Proteus mirabilis lipase (PML) features tolerance to organic solvents and great potential for biodiesel synthesis. However, the thermal stability of the enzyme needs to be improved before it can be used industrially. Various computational design strategies are emerging methods for the modification of enzyme thermal stability. In this paper, the complementary algorithm-based ABACUS, PROSS, and FoldX were employed for positive selection of PML mutations, and their pairwise intersections were further subjected to negative selection by PSSM and G_REMLIN to narrow the mutation library. Thereby, 18 potential single-point mutants were screened out. According to experimental verification, 7 mutants had melting temperature (T_m) improved, and the ΔT_m of K208G and G206D was the highest, which was 3.75 ℃ and 3.21 ℃, respectively. Five mutants with activity higher than the wild type (WT) were selected for combination by greedy accumulation. Finally, the T_m of the five-point combination mutant M10 increased by 10.63 ℃, and the relative activity was 140% that of the WT. K208G and G206D exhibited certain epistasis during the combination, which made a major contribution to the improvement of the thermal stability of M10. Molecular dynamics simulation indicated that new forces were generated at and around the mutation sites, and the rearrangement of forces near G206D/K208G might stabilize the Ca²⁺ binding site which played a key role in the stabilization of PML. This study provides an efficient and user-friendly computational design scheme for the thermal stability modification of natural enzymes and lays a foundation for the modification of PML and the expansion of its industrial applications.
Enzyme Stability ; Lipase/chemistry* ; Molecular Dynamics Simulation ; Proteus mirabilis/metabolism* ; Solvents/chemistry*

BACKGROUNDCarbapenems are used to treat severe infections caused by multi-drug-resistant organisms, however, the emergence of carbapenem-resistant bacterial isolates is becoming an increasing therapeutic challenge. Since the first Klebsiella (K.) pneumoniae carbapenemase (KPC)-producing K. pneumoniae was reported in 2001, KPC-producing isolates have been found increasingly, specially in Enterobacteriaceae. The aim of this study was to characterize the mechanisms of a carbapenem-resistant Proteus (P.) mirabilis.

METHODSA carbapenem-resistant P. mirabilis isolate was recovered from pleural drainage fluid of a patient admitted to surgical intensive care unit. Antimicrobial susceptibility testing of the isolate was performed by disk diffusion according to Clinical and Laboratory Standards Institute guidelines, and subsequent minimal inhibitory concentrations were determined with the E-test. Amplification of the bla(KPC) gene generated a positive band and the PCR products were sequenced subsequently. The plasmid of the isolate was extracted and was successfully transformed into Escherichia (E.) coli DH5α.

RESULTSThe P. mirabilis isolate was resistant to all detected antimicrobial agents except tigecycline. KPC-2 was confirmed by DNA sequence analysis. The transformant E. coli was resistant to carbapenems. Further study demonstrated that upstream and downstream regions of bla(KPC-2) were identical to that observed in K. pneumoniae submitted to GenBank from China in 2007.

CONCLUSIONCarbapenem resistance in the P. mirabilis isolate in this study is mainly due to production of KPC-2.

Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; metabolism ; China ; Klebsiella pneumoniae ; enzymology ; Proteus mirabilis ; drug effects ; enzymology ; beta-Lactamases ; metabolism

The aim of this study was to demonstrate and assess C-reactive protein (CRP) changes in dogs with induced bacterial cystitis with or without antibiotics. We also evaluated availability of CRP levels to serve as an indicator for monitoring or diagnosing bacterial cystitis. Serial CRP concentrations in dogs with induced bacterial cystitis were higher than those of controls (p < 0.001). CRP concentrations peaked on day 7 and gradually decreased thereafter. In the treatment group, CRP concentrations decreased after medication compared to the untreated group (p = 0.032). CRP levels had a linear correlation with urine white blood cell counts among all groups (r = 0.837, p < 0.001, n = 140). Compared to the negative urine culture group, dogs with positive urine culture results had higher CRP concentrations (median 43.8 mg/L vs. 5.9 mg/L; p < 0.001). Area under the receiver operating characteristic curve was 0.955; when cut-off value was 12.2 mg/L, CRP measurements were found to have a sensitivity of 92.3% and specificity of 86.4%. This result indicates that rapid increases of CRP occurred after inducing bacterial cystitis and CRP may be a useful indicator for monitoring or diagnosing canine bacterial cystitis together with sediment urinalysis and urine bacterial culture.
Amoxicillin-Potassium Clavulanate Combination/therapeutic use ; Animals ; Anti-Bacterial Agents/therapeutic use ; C-Reactive Protein/genetics/*metabolism ; Cystitis/metabolism/*veterinary ; Dogs ; Gene Expression Regulation/*physiology ; Inflammation/*metabolism ; Male ; Proteus Infections/drug therapy/metabolism/microbiology/*veterinary ; Proteus mirabilis

BACKGROUND: The aim of this study was to compare the BD Phoenix (Beckton Dickinson Diagnostic Systems, USA) extended-spectrum beta-lactamase (ESBL) test with the Clinical and Laboratory Standards Institute (CLSI) ESBL phenotypic confirmatory test by disk diffusion (CLSI ESBL test) in Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Proteus mirabilis. METHODS: We tested 224 clinical isolates of E. coli, K. pneumoniae, K. oxytoca and P. mirabilis during May 2006 to March 2007. These isolates were examined by the Phoenix and the CLSI ESBL tests simultaneously. For the isolates showing discordant results between the two tests, boronic acid disk test was performed to differentiate AmpC beta-lactamase and ESBL. RESULTS: Among the 224 clinical isolates, 75 and 79 isolates were positive for ESBL by CLSI ESBL test and Phoenix test, respectively. Having detected 4 more isolates as ESBL-producers, Phoenix test showed a 98.2% agreement with a 100% sensitivity and 97.3% specificity compared with CLSI ESBL test. Among the four false positive isolates, three were AmpC-positive but ESBL-negative. CONCLUSIONS: The BD Phoenix ESBL test was sensitive and specific, and can be used as a rapid and reliable method to detect ESBL production in E. coli, Klebsiella species, and P. mirabilis.
Automation ; Bacterial Proteins/classification/*metabolism ; Disk Diffusion Antimicrobial Tests ; Escherichia coli/drug effects/*enzymology/isolation & purification ; Humans ; Klebsiella/*enzymology ; Klebsiella oxytoca/drug effects/enzymology/isolation & purification ; Klebsiella pneumoniae/drug effects/enzymology/isolation & purification ; *Microbial Sensitivity Tests ; Proteus mirabilis/drug effects/*enzymology/isolation & purification ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; beta-Lactamases/classification/*metabolism