A voltammetric test for the rapid discrimination of ß-lactamase-producing Enterobacteriaceae in blood cultures.

The accurate identification of ß-lactamases produced by Enterobacteriaceae is a major challenge in clinical laboratories in order to optimize antimicrobial treatment and patient care. We describe here a rapid voltammetric-based method to detect and to discriminate ß-lactamase activity in Enterobacteriaceae i.e., penicillinase, cephalosporinase (inducible or overproduced), extended-spectrum beta-lactamase and carbapenemase producers. After a 2-h growth step of the sample under three separate conditions: 1) LB (Luria-Bertani) medium, 2) LB supplemented with 4 µg/mL cefotaxime and 3) LB supplemented with 4 µg/mL cefotaxime and 100 µg/mL potassium clavulanate, the ß-lactamase activity was measured by incubating a 0.5 mM nitrocefin solution for 15 min followed by the voltammetric detection of the hydrolyzed nitrocefin with disposable carbon screen-printed sensors. The development and the calibration of the method were carried out by analyzing pure cultures of fifty-seven strains with well characterized ß-lactam-resistance phenotypes. Thanks to the combination of the three currents (i1, i2, i3) recorded for each tested bacteria, the proposed procedure allowed to distinguish the different classes of ß-lactamase producers. In the second part of the study, the method was applied to the analysis of one hundred and fifteen samples Enterobacteriaceae-positive blood culture samples of bacteraemic patients. Overall data showed that the voltammetric method offered a sensitivity of 100% and a specificity of 80%. Interestingly, all of sixteen samples infected by a third-generation cephalosporins-resistant bacteria (i.e. ESBL and overproduced cephalosporinase producers) were detected. This study clearly demonstrated that the voltammetric assay is an efficient alternative technique for the rapid discrimination of ß-lactamases-producing Enterobacteriaceae in blood culture. In contrast to the approved routine assays, the electrochemical test did not require isolated colonies to be performed and was thus carried out in less than 3 h which could allow early administration of an appropriate antibiotic therapy.

A nitrocefin-based amperometric assay for the rapid quantification of extended-spectrum ß-lactamase-producing Escherichia coli in wastewaters.

A sensitive and inexpensive amperometric assay based on the electrochemical detection of the ß-lactamase activity using the nitrocefin as substrate was developed for the rapid and quantitative detection of extended spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) in urban wastewaters. The specific detection of ESBL-EC was achieved by culturing the filtered sample in a medium containing the cefotaxime supplemented or not with the potassium clavulanate inhibitor. This step was followed by the incubation of each subculture filtrate with the nitrocefin substrate which hydrolysis was monitored by amperometry using disposable carbon screen-printed sensors. Current intensities iCef and iClav correspond to the intensity of the anodic current measured (∼+ 0.2 V vs. Ag/AgCl) for the sample incubated with the cefotaxime without and with potassium clavulanate, respectively. The intensity value i = iCef - iClav was chosen as the analytical response. ESBL-EC calibration plots were established with artificially contaminated wastewater samples. This assay allowed the detection of ESBL-EC amounts as low as 10 cfu in treated effluents and 100 cfu in raw wastewaters with short time analysis of 5.5 h and 4.5 h, respectively. The amperometric method was applied to the analysis of 38 wastewater samples and the results were in good agreement with CFU counts on a selective chromogenic medium for 24 h. Owing to its rapidity, convenience, low-cost and portability, this assay is a promising tool to obtain quantitative data on antimicrobial-resistant E. coli in wastewater effluents. Furthermore, this assay might be used to improve wastewater treatment plant processes in order to minimize the release of antibiotic resistant bacteria into the aquatic environment.

Rapid amperometric detection of Escherichia coli in wastewater by measuring ß-D glucuronidase activity with disposable carbon sensors.

An assay on the indirect amperometric quantification of the ß-D-Glucuronidase (GLUase) activity was developed for the rapid and specific detection of Escherichia coli (E. coli) in complex environmental samples. The p-aminophenyl ß-D-glucopyranoside (PAPG) was selected as an electrochemical substrate for GLUase measurement and the p-aminophenol (PAP) released during the enzymatic hydrolysis was monitored by cyclic voltammetry with disposable carbon screen-printed sensors. The intensity of the measured anodic peak current was proportional to the amount of GLUase, and therefore to the number of E. coli in the tested sample. Once the substrate concentration and pH values optimized, a GLUase detection limit of 10 ng mL(-1) was achieved. Using a procedure involving a filtration step of the bacteria followed by their incubation with the substrate solution containing both the nonionic detergent Triton X-100 as permeabilization agent and the culture media Luria broth to monitor the growth, filtered bacterial cells ranging from 5 × 10(4) to 10(8) UFC/membrane were detected within 3 h. The amperometric assay was applied to the determination of fecal contamination in raw and treated wastewater samples and it was successfully compared with conventional bacterial plating methods and uidA gene quantitative PCR. Owing to its ability to perform measurements in turbid media, the GLUase amperometric method is a reliable tool for the rapid and decentralized quantification of viable but also nonculturable E. coli in complex environmental samples.

Amperometric detection of extended-spectrum ß-lactamase activity: application to the characterization of resistant E. coli strains.

The amperometric detection of extended-spectrum ß-lactamase (ESBL) with carbon screen-printed sensors was investigated in the presence of the Nitrocefin, a commercially-available ß-lactamase chromogenic cephalosporin substrate. Using an ESBL isolated from a clinical sample, it was shown for the first time that the intensity of a specific anodic pic current (EP = ∼+0.3 V vs. Ag/AgCl) resulting from the catalytic hydrolysis of the ß-lactam ring was proportional to the amount of ESBL. The proof-of-principle of a novel susceptibility assay for the rapid and accurate identification of ESBL- producing bacteria was then demonstrated. The detection scheme relied on (i) the culture of the sample in a medium containing the cefotaxime supplemented or not with the clavulanic acid inhibitor to allow the specific determination of ESBL producers (ii) followed by the incubation of the bacteria with the Nitrocefin and (iii) the measurement of the enzyme product by cyclic voltammetry. The amperometric assay was further applied to the characterization of E. coli strains and to the quantification of the ESBL producers. A detection limit of 5 × 10(4) cfu mL(-1) ESBL-producing E. coli was achieved after a 10 min incubation time. In contrast to the approved routine assays, the electrochemical approach, which did not require isolated colonies to be performed, provided quantified results regarding ESBL activity within a few hours. Finally, owing to its cost-effectiveness, portability and simplicity, this test holds great promise for clinical and environmental applications.

Development and evaluation of a method for the quantification of airborne Thermoactinomyces vulgaris by real-time PCR.

Actinomycetes are ubiquitous and some can be potentially pathogenic for humans when present in the air of some working areas. It's notably the case for Thermoactinomyces vulgaris in composting facilities where aerial concentrations can reach high values of more than 10(7) CFU·m(-3). Workers exposure to these inhalable bioaerosols can be the source of various diseases. The literature reveals a lack of knowledge about risk assessment: there is neither dose-effects relationship for most agents, or threshold limit value. The objectives of this study were to develop and standardize a method to quantify workers exposure to bioaerosols. We have developed and evaluated a method to quantify airborne T. vulgaris based on DNA extraction of aerial microbial communities and qPCR. Four DNA extraction protocols were compared, and primers and a hydrolysis probe were designed for specific amplification of the target species (gyrB gene). This method was compared to traditional methods based on viable or cultivable counting by epifluorescence microscopy or plating on selective media. The method was applied on environmental bioaerosols sampled under real exposure conditions in composting plants. We demonstrate that the method to quantify T. vulgaris in bioaerosols is specific, sensitive and repeatable. We demonstrate the occurrence and quantified T. vulgaris in the atmosphere of composting facilities with concentrations ranging from 3×10(2) to 3×10(6)×m(-3).

Mycobacterium behavior in wastewater treatment plant, a bacterial model distinct from Escherichia coli and Enterococci.

Mycobacteria are waterborne emerging pathogens causing infections in human. Mycobacteria have been previously isolated from wastewater and sludge, but their densities were not estimated due to cultural biases. In order to evaluate the impact of wastewater treatment processes on mycobacteria removal, we used a real time PCR method. First we compared six DNA extraction methods and second we used the more efficient DNA extraction procedure (i.e., enzymatic lysis combined with hexadecyltrimethylammonium bromide-NaCl procedure) in order to quantify Mycobacterium. With the aim to identify parameters that could serve as indicator of mycobacterial behavior, mycobacterial densities were measured in parallel to those of Escherichia coli and enterococci, and to concentrations of chemical parameters usually monitored in wastewater. Mycobacterium reached 5.5 × 105 ± 3.9 × 105 copies/L in the influent, but was not detected in the effluent after decantation and biofiltration. Most mycobacteria (98.6 ± 2.7%, i.e. 2.4 ± 0.7 log10) were removed by the physical-chemical decantation, and the remaining mycobacteria were removed by biofiltration. In contrast, enterococci and E. coli were lightly removed by decantation step and mainly removed by biofiltration. Our results showed that Mycobacterium corresponds to a hydrophobic behavior linked to insoluble compound removal, whereas enterococci and E. coli refer to hydrophilic behaviors linked to soluble compound removals.