, and the average liver cadmium in females with high lung-cadmium levels was 100% higher than in males in the same age range who had the same high lung-cadmium levels. Renal cadmium accumulation tended to be greater in females than in males who were in the same age range and who had similar lung-cadmium levels, a result that suggested that there was a higher absorption rate of cadmium in females. The mean value for a urinary cadmium excretion of 2.30 mug/gm creatinine was found in a subset of samples that had a mean age of 39 yr and a renal cortex cadmium concentration of 18.6 mug/gm wet tissue weight. Urinary cadmium excretion rates were correlated more strongly with lung and kidney cadmium content than with age or liver cadmium levels. The results suggest that urinary cadmium excretion may be increased in smokers and could provide some estimate of body cadmium burdens in future Australian epidemiological studies.

Simultaneous detection and differentiation of human Polyomaviruses JC and BK by a rapid and sensitive PCR-ELAHA assay and a survey of the JCV subtypes within an Australian population

Human polyomaviruses JCV and BKV can cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy (PML) and haemorrhagic cystitis. Molecular detection by polymerase chain reaction (PCR) is recognised as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, we developed a PCR assay using a single primer pair to amplify a segment of the VP1 gene of JCV and BKV. An enzyme linked amplicon hybridisation assay (ELAHA) using species-specific biotinylated oligonucleotide probes was used to differentiate between JCV and BKV. This assay (VP1-PCR-ELAHA) was evaluated and compared to a PCR assay targeting the human polyomavirus T antigen gene (pol-PCR). DNA sequencing was used to confirm the polyomavirus species identified by the VP1-PCR-ELAHA and to determine the subtype of each JCV isolate. A total of 297 urine specimens were tested and human polyomavirus was detected in 105 specimens (35.4%) by both PCR assays. The differentiation of JCV and BKV by the VP1-PCR-ELAHA showed good agreement with the results of DNA sequencing. Further, DNA sequencing of the JCV positive specimens showed the most prevalent JCV subtype in our cohort was 2a (27%) followed by 1b (20%), 1a (15%), 2c (14%), 4 (14%) and 2b (10%). The results of this study show that the VP1-PCR-ELAHA is a sensitive, specific and rapid method for detecting and differentiating human polyomaviruses JC and BK and is highly suitable for routine use in the clinical laboratory. (C) 2004 Wiley-Liss, Inc.

The measurement of nicotine in human plasma by high-performance liquid chromatography-electrospray-tandem mass spectrometry

The authors describe a reverse-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry method for the measurement of nicotine in human plasma. Samples (500 muL) with added deuterium-labeled d(3)-nicotine as an internal standard (IS) were treated with a 2-step process of ether extraction (6 mL) followed by back-extraction into 0.1% formic acid (50 muL). Chromatography was performed on a phenyl Novapak column with a mobile phase consisting of 50% 10 mM ammonium fortriate (pH 3.3) and acetonitrile (50:50, vol/vol). A flow rate of 0.2 mL/min resulted in a total analysis time of 5 minutes per sample. Mass spectrometric detection was by selected reactant monitoring (nicotine m/z 163.2 --> 130.2; IS m/z 166.2 --> 87.2). The assay was linear from 0.5 to 100 mug/L (r > 0.993, n = 9). The accuracy and imprecision of the method for quality control sampleswere 87.5% to 113% and < 10.2%, respectively. Interday accuracy and imprecision at the limit of quantification (0.5 mug/L) was 113% and 7.2% (n = 4). The process efficiency for nicotine in plasma was > 75%. The method described has good process efficiency, stabilized nicotine, avoided concentration steps, and most importantly minimized potential contamination. Further, we have established that water-based standards and controls are interchangeable with plasma-based samples. This method was used successfully to measure the pharmacokinetic profiles of subjects involved in the development of an aerosol inhalation drug delivery system.

Effects of chronic exposure to low-level cadmium on renal tubular function and CYP2A6-mediated coumarin metabolism in healthy human subjects

Relationships between cadmium (Cd) body burden, kidney function and coumarin metabolism were investigated using two groups of 197 and 200 healthy Thais with men and women in nearly equal numbers. A mean age of one group was 30.5 years and it was 39.3 years for the other group. Of 397, 20 subjects (5%) excreted urine Cd between 1.4 mug/g and 3.8 mug/g creatinine and these subjects faced 10-15% increase in the probability of having abnormal urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG-uria). The prevalence of NAG-uria varied with Cd body burden in a dose-dependent manner (chi(2) = 22, P < 0.008). Also NAG-nuria was one of the three kidney effect markers tested that showed the greatest strength of correlation with urine Cd in both men and women (r = 0.48 P < 0.001). In addition, urine Cd excretion of men and women showed a positive correlation (r = 0.46 to 0.54. P < 0.001) with urine 7-hydroxycoumarin (7-OHC) excretion which was used as a marker of liver cytochrome P450 2A6 (CYP2A6) enzyme activity. Urinary CA excretion accounted for 25% of the total variation in urine 7-OHC excretion (P < 0.001). These data suggest that Cd may increase the expression of CYP2A6 in liver, resulting in enhanced coumarin metabolism in subjects with high Cd body burden. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Central and peripheral mediation of human force sensation following eccentric or concentric contractions

Fatigue was induced in the triceps brachii of the experimental arm by a regimen of either eccentric or concentric muscle actions. Estimates of force were assessed using a contralateral limb-matching procedure, in which target force levels (25 %, 50 % or 75 % of maximum) were defined by the unfatigued control arm. Maximum isometric force-generating capacity was reduced by 31 % immediately following eccentric contractions, and remained depressed at 24 (25 %) and 48 h (13 %) post-exercise. A less marked reduction (8.3 %) was observed immediately following concentric contractions. Those participants who performed prior eccentric contractions, consistently (at all force levels), and persistently (throughout the recovery period), overestimated the level of force applied by the experimental arm. In other words, they believed that they were generating more force than they actually achieved. When the forces applied by the experimental and the control arm, were each expressed as a proportion of the maximum force that could be attained at that time, the estimates matched extremely closely. This outcome is that which would be expected if the estimates of force were based on a sense of effort. Following eccentric exercise, the amplitude of the EMG activity recorded from the experimental arm was substantially greater than that recorded from the control arm. Cortically evoked potentials recorded from the triceps brachii (and extensor carpi radialis) of the experimental arm were also substantially larger than those elicited prior to exercise. The sense of effort was evidently not based upon a corollary of the central motor command. Rather, the relationship between the sense of effort and the motor command appears to have been altered as a result of the fatiguing eccentric contractions. It is proposed that the sense of effort is associated with activity in neural centres upstream of the motor cortex.

The effects of viscous loading of the human forearm flexors on the stability of coordination

This experiment investigated whether the stability of rhythmic unimanual movements is primarily a function of perceptual/spatial orientation or neuro-mechanical in nature. Eight participants performed rhythmic flexion and extension movements of the left wrist for 30 s at a frequency of 2.25 Hz paced by an auditory metronome. Each participant performed 8 flex-on-the-beat trials and 8 extend-on-the-beat trials in one of two load conditions, loaded and unload. In the loaded condition, a servo-controlled torque motor was used to apply a small viscous load that resisted the flexion phase of the movement only. Both the amplitude and frequency of the movement generated in the loaded and unloaded conditions were statistically equivalent. However, in the loaded condition movements in which participants were required to flex-on-the-beat became less stable (more variable) while extend-on-the-beat movements remained unchanged compared with the unload condition. The small alteration in required muscle force was sufficient to result in reliable changes in movement stability even a situation where the movement kinematics were identical. These findings support the notion that muscular constraints, independent of spatial dependencies, can be sufficiently strong to reliably influence coordination in a simple unimanual task.

Excitability changes in human forearm corticospinal projections and spinal reflex pathways during rhythmic voluntary movement of the opposite limb

Rhythmic movements brought about by the contraction of muscles on one side of the body give rise to phase-locked changes in the excitability of the homologous motor pathways of the opposite limb. Such crossed facilitation should favour patterns of bimanual coordination in which homologous muscles are engaged simultaneously, and disrupt those in which the muscles are activated in an alternating fashion. In order to examine these issues, we obtained responses to transcranial magnetic stimulation (TMS), to stimulation of the cervicomedullary junction (cervicomedullary-evoked potentials, CMEPs), to peripheral nerve stimulation (H-reflexes and f-waves), and elicited stretch reflexes in the relaxed right flexor carpi radialis (FCR) muscle during rhythmic (2 Hz) flexion and extension movements of the opposite (left) wrist. The potentials evoked by TMS in right FCR were potentiated during the phases of movement in which the left FCR was most strongly engaged. In contrast, CMEPs were unaffected by the movements of the opposite limb. These results suggest that there was systematic variation of the excitability of the motor cortex ipsilateral to the moving limb. H-reflexes and stretch reflexes recorded in right FCR were modulated in phase with the activation of left FCR. As the f-waves did not vary in corresponding fashion, it appears that the phasic modulation of the H-reflex was mediated by presynaptic inhibition of Ia afferents. The observation that both H-reflexes and f-waves were depressed markedly during movements of the opposite indicates that there may also have been postsynaptic inhibition or disfacilitation of the largest motor units. Our findings indicate that the patterned modulation of excitability in motor pathways that occurs during rhythmic movements of the opposite limb is mediated primarily by interhemispheric interactions between cortical motor areas.

Spatial, temporal, and human dimensions of air pollution in Brisbane

Arriving in Brisbane some six years ago, I could not help being impressed by what may be prosaically described as its atmospheric amenity resources. Perhaps this in part was due to my recent experiences in major urban centres in North America, but since that time, that sparkling quality and the blue skies seem to have progressively diminished. Unfortunately, there is also objective evidence available to suggest that this apparent deterioration is not merely the result of habituation of the senses. Air pollution data for the city show trends of increasing concentrations of those very substances that have destroyed the attractiveness of major population centres elsewhere, with climates initially as salubrious. Indeed, present figures indicate that photochemical smog in unacceptably high concentrations is rapidly becoming endemic also over Brisbane. These regrettable developments should come as no surprise. The society at large has not been inclined to respond purposefully to warnings of impending environmental problems, despite the experiences and publicity from overseas and even from other cities within Australia. Nor, up to the present, have certain politicians and government officials displayed stances beyond those necessary for the maintenance of a decorum of concern. At this stage, there still exists the possibility for meaningful government action without the embarrassment of losing political favour with the electorate. To the contrary, there is every chance that such action may be turned to advantage with increased public enlightenment. It would be more than a pity to miss perhaps the final remaining opportunity: Queensland is one of the few remaining places in the world with sufficient resources to permit both rational development and high environmental quality. The choice appears to be one of making a relatively minor investment now for a large financial and social gain the near future, or, permitting Brisbane to degenerate gradually into just another stagnated Los Angeles or Sydney. The present monograph attempts to introduce the problem by reviewing the available research on air quality in the Brisbane area. It also tries to elucidate some seemingly obvious, but so far unapplied management approaches. By necessity, such a broad treatment needs to make inroads into extensive ranges of subject areas, including political and legal practices to public perceptions, scientific measurement and statistical analysis to dynamics of air flow. Clearly, it does not pretend to be definitive in any of these fields, but it does try to emphasize those adjustable facets of the human use system of natural resources, too often neglected in favour of air pollution control technology. The crossing of disciplinary boundaries, however, needs no apology: air quality problems are ubiquitous, touching upon space, time and human interaction.