Bacteriologic analyses of bile and brown pigment stones in patients with acute cholangitis.

BACKGROUND: Bacteria play an important role in the formation of brown pigment stones through adherence and biofilm formation. Scanning electron microscopy of cross sections of these stones reveals a laminated appearance and various bacteria in the different layers. Our postulation was that different bacteria might be involved at different stages of stone formation. METHODS: By using standard bacteriologic cultures, the composition, morphology, and antibiotic sensitivity patterns of bacteria isolated from paired stone were compared with bile samples from 70 patients with acute cholangitis. A further comparison was made between bacteria isolated from the periphery and center of 3 randomly selected brown pigment stones. RESULTS: Ninety-one percent of bile and 99% of stone samples yielded positive cultures, with a total of 151 and 149 bacteria isolated from bile and stones, respectively. In 22 patients (33%), the bacteria isolated from the paired bile and stone samples were totally different. The mean percentage similarity of bacteria isolated from bile and stones was 39% (range 0%-100%). Of the 59 pairs of similar bacteria isolated, the antibiotic sensitivity patterns were different in 24 (41%) cases. Of the 3 brown stones studied, either different bacterial species or the same bacteria but different strains with different antibiotic sensitivities were isolated from the center and periphery of the stones. CONCLUSIONS: Bacteria present in the different layers of brown pigment stones may represent the bacterial flora in bile at different times. Simple bile culture may not identify bacteria trapped inside the stone.

Expression of bacterial beta-glucuronidase in human bile: an in vitro study.

BACKGROUND: Bacterial beta-glucuronidase causes deconjugation of bilirubin diglucuronide resulting in the precipitation of calcium bilirubinate, which contributes to biliary sludge and stone formation. This process is attributed to enzyme activity produced by the aerobic enterobacteriaceae such as Escherichia coli and Klebsiella sp. The presence of Clostridium sp. was detected in 48 of 56 intrahepatic stones by using polymerase chain reaction techniques and cultured Clostridium perfringens from 14 of 18 unblocked biliary stents. Such bacteria are reported to produce beta-glucuronidase activity. The aim of this study was to determine the proportion of biliary bacteria isolated from pigment stones and stents that produce beta-glucuronidase and to compare the enzyme activity expressed by the different bacteria in human bile. METHODS: A total of 202 bacteria were isolated from blocked and unblocked biliary stents and pigment ductal stones recovered from patients. Of these, 61 bacteria expressed beta-glucuronidase activity in brain heart infusion broth. These 61 bacteria were subsequently grown in human bile under aerobic or anaerobic conditions to the early stationary phase and assayed for beta-glucuronidase activity by using rho-nitrophenyl beta-D glucuronide as substrate. Results were normalized and reported as units of enzyme activity per milligram protein of the bacteria. RESULTS: C. perfringens produced beta-glucuronidase enzyme activity that was 34-fold higher than that for E coli, Staphylococcus, Corynebacterium sp., Bacillus sp., Enterococcus sp., Acinetobacter sp., Streptococcus sp., and Klebsiella sp. CONCLUSION: C. perfringens with its higher enzyme activity is more important in the deconjugation of bilirubin diglucuronide than E coli and Klebsiella sp.

Effect of antibiotic-loaded hydrophilic stent in the prevention of bacterial adherence: a study of the charge, discharge, and recharge concept using ciprofloxacin.

BACKGROUND: Ciprofloxacin prophylaxis significantly prolonged stent patency in cats, but human studies produced conflicting results, possibly due to varying drug levels in bile. The uptake (charge) and release (discharge) of ciprofloxacin from a hydrophilic stent (HS) in an antibiotic solution and the effect of a ciprofloxacin-loaded stent (CHS) in inhibiting Escherichia coli adherence were tested. The adjuvant effect of ciprofloxacin perfusion (recharge) in the inhibition of E coli adherence was also tested. METHODS: Uptake: segments of HS were immersed in 5 mL of ciprofloxacin solutions for 24 hours. Ciprofloxacin remaining in solution was measured to determine the uptake by the HS. Release: CHS were placed in 5 mL water for 24 hours, and released ciprofloxacin was measured. CHS were placed on culture plates with E coli and incubated; diameters of inhibited zones were measured. CHS 0.5 cm in length were incubated in separate 5 mL E coli suspension (10(7) colony forming units [CFU]/mL) in 2% ox bile for 4 hours. E coli adhered on CHS were measured and compared with control HS. An E coli (10(6) CFU/mL) suspension was perfused through a modified Robbins device (MRD)-containing CHS. Stents were removed at regular intervals and processed to determine the adherence of E coli; non-loaded HS served as controls. The experiment was repeated by using CHS together with perfusion of ciprofloxacin solution (0.3 microg/mL) into the MRD for up to 7 days; normal saline solution was used as a control in a second MRD. Stents were removed daily to determine the adherence of E coli. RESULTS: Uptake and release of ciprofloxacin by HS and CHS, respectively, were related to concentration of ciprofloxacin. Between 50% to 90% of the drug was released in 24 hours. Zonal inhibition of E coli growth was proportional to the concentration of ciprofloxacin on the CHS. There was an initial 10-fold reduction in attached E coli on CHS compared with controls, but this effect diminished after 24 hours. With ciprofloxacin perfusion, there was a 100-fold reduction in adhered E coli on CHS, although there was no change in E coli concentration in bile. CONCLUSIONS: There was a free exchange (uptake and release) of ciprofloxacin along a concentration gradient between the antibiotic solution and HS. CHS reduced the number of adhered E coli, but the effect was short-lived. Perfusion of ciprofloxacin offers an adjuvant benefit by enhancing inhibition of E coli adherence on CHS.

rDrak1, a novel kinase related to apoptosis, is strongly expressed in active osteoclasts and induces apoptosis.

This is the first report of a novel serine/threonine kinase, rabbit death-associated protein (DAP) kinase-related apoptosis-inducing protein kinase 1 (rDRAK1), involved in osteoclast apoptosis. We searched for osteoclast-specific genes from a cDNA library of highly enriched rabbit osteoclasts cultured on ivory. One of the cloned genes has a high homology with human DRAK1 (hDRAK1), which belongs to the DAP kinase subfamily of serine/threonine kinases. By screening a rabbit osteoclast cDNA library and 5'-RACE (rapid amplification of cDNA ends), we obtained a full length of this cDNA, termed rDRAK1. The sequencing data indicated that rDRAK1 has 88.0, 44.6, 38.7, and 42.3% identity with hDRAK1, DAP kinase, DRP-1, and ZIP (zipper-interacting protein) kinase, respectively. To clarify the role of DRAK1 in osteoclasts, we examined the effect of three osteoclast survival factors (interleukin-1, macrophage colony-stimulating factor, and osteoclast differentiation-inducing factor) on rDRAK1 mRNA expression and the effect of rDRAK1 overexpression on osteoclast apoptosis. The results suggested that these three survival factors were proved to inhibit rDRAK1 expression in rabbit osteoclasts. After transfection of a rDRAK1 expression vector into cultured osteoclasts, overexpressed rDRAK1 was localized exclusively to the nuclei and induced apoptosis. Hence, rDRAK1 may play an important role in the core apoptosis program in osteoclast.

Direct-method-aided phasing of MAD data.

The direct methods of breaking the phase ambiguity intrinsic in one-wavelength anomalous scattering (OAS) data and MAD phasing are powerful methods in their own rights. In a different context, in addition to their success in phasing OAS data, direct methods can also be useful in the treatment of MAD data. The idea has been tested with the MAD data at 2.5 A resolution from the protein human adenosine kinase [Mathews et al. (1998), Biochemistry, 37, 15607--15620]. The results showed that the incorporation of direct methods in MAD phasing led to a significant improvement of phases over those obtained from the conventional MAD phasing method alone, as indicated by improved map correlation coefficients (with the existing model), reduced phase errors by 4.5 degrees and improved map connectivity.