Development of an "early warning" sensor for encrustation of urinary catheters following Proteus infection.

Biofilm formation in long-term urinary catheterized patients can lead to encrustation and blockage of urinary catheters with serious clinical complication. Catheter encrustation stems from infection with urease-producing bacteria, particularly Proteus mirabilis. Urease generates ammonia from urea, and the elevated pH of the urine results in crystallization of calcium and magnesium phosphates, which block the flow of urine. The aim of this research is to develop an "early warning" silicone sensor for catheter encrustation following bacterial infection of an in vitro bladder model system. The in vitro bladder model was infected with a range of urease positive and negative bacterial strains. Developed sensors enabled catheter blockage to be predicted ~17-24 h in advance of its occurrence. Signaling only occurred following infection with urease positive bacteria and only when catheter blockage followed. In summary, sensors were developed that could predict urinary catheter blockage in in vitro infection models. Translation of these sensors to a clinical environment will allow the timely and appropriate management of catheter blockage in long-term catheterized patients.

Clinical characteristics and outcomes of patients with extended-spectrum ß-lactamase-producing bacteremias in the emergency department.

Extended-spectrum ß-lactamase (ESBL)-producing bacteria have been spreading from hospitals to communities. Despite this, there are limited emergency department (ED) patient-based studies about these bacteremias. A retrospective matched case-control study with a ratio of 1:3 was conducted at a university hospital. The case group consisted of patients aged >16 years with ESBL-producing bacteremias in the ED. Patients matched for age and sex with non-ESBL-producing bacteremias were sampled as the control group. Finally, 64 episodes of ESBL-producing Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis bacteremias were included in our study. The median age of case patients was 71 years, and 29 (45.3%) were males. The most common type of infection was urinary tract infection (71.9%), followed by intra-abdominal infection (12.5%). Inappropriate empirical antibiotics therapy was prescribed in 87.5% of case patients, which was significantly higher than the control group (13.0%; p < 0.001). Patients with inappropriate empirical antibiotics had a significantly longer hospital stay than those with appropriate empirical antibiotics (p < 0.001). Multivariate analysis showed that hospital-acquired infection, urinary catheterization, and previous antibiotics use were independent risk factors for the acquisition of ESBL-producing bacteremia. The 28-day mortality rate of case patients was 18.8%. Whether they received appropriate empirical antibiotics treatment or not, there was no statistical difference in the mortality of patients with ESBL-producing bacteremias (p = 0.167). To face these emerging multidrug-resistant bacteria and to guide the empirical antibiotics therapy, it is crucial for emergency physicians to recognize the characteristics and risk factors for ESBL-producing organisms.

Characterization of CMY-type beta-lactamases in clinical strains of Proteus mirabilis and Klebsiella pneumoniae isolated in four hospitals in the Paris area.

We isolated five clinical strains (three Proteus mirabilis and two Klebsiella pneumoniae) with beta-lactam resistance phenotypes consistent with production of an AmpC-type beta-lactamase. The predicted amino acid sequences of the enzymes were typical of class C beta-lactamases. The enzymes were identified as CMY-2, CMY-4 and a new CMY-variant beta-lactamase, CMY-12. The AmpC beta-lactamases from the two K. pneumoniae isolates were found to be encoded on self-transferable plasmids. The genes encoding the AmpC-type beta-lactamase produced by the three P. mirabilis isolates were chromosomal. Four of the five clinical isolates were from patients transferred from Greece, Algeria and Egypt; one of the K. pneumoniae strains was recovered from a French patient. PFGE analysis and rep-PCR fingerprinting showed that the two P. mirabilis isolates from Greek patients were closely related.

Investigation of the types and characteristics of the proteolytic enzymes formed by diverse strains of Proteus species.

Many diverse clinical isolates of Proteus mirabilis (48 strains), P. penneri (25), P. vulgaris biogroup 2 (48) and P. vulgaris biogroup 3 (21) from man were examined for their ability to produce proteolytic enzymes and the nature and characteristics of the proteases were studied. All the P. penneri isolates, most (94-90%) of the P. mirabilis and P. vulgaris biogroup 2 isolates, but only 71% of the P. vulgaris biogroup 3 isolates, secreted proteolytic enzymes. These were detected most readily at pH 8 with gelatin as substrate. A strong correlation was found between the ability of a strain to form swarming growth and its ability to secrete proteases. Non-swarming isolates invariably appeared to be non-proteolytic. However, some isolates, particularly of P. vulgaris biogroup 3, were non-proteolytic even when they formed swarming growth. Analysis of the secreted enzymes of the different Proteus spp. on polyacrylamide-gelatin gels under various constraints of pH and other factors showed that they were all EDTA-sensitive metalloproteinases. Analysis of the kinetics of production of the proteases revealed the formation of an additional protease of undefined type and function that was cell-associated and formed before the others were secreted. The secreted protease was subsequently modified to two isoforms whose mass (53-46 kDa) varied with the Proteus spp. and the strain. There was no evidence that the secreted proteases of strains of Proteus spp. were of types other than metalloproteinases.

Proteinases of Proteus spp.: purification, properties, and detection in urine of infected patients.

The proteinases secreted by pathogenic strains of Proteus mirabilis, P. vulgaris biotype 2, P. vulgaris biotype 3, and P. penneri were purified with almost 100% recovery by affinity chromatography on phenyl-Sepharose followed by anion-exchange chromatography. The proteinase purified from the urinary tract pathogen P. mirabilis, which we had previously shown to degrade immunoglobulins A and G, appeared as a composite of a single band and a double band (53 and 50 kDa, respectively) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The other Proteus proteinases had similar patterns but slightly different mobilities. In each case all proteinase activity in culture supernatants was demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be associated with only the triple-band complex; all three bands were proteolytically active. The P. mirabilis proteinase was resistant to inhibitors of both serine and thiol proteinases but strongly inhibited by metal chelators, although it was not affected by phosphoramidon, an inhibitor of the thermolysin group of bacterial metalloproteinases. Active proteinase was detected in urine samples from P. mirabilis-infected patients; this is consistent with our detection of immunoglobulin A fragments of a size suggestive of P. mirabilis proteinase activity.

Experimental infections for the evaluation of beta-lactamase resistance.

The antibacterial activity and pharmacokinetics of the beta-lactamase-stable cephalosporin cefuroxime and the gram-negative beta-lactamase-susceptible cephalosporin cefazolin were compared in two contrasting infection models in which Proteus morganii 82, which produces chromosomally mediated beta-lactamase, was the pathogen. In the rat paw model, characterized by high numbers of localized bacteria, cefazolin was destroyed at the site of infection and consequently did not produce a therapeutic response. In the mouse intraperitoneal model cefazolin was also inactive, despite peritoneal concentrations being unaffected by high counts of the beta-lactamase-producing P. morganii in the body cavity. In contrast the pharmacokinetics of cefuroxime was unaffected by the presence of the beta-lactamase-producing P. morganii, and good therapeutic responses were seen in both models.

Pharmacokinetics and antibacterial efficacy in vivo of beta-lactam combinations against Proteus vulgaris.

The pharmacokinetics and antibacterial efficacy of mezlocillin, cefotaxime, cefoperazone and the mezlocillin/cefalosporin combinations, respectively, were studied by adopting the granuloma pouch model in rats. Exudate concentrations of mezlocillin were higher after combined i.v. injection with a cefalosporin as determined microbiologically and by high performance liquid chromatography (HPLC). Cefoperazone levels, however, were not affected. Not metabolized cefotaxime concentrations as determined by HPLC were also not increased following simultaneous injection with mezlocillin. Cefotaxime metabolite concentrations, however, were generally higher than unchanged cefotaxime and increased upon repeated administration of cefotaxime alone and to a greater extent when combined with mezlocillin. Antibacterial efficacy of mono- or combined chemotherapy was correlated to beta-lactamase inducibility of the test strains insofar as drugs acting as good or moderate enzyme inducers were ineffective in vivo. The combined therapy of mezlocillin with cefotaxime was effective in this case. This result was also correlated to the bioavailability of the beta-lactams in infected pouches. Due to the degree of beta-lactamase inducibility and production, drug levels were either decreased or not detectable.

Urease stones.

Urinary stones form as a consequence of urinary supersaturation. Supersaturation occurs as a result of elevated concentrations of urinary solutes. Dietary, metabolic, endocrine, hereditary, and infectious processes alter urinary solute concentrations. Struvite (MgNH4PO. 6H2O) and carbonate-apatite [Ca10(PO4)6CO3] stones form in urine that becomes supersaturated as a by-product of the hydrolysis of urea by the bacterial enzyme urease. Urease-induced stones manifest primarily as branched renal calculi and as bladder calculi. Conventional therapy has usually consisted of surgical removal of the stone combined with a short course of antimicrobial therapy. Such treatment is curative in about 50% of cases. Recurrent stone formation and progressive pyelonephritis occur in those who are not cured. Adjunctive medical treatment with acetohydroxamic acid or hydroxyurea lessens the risk of calculogenesis and decreases growth of residual stones in patients who are not cured by conventional therapy. Patients with urea-splitting urinary infection and renal stones have a major life-threatening disease. The morbidity and expense that result from this disease are great. Long-term (perhaps lifetime) chemotherapy with antimicrobial agents and/or urease-inhibiting drugs combined with judicious and expert surgical intervention can be expected to significantly improve the plight of these unfortunate patients.

[Spontaneous Peripheral Proteolysis/1st Communication: method and results of clinical examinations (author's transl)]. / Spontane periphere Proteolyse. 1. Mitteilung: Methodik und klinische Untersuchungsergebnisse

With a modificated Astrup-fibrin plate-method also an inhibition of proteolysis can be registrated. In various medical areas a spontaneous peripheral proteolysis had been found, especially so in chronical bacterial infections.

Effect of antibacterial therapy on renal lysozyme levels in rats developing bacterial pyelonephritis.

In animals developing unilateral Proteus mirabilis-induced pyelonephritis, the total soluble renal lysozyme (SRL) of both kidneys undergoes a biphasic elevation. The second phase of elevated SRL is associated with the onset of chronicity in the infected kidney. To discover whether effective antibacterial therapy altered the second elevation of SRL, levels of SRL were determined in rats developing unilateral chronic pyelonephritis with and without effective regimens of antibacterial agents. Therapeutic doses of ampicillin and nitrofurantoin caused elevations of SRL in both kidneys of infected animals, but these differences were not statistically significant (P > 0.05). Both agents produce elevations of SRL in uninfected animals which were significant (P < 0.05) when compared with normal animals. Kanamycin sulfate at a therapeutic dose induced great elevations of SRL in kidneys of both infected and uninfected animals. It is concluded that infection per se is not the cause of the elevated SRL.

Renal lysozyme levels in animals developing Proteus mirabilis-induced pyelonephritis.

In animals developing experimentally induced unilateral pyelonephritis, both the infected kidney (IK) and the contralateral noninfected kidney (NIK) showed an immediate increase in renal lysozyme activity of about 5 days' duration after the unilateral injection of viable Proteus mirabilis into the renal cortex. Lysozyme activities of the NIK were consistently higher than those of the IK. This initial increase was followed by a second increase which lasted throughout the period of observation (17 days), and enzyme activities of the NIK were consistently higher than those of the IK. In saline punctured kidneys of control animals, both the saline punctured kidney (SP) and the non-saline punctured kidney (NSP) showed only the immediate increase in renal lysozyme activity, which persisted until the SP was completely healed. These enzyme activities were less than those observed in the infected animals, but the response of the NSP was greater than that of the SP. Trauma not directed to the kidney does not produce a similar response of renal lysozyme. The elevated renal lysozyme of the NIK could not be shown to protect it from bacterial infection.