Adult trauma patients admitted to the 22-bed surgical ICU from the university hospital of Bicêtre (AP-HP, France) with an Injury Severity Score (ISS) 13 higher than 16 and requiring an indwelling urethral catheter were eligible for enrolment. Patients were not included if (i) antibiotic therapy was predicted to be necessary for clinical care when admitted to the ICU (except for prophylactic antibiotics), (ii) hospitalization occurred before ICU admission, (iii) the expected duration of stay in the ICU was shorter than 5 days, (iv) they were less than 18 years of age or pregnant, or (v) they had genitourinary trauma. The ethics committee of the Lariboisière Hospital (Paris, France) agreed to the protocol used in this research. The informed written consent was deemed non applicable as patient samples were taken as part of standard care.

The demographic characteristics of each patient, including age and gender, were recorded. The severity of illness was assessed by Simplified Acute Physiology Score (SAPS) II 14 , Systemic Organ Failure Assessment (SOFA) score 15 , and ISS at admission to the ICU, and (for the SOFA score) at five days. The following clinical and laboratory variables were recorded at the time of urine collection, at the first and the fifth days of ICU hospitalisation: (i) interventions including antibiotic administration from the first to the fifth day (antibiotic used, dose and duration which allowed us to determine whether antibiotics were given for prophylaxis or treatment), invasive mechanical ventilation and catecholamine infusion; (ii) biochemical data including arterial lactate (normal values: 0.5 to 2.2 mmol/L), plasma pH (7.40 ± 0.2), 24-hour urine flow, serum creatinine (normal values: 60 to 120 μmol/L), creatinine clearance (normal values > 80 mL/min), blood urea (normal values: 4 to 7 mmol/L), plasma creatinine kinase level (normal values < 250 U/L) ; (iii) and urinary bacteriology.

Sterile urine samples over 24 hours were collected with an indwelling urinary catheter at day 1 (D1) and day 5 (D5) of the stay in the ICU. After centrifugation and filtration (0.22 μm pore size), urine samples were stored at −20°C.

Sterile urine from 6 healthy male volunteers (HV) aged between 22 and 50 years old [median of 28 years, (IQR, 25.5, 32.5)] with no history of UTIs or antibiotic use during the previous two months was collected. Furthermore, HV were free of iron intake. Before being stored at 4°C if used in the following 48 hours, or at – 20°C, midstream urine from at least three of the six HV were pooled. Twelve urine batches were studied.

The volume, biochemistry sodium (normal values: 50 to 200 mmol/24 hours), potassium (normal values: 50 to 150 mmol/24 hours), urea (normal values: 200 to 500 mmol/24 hours) and creatinine (normal values: 9 to 16 mmol/24 hours), pH (normal values: 5 to 7), osmolality (normal values: 700 to 1000 mosm/L, glucose, protein (normal values < 0.1 g/24 hours) and urinary sediment were recorded for each urine sample. Urinary glucose concentration higher than 0 mmol/l was considered as abnormal, based on the pathology lab‘s definition. The concentration of free iron was measured by spectrophotometry on a Modular PP analyser (Fer/Roche®; Roche Diagnostic) in urine of patients and HV. Urine samples were firstly acidified to a pH <2 to separate iron from carrier proteins. As described previously 16 , ascorbate was added to allow the reduction of Fe³⁺. Then, FerroZine was used to determine the free iron concentration.

The urinary concentrations of nitrates were measured as described previously 17 . The levels of amino acids were determined using High Performance Level Chromatography (HPLC) on 24 samples (9 from urine of HV, 15 from 8 patients (8 samples collected on day 1, and 7 on day 5) 18 . The concentrations of norepinephrine, dopamine and epinephrine, which are known to influence bacterial growth, were measured on 24 samples from patients (12 samples collected on day 1 and 12 collected on day 5). Catecholamines were extracted from urine by ion exchange chromatography followed by alumina adsorption. Extracted catecholamines were separated and quantified by an HPLC method with amperometric detection 19 20 . This method is specific for catecholamines and does not detect oxidized derivatives such as o-quinone or adrenochrome. The concentrations of urinary catecholamines of trauma patients were compared with reference values measured in a healthy population (dopamine < 630 μg/g of creatinine, epinephrine <16 μg/g of creatinine and norepinephrine < 77μg/g of creatinine) 19 .

Two strains of E. coli were studied. E. coli CFT073, the prototype of the uropathogenic (UPEC) strains, was originally isolated from the blood and urine of a woman admitted to the University of Maryland Medical System (USA) 21 . E. coli CFT073 is fully sensitive to antibiotics. The second strain is E. coli R1B8J20 isolated during a septicaemia recorded in the prospective COLIBAFI study 22 . E. coli R1B8J20 is resistant to beta-lactams due to over-production of AmpC cephalosporinase. The minimal inhibitory concentrations for the following antibiotics, determined using an E-test method, were: ceftazidime >256 mg/L, cefoxitin >192 mg/L, cefepime = 16 mg/L, cefotaxime = 12 mg/L, amoxicillin-clavulanic acid >256 mg/L. This strain was also resistant to quinolones, kanamycin, streptomycin and sulfonamides. E. coli R1B8J20 allowed an evaluation of bacterial growth in urine of patients who had received antibiotics.

Both E. coli strains were cultured in 4 ml of Luria Bertani medium for 18 hours at 37°C. The overnight cultures were centrifuged (4500 rpm during 15 min) and washed with 4 ml of isotonic saline serum. Forty ml of urine were inoculated with the volume of the overnight culture required to obtain a starting optical density of 0.01 at 600 nm (OD₆₀₀), which corresponded to 2×10⁶ bacteria/mL, and incubated at 37°C with an agitation at 180 rpm. Optical density was measured at intervals with an Ultrospec 1100 pro (Pharmacia^®) spectrophotometer, until the culture reached stationary phase or at least over 420 minutes.

Experiments were normally carried out using E. coli CFT073. When the patient received prophylactic antibiotics or therapeutic antibiotic therapy before the fifth day that was not predicted at admission, the resistant E. coli R1B8J20 isolate was also studied. Experiments were done in replicates.

To study the role of iron in bacterial proliferation, E. coli CFT073 growth in urine from trauma patients was studied after addition of an iron chelator, desferrioxamine (0.1 mM), using three urine samples collected on the first day from patients who were not on antibiotics. The inhibitory effect of desferrioxamine was expressed by percentage of growth inhibition after 360 minutes of growth.

The effect of norepinephrine was studied after addition of 1 μM, 10 μM or 50 μM to HV urine samples, using the method described by Freestone et al. 23 . These experiments were carried out using two independent HV urine samples.

The clinical and biological characteristics recorded on days 1 and 5 were compared using the Student t test (MedCalc). The biochemical characteristics of urine from trauma patients on days 1 and 5 were compared with the biochemical characteristics of urine from HV. When statistical distribution was Gaussian, mean difference in means and 95% confidence interval (CI) are shown. In cases of non Gaussian statistical distribution, the median difference and non parametric Hodges-Lehman 95% CI are shown. The growth of E. coli CFT073 in urine from patients without antibiotics on day 1 was compared with E. coli CFT073 growth in HV urine, using an analysis of variance for repeated measures (Statview). The same analysis was carried out for urine collected on day 5, and for the resistant strain E. coli R1B8J20. Finally, growth in urines from the first and the fifth days was compared. Spearman’s nonparametric correlation coefficient was used to correlate clinical or biological parameters and growth (Statview). The results are expressed in mean ± standard deviations. Statistical significance was fixed at 0.05 (two-tailed).

Twenty-two patients with severe trauma were recruited to the study over a period of 11 months.

Of these 22 patients, sixteen received prophylactic antibiotics on day 1, and one had a positive bacteriological urine culture (Proteus mirabilis 10⁵/mL) (Figure 1). On day 5, two patients had received antibiotics from day 2 for a hospital-acquired infection, three patients had positive bacteriological samples (>10⁵/mL of P. mirabilis, coagulase-negative Staphylococcus, Enterococcus faecalis) and 4 of 22 samples were not available because the indwelling catheter was removed before day 5 (Figure 1).

The demographic and clinical characteristics are summarized in Table 1. On day 5, the majority of patients showed signs of recovery, demonstrated by a decrease in the SOFA score and the number of patients requiring life support. The significant improvement in biological parameters such as the creatinine clearance and the serum lactate level also demonstrated this trend (Table 2).

MV Mechanical ventilation, ISS Injury Severity Score, SAPS II Simplified Acute Physiology Score, SOFA Systemic Organ Failure Assessment, D1 on day 1, D5 on day 5.

D1 on day 1, D5 on day 5.

On day 1, for urine from five patients who were not treated with an antibiotic, the growth of the susceptible strain, E. coli CFT073, was statistically higher than in HV urine (p = 0.0028) (Figure 2). For the 16 patients who received prophylactic antibiotics on day 1, the growth of E. coli R1B8J20, the resistant strain, was studied in 5 urine samples. Growth was inhibited in 1 sample. The 4 remaining urine samples supported a significantly better growth than HV urine (p = 0.0371) (Figure 3). When tested in HV urine or in TP urine, there was no difference in growth between the two strains (data not shown).

Growth of E. coli CFT073 was evaluated for the 13 urine samples from patients without bacteriuria and without antibiotic on day 5. The growth in these 13 samples was statistically greater than in HV urine (p = 0.0108) (Figure 4). Results similar to the E. coli CFT073 strain were found for E. coli R1B8J20 grown in the 8 tested samples from the 13 patients without antibiotics and compared to HV urine growth (p = 0.0478). One of the 2 patients with antibiotics on day 5 had received gentamicin; as both strains of E. coli were susceptible to this antibiotic, only the sample from the patient who did not received gentamicin was evaluated. The bacterial growth was higher than in HV urine (data not shown).

To test the role of iron in the bacterial growth, we carried out inhibition experiments using an iron chelator in urine from 3 patients who did not received antibiotics at day 1. Addition of desferrioxamine (0.1 mM) in trauma urine suppressed growth, with a decrease in the number of bacteria at the stationary phase of around 40% (± 20%).

The addition of norepinephrine (1 μM, 10 μM and 50 μM) to HV urine does not change E. coli CFT073 strain proliferation in our experimental conditions (data not shown).

On day 1, the levels of urea and creatinine, and the urinary osmolarity, were significantly lower in the urine of trauma patients (TP urine) than in HV urine (Table 3). Moreover, all of the patients had a glycosuria, and 80% of them had subnormal levels of urinary iron (Table 4). Seventy-seven per cent of the patients had received a norepinephrine infusion on day 1. The urinary norepinephrine concentration was above normal values in 75% of patients receiving norepinephrine on day 1 and in 40% of patients who had not received catecholamines (Table 4).

D1 on day 1.

^a Medians difference and Hodges-Lehmann 95% Confidence Interval.

D1 on day 1, D5 on day 5.

On day 5, glycosuria was present in all patients, and proteinuria was higher in TP urine than in HV urine. There was no difference in urinary osmolarity, and urinary iron and urea concentrations between TP and HV urines (Tables 4 and 5). Less than 45% of the patients had a urinary concentration of norepinephrine above normal values (Table 4), and three fourth of them still received catecholamines.

Values are expressed as means ± SD, D5 on day 5, HV healthy volunteers.

^a Medians difference and Hodges-Lehmann 95% Confidence Interval.

Analysis of amino acids revealed an increase of urinary excretion of glutamic acid, valine, methionine, leucine, tyrosine, histidine and phenylalanine during the first 24 hours after trauma (Table 6). By the fifth day post-trauma, the excretion of almost all amino acids had significantly increased (Table 7).

D1 on day 1, HV healthy volunteers.

^a Amino acids known to be particularly important during growth in urine for E. coli (personal data and 11 17 18 24 .

^b Medians difference and Hodges-Lehmann 95% Confidence Interval.

D5 on day 5, HV healthy volunteers.

^a Amino acids known to be particularly important during growth in urine for E. coli (personal data and 11 17 18 24 .

^b Medians difference and Hodges-Lehmann 95% Confidence Interval.