Toxins, butyric acid, and other short-chain fatty acids are coordinately expressed and down-regulated by cysteine in Clostridium difficile.

It was recently found that a mixture of nine amino acids down-regulate Clostridium difficile toxin production when added to peptone yeast extract (PY) cultures of strain VPI 10463 (S. Karlsson, L. G. Burman, and T. Akerlund, Microbiology 145:1683-1693, 1999). In the present study, seven of these amino acids were found to exhibit a moderate suppression of toxin production, whereas proline and particularly cysteine had the greatest impact, on both reference strains (n = 6) and clinical isolates (n = 28) of C. difficile (>99% suppression by cysteine in the highest toxin-producing strain). Also, cysteine derivatives such as acetylcysteine, glutathione, and cystine effectively down-regulated toxin expression. An impact of both cysteine and cystine but not of thioglycolate on toxin yield indicated that toxin expression was not regulated by the oxidation-reduction potential. Several metabolic pathways, including butyric acid and butanol production, were coinduced with the toxins in PY and down-regulated by cysteine. The enzyme 3-hydroxybutyryl coenzyme A dehydrogenase, a key enzyme in solventogenesis in Clostridium acetobutylicum, was among the most up-regulated proteins during high toxin production. The addition of butyric acid to various growth media induced toxin production, whereas the addition of butanol had the opposite effect. The results indicate a coupling between specific metabolic processes and toxin expression in C. difficile and that certain amino acids can alter these pathways coordinately. We speculate that down-regulation of toxin production by the administration of such amino acids to the colon may become a novel approach to prophylaxis and therapy for C. difficile-associated diarrhea.

Correlation between surface and air counts of particles carrying aerobic bacteria in operating rooms with turbulent ventilation: an experimental study.

Airborne contamination with bacteria-carrying particles (cfu/m3) and their sedimentation rate (cfu/m2/h) was compared in an operating room (OR) equipped with two turbulent ventilation systems. One was a thermally based system with inlet of cool clean air at the floor level and evacuation of the air at the ceiling by convection (17 air changes/h). The other was a conventional plenum pressure system with air supply at the ceiling and evacuation at the floor level (16 air changes/h). The study was made during rigidly standardised sham operations (N = 20) performed in the same OR by the same six member team wearing non-woven disposable or cotton clothing. Airborne contamination in the wound and instrument areas was related to the surface contamination rate in the same areas and in addition, on the patient chest and in the periphery of the OR. With the exception of the periphery of the OR, the surface and air contamination rates were highly correlated in both ventilation systems (P = 0.02-0.0006, r2 = 0.52-0.79). This was also true particularly when disposable clothing was used while the correlation was weaker in cotton clothing experiments. An equation describing the relation between surface and air counts is given. Typically, the surface counts were numerically 16-fold the air counts, i.e., the number of colonies sedimenting on four 14 cm-diameter agar plates during 1 h will almost equal the number of airborne cfu per m3. We propose, that sedimentation plates represent not only a technically easier method than air sampling but when correctly used, are also the most realistic indicator of airborne bacterial OR contamination in areas critical for surgery.

Cytokine gene expression during experimental Escherichia coli pyelonephritis in mice.

PURPOSE: We studied nine inflammatory and immunoregulatory cytokines in acute pyelonephritis and urethral obstruction in mice to better understand the processes underlying kidney inflammation and scarring. MATERIALS AND METHODS: Experimental acute pyelonephritis was established in Bki NMRI outbred mice by bladder inoculation of Escherichia coli, followed by 6 h urethral obstruction. The numbers of cytokine mRNA expressing cells for interleukin-1 (IL-1), IL-4, IL-6, IL-10, IL-12, tumor necrosis factor alpha (TNF-alpha), TNF-beta, transforming growth factor beta (TGF-beta) and interferon gamma (IFN-gamma) were determined in the kidneys and spleens from the infected, non-infected but obstructed and untouched mice using in situ hybridization with radio-labelled oligonucleotide probes at 12 h, 48 h and 6 d after release of the urethral obstruction. RESULTS: Kidney cell expression of IL-1, IL-6 and TNF-alpha mRNA was observed already at 12 h and persisted on day 6 in the infected animals. A significant proinflammatory cytokine response occurred also in the non-infected obstructed animals, albeit later and at lower levels. A marked increase of IL-4, IL-10, TGF-beta and IFN-gamma mRNA producing cells was also found in the kidneys of these two groups again with higher levels in the infected animals. Very high numbers of splenocytes expressing mRNA for IL-1 were observed especially in the infected animals. A high proportion of splenocytes further expressed mRNA for IL-6, TNF-alpha, IL-4, IL-10, IFN-gamma and TGF-beta, again with highest numbers in the infected group of animals. CONCLUSIONS: The present study extends previous knowledge about the local and systemic cytokine expression profiles during acute pyelonephritis and after urethral obstruction. Of particular interest was the marked kidney cell expression of mRNA for TGF-beta, presumed to be important both for obstructive and post-infectious renal scarring.

Interleukin-1 alpha and interleukin-6 in the urine, kidney, and bladder of mice inoculated with Escherichia coli.

After bladder inoculation of mice using the pyelonephritogenic Escherichia coli strain DS17, urinary interleukin-1 alpha (IL-1 alpha) peaked at 0.5 h post infection (mean 233 pg/ml), interleukin-6 (IL-6) at 2 h (mean 572 pg/ml), and leukocyturia at 4 h, all three persisting for more than 24 h. In the kidneys IL-1 alpha peaked at 2 h, persisted over 24 h (mean 900-1,000 pg/ml), and decreased over 2-6 days post inoculation to a mean value of 208 pg/ml. Control kidneys showed low IL-1 alpha values. IL-6 in the kidneys peaked at 5 h (mean 9,999 pg/ml) but normalized, i.e., similar to control kidneys, by 48 h (mean 105 pg/ml). A similar cytokine response, but with tenfold lower levels, was found in the bladder tissue. The isogenic P-negative E. coli mutant DS178 elicited lower IL-6 in the kidneys at 5 h, but persisted in higher numbers in the kidneys at 6 days. Bacterial counts and cytokine levels correlated both in the kidneys and in bladder tissue, (r = 0.50-0.76, P < 0.001). Characterization of the immune response gives a better understanding of the relative importance of different bacterial characteristics for the local inflammatory process and is needed for studies of its pharmacological down-regulation.

Elevated cytokine levels in tracheobronchial aspirate fluids from ventilator treated neonates with bronchopulmonary dysplasia.

UNLABELLED: Bronchopulmonary dysplasia (BPD) is a chronic lung disease often occurring in ventilator-treated very low birth weight infants. The aetiology of BPD is multifactorial and pulmonary immaturity, high oxygen concentrations, peak inspiratory pressure levels and large tidal volumes during prolonged mechanical ventilation are important factors. We measured in tracheobronchial aspirate fluid (TAF) the concentrations of the pro-inflammatory cytokines tumour necrosis factor alpha, interleukin-1 beta (IL-1 beta), IL-6, IL-8, and IL-1 receptor antagonist in infants requiring artificial ventilation for BPD (n = 17) or respiratory distress syndrome (RDS) (n = 15) or postoperatively after surgery (n = 15). The median levels of all studied cytokines in TAF were higher in infants with BPD without local or systemic corticosteroid treatment compared to the median TAF levels of BPD neonates treated with corticosteroids (P = 0.06-P < 0.01). The neonates with BPD not treated with corticosteroids also showed higher levels of the five studied cytokines in TAF compared to infants on short-time ventilator treatment (P < 0.01-P < 0.001) and compared to neonates with RDS (P = 0.07-P < 0.001). The corticosteroid treated neonates with BPD had TAF cytokine levels approaching those of the control neonates. CONCLUSION: Tumour necrosis factors alpha, IL-1 beta, IL6, IL8 and IL1ra were markedly elevated in tracheobronchial aspirate fluids from neonates with bronchopulmonary dysplasia. Corticoid treatment seemed to reduce these levels.

Interleukin-6 and interleukin-8 in the urine of children with acute pyelonephritis.

Interleukin-6 (IL-6) and interleukin-8 (IL-8) are important mediators of the inflammatory response in serious bacterial infections. We studied the levels of these two cytokines (standardised for urinary creatinine) in the urine of infants and children during and 6 weeks after acute pyelonephritis and in non-renal febrile controls and healthy children without apparent infection. IL-6 was detected in the urine of 52% of children with pyelonephritis compared with 15% of other children (P < 0.001). The median urinary IL-6 level in acute pyelonephritis was 4 pg/mumol compared with undetectable levels in the control group (P < 0.001). IL-8 was detected in 98% of children with pyelonephritis and 42% of other children (P < 0.001). The median concentration of IL-8 was 188 pg/mumol in pyelonephritis; it was undetectable in controls (P < 0.001). IL-8 levels were higher in children less than 1 year of age (P < 0.001).

Urine interleukin-6 and interleukin-8 in children with acute pyelonephritis, in relation to DMSA scintigraphy in the acute phase and at 1-year follow-up.

The relationship between urine interleukin-6 (IL-6) and interleukin-8 (IL-8)/creatinine quotients and 99mTc-dimercaptosuccinic acid (DMSA) scintigraphy, performed within 10 days of acute first-time pyelonephritis and after 1 year, was studied in 41 children. The urine IL-6 and IL-8/creatinine quotients were also related to the urine N-acetyl-beta-D-glucosaminidase (NAG) and albumin/creatinine quotients. Presence of DMSA uptake defects, reflecting local inflammation, in children in the acute phase of pyelonephritis, were associated with elevated urine IL-6/creatinine quotients (median 27 pg/mumol); in children without DMSA changes there was no increase in quotients (median non-detectable) (P < 0.05). Persistent DMSA changes at the 1-year follow-up, probably reflecting renal scarring, were only seen in children with increased urine IL-6/creatinine quotients in the acute phase (P < 0.01). No correlation was found between urine IL-8 and DMSA uptake defects. Vesicoureteral reflux (VUR) at 6-8 weeks did not correlate with the urine cytokine levels in the acute phase. The urine excretion of NAG and albumin, reflecting renal dysfunction, was associated with values of both urine IL-6 and IL-8/creatinine quotients, but not with DMSA defects or VUR. Thus, the initial urine IL-6/creatinine quotients might be used as an indicator of risk for persistent renal damage in acute pyelonephritis.

Fosfomycin/metronidazole compared with doxycycline/metronidazole for the prophylaxis of infection after elective colorectal surgery. A randomised double-blind multicentre trial in 517 patients.

OBJECTIVE: To find out if fosfomycin together with metronidazole was any better than doxycycline with metronidazole for the prophylaxis of infection before elective colorectal operations. DESIGN: Multicentre, double blind, random control trial. SETTING: Nine Swedish hospitals. SUBJECTS-559 patients admitted for elective colorectal operations. INTERVENTIONS: Fosfomycin 8 g and metronidazole 1 g before operation and fosfomycin 8 g eight hours afterwards, or doxycycline 400 mg and metronidazole 1 g before operation, and placebo eight hours afterwards. MAIN OUTCOME MEASURES: Incidence of all types of infection, mortality, and side effects. RESULTS: There were no significant differences between the groups for any of the outcome measures studied, the overall abdominal infection rates (wound, deep, and septicaemia) being 4.6% and 7.4%, and the remote infection rates (pneumonia, urinary tract, and central venous line) 15.1% and 12.8%, respectively. Of the predictors studied, only duration of operation was significantly related to risk of infection. CONCLUSION: The combination of fosfomycin and metronidazole was as safe and effective as that of doxycycline and metronidazole in preventing infections after elective colorectal operations.

[Lysozyme--an enzyme of both historical and current interest as a therapeutical agent]. / Lysozym--ett enzym av historiskt och åter aktuellt intresse som läkemedel.

Lysozyme, a bacteriolytic protein discovered by Fleming in 1922 and found to be phylogenetically ancient and almost ubiquitous among living organisms, is probably the most studied enzyme in biology and medicine. Evidence of its involvement in resistance to bacterial infection is compelling but remains indirect. Muramyl peptides (fragments of bacterial cell wall peptidoglycan) exert many effects on the immune system and the CNS, and appear to contribute to non-specific resistance to infection, fever, fatigue, and the pathogenesis of bacterial infection. Synthetic muramyl peptide analogues are currently used as adjuvants in vaccine trials in humans. Several pathological conditions are associated with changes in lysozyme concentrations, and egg-white lysozyme treatment has been tried on a small scale. With the cloning of the human lysozyme gene in yeast cells the enzyme can now be produced on a large scale, which will enable its therapeutic applications to be evaluated.

The significance of timing of metronidazole prophylaxis and addition of fosfomycin in colorectal surgery. An experimental study in rats.

A novel approach to antibiotic prophylaxis in colonic surgery, suggested by our previous clinical experience, was experimentally tested. Intravenous administration of metronidazole or fosfomycin at greater than or equal to 4 hours before surgery effectively prevented lethal infectious complications, whereas metronidazole prophylaxis begun at induction of anaesthesia proved to be much less efficacious (4% and 43% deaths vs. 50% among controls, p less than 0.001 and p greater than 0.7, respectively). The in vivo efficacy of antibiotic prophylaxis correlated better with mucosal counts of Clostridium and Bacteroides than with luminal counts of aerobic or anaerobic bacteria. Earlier initiation of intravenous metronidazole prophylaxis thus markedly increased its in vivo efficacy, apparently due to preoperative mucosal suppression of endogenous potential pathogens. This 'early timing' approach to systemic prophylaxis against infection in abdominal surgery warrants further evaluation both for other antibiotics and clinically.

Serum and tissue pharmacokinetics of intravenous metronidazole in surgical patients.

Intravenous administration of 1 g metronidazole during 30 min to 12 patients undergoing colorectal surgery yielded mean serum levels of 20.4 (+/- 4.9 SD) mg/l at 1 h, 15.6 (+/- 4.1) at 4 h and 3.9 (+/- 2.0) at 24 h after infusion. The mean elimination half-life of metronidazole was 8 h and the mean apparent volume of distribution 42 l. At 1-4 h after the infusion the median tissue/serum drug concentration ratio was 0.94 for rectus abdominis muscle and 0.76 for colonic mucosa. For adipose tissue the corresponding ratio was significantly lower (0.21, p less than 0.01) and highly unpredictable. Since in gastrointestinal surgery most wound infections develop in the cut surface of the abdominal fat, failure of metronidazole to reach bactericidal concentrations in adipose tissue could be a risk factor in this respect, contributing to the variable reported results of metronidazole prophylaxis.

Failure of single-dose metronidazole prophylaxis in colorectal surgery. No benefit from high dosage or combination with nalidixic acid.

In an open prospective study of postoperative infectious complications, patients undergoing elective colorectal surgery were randomly allocated to one of three groups receiving parenteral single-dose antimicrobial prophylaxis (1 g or 3 g metronidazole or 1 g metronidazole + 3 g nalidixic acid). Because of an unacceptably high rate of surgical infection in all three groups (36%, 29% and 39%, p greater than 0.1) among the first 103 evaluable patients, the study was discontinued. Nalidixic acid--though effective in in vitro tests of bacterial susceptibility--thus was found to be of little or no value as prophylaxis against Gram-negative infection. As the observed infection rate when metronidazole was given at the start of operation was seven-fold that previously found in the same department when 1 g metronidazole was administered 3-4 hours preoperatively (28/103 vs. 2/50, p less than 0.01), the timing of metronidazole prophylaxis was assumed to be potentially important for its ability to protect also against aerobic postoperative infection.

Use of indwelling urinary tract catheters in Swedish hospitals.

In a survey of 67,774 patient days in 27 Swedish acute care hospitals, an indwelling urinary tract catheter (IUTC) was in place during 12% of patient days. There were marked differences between services (from 2% in rheumatology to 49% in urology), but also between hospitals for the same type of service (general surgery, internal medicine, gynecology, orthopaedic surgery) concerning proportion IUTC days, average duration of catheterization and the indications for IUTC use. The great local variation in IUTC usage for a particular service was usually not correlated with type of hospital (district, county, or regional/teaching), ie, with the mix of patients studied. We propose that the results mainly reflect a variable local impact of infection control and that a much more restrictive use of IUTCs is possible in many wards. This condition would imply a considerable potential for reducing the incidences of urinary tract infection and bacteremia and thus, mortality among hospitalized patients in Sweden.

Significance of the sulfonamide component for the clinical efficacy of trimethoprim-sulfonamide combinations.

The reasons for combining trimethoprim (TMP) with sulfonamides (SUL) are still mainly theoretical but are supported by results from experimental infections and treatment of specific pathogens in humans, such as Branhamella catarrhalis, Neisseria gonorrhoeae, Brucella, Nocardia asteroides and perhaps Bordetella pertussis and Chlamydia trachomatis. Addition of SUL to TMP confers a therapeutic advantage also in patients with complicated urinary tract infection but probably not in young women with acute cystitis. Conditions that may enable TMP-SUL synergy in vivo can be expected to occur only in occasional cases of infection due to staphylococci, streptococci, Haemophilus or enteric bacteria. This fact together with ethical problems and availability of alternative therapies make further evaluations of the clinical significance of the SUL component of TMP-SUL very difficult. Although the use of TMP alone has shown promise in exacerbations of chronic bronchitis the role of the SUL component in TMP-SUL treatment of infections outside the urinary tract remains to be defined in comparative clinical trials.

Pathogenesis of Escherichia coli cystitis and pyelonephritis: apparent lack of significance of bacterial motility and chemotaxis towards human urine.

All 45 microscopically motile urinary isolates tested here (37 Escherichia coli, two Enterobacter cloacae, two Citrobacter freundii, three Proteus mirabilis and one Proteus morganii) were strongly attracted to fresh human urine in a capillary chemotaxis assay. This observation suggested that urine taxis of gram-negative bacteria promotes their invasion of the human lower urinary tract and their ascension to the kidney(s). However, the incidence of motile isolates and their activity in urine taxis assays were similar for fecal E. coli isolates, for isolates from patients with recurrent cystitis and from patients with presumed pyelonephritis (E. coli blood isolates with concomitant E. coli bacteriuria). Thus, the present study of E. coli did not support the hypothesis that bacterial motility is a virulence factor in urinary tract infection.

Single-dose intravenous metronidazole v. doxycycline prophylaxis in colorectal surgery. An open prospective, randomized trial.

In an open prospective, randomized study of antimicrobial prophylaxis in colorectal surgery, using a single intravenous dose, metronidazole (1 g) was more effective than doxycycline (0.2 g). The difference in infection rates (2/41 = 5% v. 7/33 = 21%) was statistically significant. Prolonged administration of either agent (2-7 days) yielded results comparable to those with single doses. Five emergency cases were admitted to the study. In two of them, a metronidazole-based therapeutic regimen prevented postoperative infection, whereas infection occurred in all 3 patients given doxycycline therapy. The total infection rate after elective surgery was 4% with metronidazole and 25% with doxycycline prophylaxis. The infections in the metronidazole group were superficial and caused by Escherichia coli, whereas half of the infections in the doxycycline group were intra-abdominal and due to intestinal aerobic and/or anaerobic bacteria. In contrast to doxycycline, therefore, metronidazole prevented postoperative anaerobic infection and was associated with low incidence of aerobic infection.

Autolysis of Neisseria gonorrhoeae.

Autolysis of Neisseria gonorrhoeae was studied under different conditions. It was found that low pH and temperature, as well as the presence of divalent cations, spermine, sucrose, and polyvinylpyrrolidone, stabilized nongrowing gonococci. Ethylenediaminetetraacetic acid alone promoted lysis, whereas lysozyme had only a limited additive effect. The autolytic behavior of gonococci appears to be connected with their prolonged cell division process. The relative dependence on the outer membrane and the peptidoglycan layer for the mechanical stability of gonococci is discussed.