Affinity purification of Car9-tagged proteins on silica matrices: Optimization of a rapid and inexpensive protein purification technology.

Car9, a dodecapeptide identified by cell surface display for its ability to bind to the edge of carbonaceous materials, also binds to silica with high affinity. The interaction can be disrupted with l-lysine or l-arginine, enabling a broad range of technological applications. Previously, we reported that C-terminal Car9 extensions support efficient protein purification on underivatized silica. Here, we show that the Car9 tag is functional and TEV protease-excisable when fused to the N-termini of target proteins, and that it supports affinity purification under denaturing conditions, albeit with reduced yields. We further demonstrate that capture of Car9-tagged proteins is enhanced on small particle size silica gels with large pores, that the concomitant problem of nonspecific protein adsorption can be solved by lysing cells in the presence of 0.3% Tween 20, and that efficient elution is achieved at reduced l-lysine concentrations under alkaline conditions. An optimized small-scale purification kit incorporating the above features allows Car9-tagged proteins to be inexpensively recovered in minutes with better than 90% purity. The Car9 affinity purification technology should prove valuable for laboratory-scale applications requiring rapid access to milligram-quantities of proteins, and for preparative scale purification schemes where cost and productivity are important factors.

Evaluation of a novel immunochromatographic lateral flow assay for rapid detection of OXA-48, NDM, KPC and VIM carbapenemases in multidrug-resistant Enterobacteriaceae.

PURPOSE: The global spread and increasing clinical impact of carbapenemase-producing bacteria are alarming. Rapid diagnostic techniques for carbapenemase detection are of the utmost importance to prevent delays in efficient antibiotic therapy and the control of spread in hospitals. Recently, multiplex immunochromatographic lateral flow tests (ICTs) for the fast detection of carbapenemase-producers have become commercially available. We evaluated a novel multiplex ICT for the rapid detection of OXA-48, KPC, NDM and VIM carbapenemases. METHODOLOGY: One hundred well-characterized multidrug-resistant Enterobacteriaceae were analysed by RESIST-4 (Coris, BioConcept, Gembloux, Belgium). The reference standard included confirmation at the molecular level at the German National Reference Laboratory for multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany).Results/Key findings. The tested ICT was highly reliable, showing 100 % sensitivity and 100 % specificity. CONCLUSION: As it is fast, easy to perform and has few technical requirements, the ICT represents a good opportunity to improve turnaround times and patient care for the clinical microbiology laboratory.

Detection of cfxA2, cfxA3, and cfxA6 genes in beta-lactamase producing oral anaerobes.

Purpose The aim of this study was to identify ß-lactamase-producing oral anaerobic bacteria and screen them for the presence of cfxA and BlaTEM genes that are responsible for ß-lactamase production and resistance to ß-lactam antibiotics. Material and Methods Periodontal pocket debris samples were collected from 48 patients with chronic periodontitis and anaerobically cultured on blood agar plates with and without ß-lactam antibiotics. Presumptive ß-lactamase-producing isolates were evaluated for definite ß-lactamase production using the nitrocefin slide method and identified using the API Rapid 32A system. Antimicrobial susceptibility was performed using disc diffusion and microbroth dilution tests as described by CLSI Methods. Isolates were screened for the presence of the ß-lactamase-TEM (BlaTEM) and ß-lactamase-cfxA genes using Polymerase Chain Reaction (PCR). Amplified PCR products were sequenced and the cfxA gene was characterized using Genbank databases. Results Seventy five percent of patients carried two species of ß-lactamase-producing anaerobic bacteria that comprised 9.4% of the total number of cultivable bacteria. Fifty one percent of ß-lactamase-producing strains mainly Prevotella, Porphyromonas, and Bacteroides carried the cfxA gene, whereas none of them carried blaTEM. Further characterization of the cfxA gene showed that 76.7% of these strains carried the cfxA2 gene, 14% carried cfxA3, and 9.3% carried cfxA6. The cfxA6 gene was present in three Prevotella spp. and in one Porphyromonas spp. Strains containing cfxA genes (56%) were resistant to the ß-lactam antibiotics. Conclusion This study indicates that there is a high prevalence of the cfxA gene in ß-lactamase-producing anaerobic oral bacteria, which may lead to drug resistance and treatment failure.

A beta-lactamase belonging to group 2e from oral clinical isolates of Prevotella intermedia.

A beta-lactamase in oral clinical isolates of Prevotella intermedia that hydrolyzed cefuroxime and cephalothin with rates of 600 and 53.3 respectively, relative to that for cephaloridine (100), was characterized as a 2e-cephalosporinase. Inhibition was observed by clavulanic acid (IC50 0.72 microM), tazobactam (IC50 0.21 microM) and sulbactam (IC50 0.07 microM) and was not inhibited by cloxacillin, EDTA, NaCl or p-CMB. The pI and pH optima were 4.7 and 5.4, respectively.

beta-Lactamase recovery from E. coli cell lysate via two-phase electrophoresis.

beta-Lactamase was recovered from Escherichia coli cell lysate by a novel cell debris removal method using two-phase electrophoresis. The cells were harvested by centrifugation after fermentation, resuspended in a low ionic strength electrophoresis buffer, lysed, and combined with a poly(ethylene glycol)/dextran aqueous two-phase system in the same buffer. The cell lysate was subjected to a 40 V/cm electric field oriented perpendicular to the phase interface for 90 min. Experiments were conducted both with and without a nucleic acid precipitation step using poly(ethylene imine) (PEI). For PEI-treated lysate at pH 5, the positively charged beta-lactamase was directed to the upper phase, while negatively charged contaminants (including cell debris, nucleic acid/PEI precipitates, and negatively charged proteins) were directed to the lower phase with the applied field. beta-Lactamase yield in the upper phase was 81%, while cell debris and nucleic acids partitioned almost exclusively to the lower phase. For untreated lysate, beta-lactamase did not move in the electric field due to strong interaction with nucleic acids in solution.

Effect of column dimensions and flow rates on size-exclusion refolding of beta-lactamase.

We have investigated the effect of changing the column diameter and length on the size exclusion chromatography (SEC) refolding of beta-lactamase from Escherichia coli-derived inclusion bodies (IBs). Inclusion bodies were recovered and solubilised in 6 M GdnHCl and 5 mM DTT. Up to 16 mg of denatured, solubilised beta-lactamase was loaded onto size exclusion columns packed with Sephacryl S-300 media (fractionation range: 10(4)-1.5 x 10(6) Da). beta-Lactamase was refolded by eluting the loaded sample with 1 M urea in 0.05 M phosphate buffer, pH 7 at 23 degrees C. The following columns were studied: 26 x 400, 16 x 400 and 26 x 200 mm, with a range of mobile phase flow rates from 0.33 to 4.00 ml/min. beta-Lactamase was successfully refolded in all three columns and at all flow rates studied. The beta-lactamase activity peak coincided with the major protein peak. Reducing the column diameter had little effect on refolding performance. The enzyme activity recovered was relatively independent of the mobile phase linear velocity. Reducing the column length gave a poorer resolution of the protein peaks, but the enzyme activity peaks were well resolved. Calculation of the partition coefficients for beta-lactamase activity showed that the 26 x 400 column gave the greatest refolding performance.

Recovery of a foreign protein from the periplasm of Escherichia coli by chemical permeabilization.

We have applied the technique of protein release by chemical permeabilization to recover a foreign protein in active form from the periplasm of a recombinant strain of Escherichia coli. The two agents used in our chemical permeabilization scheme, guanidine hydrochloride and Triton X-100, have different modes of action, allowing selectivity in protein release based on intracellular location under different treatment conditions. Specifically, treatment of E. coli C600-1 cells by guanidine alone resulted in 40-fold purification of recombinant beta-lactamase, which is periplasmically expressed in this host. Achieving such high purification in the cell disruption stage could alleviate some of the problems associated with recovery of intracellular products, such as low expression or the need to solubilize cytoplasmic inclusion bodies. Recovery of periplasmic proteins by chemical permeabilization is simpler than by osmotic shock and is less expensive than using enzymatic digestion.

Detection of cfxA2, cfxA3, and cfxA6 genes in beta-lactamase producing oral anaerobes

ABSTRACT Purpose The aim of this study was to identify β-lactamase-producing oral anaerobic bacteria and screen them for the presence of cfxA and BlaTEM genes that are responsible for β-lactamase production and resistance to β-lactam antibiotics. Material and Methods Periodontal pocket debris samples were collected from 48 patients with chronic periodontitis and anaerobically cultured on blood agar plates with and without β-lactam antibiotics. Presumptive β-lactamase-producing isolates were evaluated for definite β-lactamase production using the nitrocefin slide method and identified using the API Rapid 32A system. Antimicrobial susceptibility was performed using disc diffusion and microbroth dilution tests as described by CLSI Methods. Isolates were screened for the presence of the β-lactamase-TEM (BlaTEM) and β-lactamase-cfxA genes using Polymerase Chain Reaction (PCR). Amplified PCR products were sequenced and the cfxA gene was characterized using Genbank databases. Results Seventy five percent of patients carried two species of β-lactamase-producing anaerobic bacteria that comprised 9.4% of the total number of cultivable bacteria. Fifty one percent of β-lactamase-producing strains mainly Prevotella, Porphyromonas, and Bacteroides carried the cfxA gene, whereas none of them carried blaTEM. Further characterization of the cfxA gene showed that 76.7% of these strains carried the cfxA2 gene, 14% carried cfxA3, and 9.3% carried cfxA6. The cfxA6 gene was present in three Prevotella spp. and in one Porphyromonas spp. Strains containing cfxA genes (56%) were resistant to the β-lactam antibiotics. Conclusion This study indicates that there is a high prevalence of the cfxA gene in β-lactamase-producing anaerobic oral bacteria, which may lead to drug resistance and treatment failure.

Effectiveness of a new decolonisation regimen for eradication of extended-spectrum ß-lactamase-producing Enterobacteriaceae.

Gram-negative bacteria expressing extended-spectrum ß-lactamases (ESBL) have emerged worldwide. ESBL colonisation can persist for years and may favour ESBL transmission. Interventions include contact isolation precautions and restriction of antibiotic use, but decolonisation (DC) for ESBL is not established. We performed a prospective controlled open-label cohort-study from 1/2000 to 1/2008 to determine the effectiveness of a standardised DC programme. ESBL-positive patients routinely underwent screening from rectum, throat, and urine. DC included: chlorhexidine 0.2% mouth rinse three times daily (throat colonisation), paromomycin 4 × 1 g daily (intestinal colonisation), and oral antibiotics for urinary tract colonisation. ESBL elimination was defined as ≥ 1 set of negative follow-up screenings (throat, rectal, urine). Of 100 enrolled patients, 83% of patients were infected and 17% colonised with ESBL. Escherichia coli (71%) and Klebsiella pneumoniae (25%) were the most frequent pathogens. Overall, 76% (76/100) of patients became negative for ESBL at follow-up. Fifty-five percent (42/76) of the successfully treated patients received systemic treatment for infection. Of those who completed DC, 83% (15/18) were free of ESBL at follow-up. DC success correlated with the number of risk factors and colonised sites. DC may be beneficial in a selected group of patients, potentially shortening duration of ESBL colonisation and subsequently reducing the risk for transmission.

Expression, purification and crystallization of an extended-spectrum beta-lactamase from Klebsiella oxytoca.

OXY-1a is an extended-spectrum beta-lactamase from the conditional pathogenic bacterium Klebsiella oxytoca. OXY-1a is responsible for the antibiotic resistance of this pathogen. A soluble form of OXY-1a with a His tag at its C-terminus was overexpressed in Escherichia coli. The recombinant protein was purified and crystallized at room temperature using PEG 4000 as the main precipitant. Two crystal forms were obtained from the same growth conditions. One was orthorhombic, with crystals that diffracted to better than 1.9 A, while the other was tetragonal, with crystals that only diffracted to about 3.0 A. Complete data sets were collected from both crystal forms. The orthorhombic crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.54, b = 73.43, c = 84.56 A, while the tetragonal crystal has unit-cell parameters a = b = 73.72, c = 96.81 A. The asymmetric unit of the orthorhombic crystal is estimated to contain one OXY-1a molecule, giving a crystal volume per protein weight (V(M)) of 2.25 A(3) Da(-1) and a solvent content of 45%.

Decreased outer membrane permeability in imipenem-resistant mutants of Pseudomonas aeruginosa.

The outer membrane of imipenem-resistant mutants of Pseudomonas aeruginosa was shown to have decreased permeability to imipenem but not to cephaloridine. These experiments were performed with intact cells and liposomes containing imipenem-hydrolyzing beta-lactamase derived from Pseudomonas maltophilia, in both cases utilizing an imipenem concentration of 50 microM. In contrast, liposome swelling assays using imipenem at 8 mM detected no significant difference between the imipenem-resistant mutants and their parents. It was found that the outer membrane of P. aeruginosa has a saturable specific channel through which imipenem travels mainly at low concentrations, whereas at high concentrations this pathway is obscured by diffusion through nonspecific porin channels.