-glucuronidase activity determination as an indirect estimate of Escherichia coli: development of a miniaturized assay and its application to seawater samples

Background: The search of rapid methods for the detection of Escherichia coli in coastal marine waters is a topic of scientifi c interest to evaluate potential risks to human health related to their low bacteriological quality. The context and purpose of the study: A miniaturized assay for the analytical determination of β− glucuronidase activity in seawater as a selective marker of Escherichia coli was developed by using the chromogenic Indoxyl-β-D-glucuronide (IBDG) substrate. This compound is specifi cally cleaved by E. coli, releasing an insoluble chromophoric blue-indigo product that precipitates and is measured at 450 nm wavelength by a microplate reader. Results: After its preliminary optimization, the enzymatic assay was applied to the analysis of seawater samples and enabled to discriminate them according to their pollution level. Main fi ndings: The fi rst obtained data proved the suitability of the developed miniaturized enzymatic assay for fecal contamination monitoring. It can be used for the detection and indirect quantifi cation of E. coli, without the need for confi rmatory steps. Conclusions: This study suggests that the proposed analytical protocol is suitable for E. coli monitoring in seawater, and provides in a short time (i.e. 2 hours from sampling) results which are in agreement with the standard culture counts. Brief summary: The results obtained with the developed IBDG protocol encourage its use for environmental quality assessment. Any potential implications: The possibility to obtain near “real-time” data on the occurrence and distribution of anthropogenic inputs makes this method a simple and quick tool for early warning detection of marine fecal pollution. Research Article -glucuronidase activity determination as an indirect estimate of Escherichia coli: development of a miniaturized assay and its application to seawater samples Caruso G1*, Caruso R2, Monticelli LS1, Maimone G1 and Crisafi E1 1National Research Council, Institute for Coastal Marine Environment (CNR-IAMC), Messina, Italy 2Hospital Agency A.O.U. “G. Martino”, Messina, Italy Dates: Received: 25 November, 2017; Accepted: 12 December, 2017; Published: 16 December, 2017 *Corresponding author: Gabriella Caruso, Graduate in Biological Sciences, Specialist in Applied Microbiology. Researcher at: CNR-IAMC, Spianata S. Raineri 86, 98122 Messina, Italy, Tel: +39-0906015423; Fax: +39-090669007; E-mail:


Introduction
Multiple human activities that coexist in coastal areas cause signifi cant threats to both the whole marine environment and human health [1]. The overall quality of bathing sites may be affected by many pollution sources such as urbanization, agricultural activities, harbour activities, waste discharges, industry; a complete environmental management needs the availability of specifi c and quick tools, able to provide integrated information on the level of contamination and pollution and its consequences for the health of coastal areas [2][3][4]. Since areas. In seawater quality monitoring, rapid microbiological methods relying on biochemical or molecular approaches offer new interesting perspectives for the determination of Escherichia coli, which is universally recognised as the most suitable indicator of fecal contamination [5][6][7][8][9]. Typically, 90 to 95% of E. coli strains possess the gene for -glucuronidase, and therefore this enzyme activity has been traditionally regarded as a selective marker for rapid detection of this microorganism [10]. Since the last decade, a variety of chromogenic and fl uorogenic substrates have been formulated and proposed in the bacterial diagnostic as an attractive tool to quickly identify specifi c microorganisms [11][12], by their inclusion as components in both solid and liquid culture media. They proved to be successful for the analysis of water, food and clinical samples, providing sensitive and specifi c results [13].
Like fl uorogenic substrates, widely applied in the monitoring of aquatic environmental health [7,[14][15], chromogenic substrates produce a visible signal when cleaved by the specifi c enzyme that is present or is being expressed by a specifi c microorganism. In the case of chromogenic compounds, however, the reaction product precipitates in the assay mixture and can be easily quantifi ed by a photometric method; this results in a signifi cant advantage, because it avoids background fl uorescence due to the spreading of the fl uorescent reaction product into the culture medium, which is frequently reported as a source of biases when using fl uorogenic substrates [16][17][18].
Experiments were carried out at the laboratory of the CNR-IAMC, with the aim of developing a miniaturized method for the determination of -glucuronidase activity rates as an indirect estimate of E. coli, by using the chromogenic compound Indoxyl--D-glucuronide (IBDG). This compound is specifi cally cleaved by E. coli, producing after reaction a waterinsoluble blue-indigo aglycone product that precipitates and is measured at 450 nm wavelength; this makes the observation of the reaction product easy, also under normal daylight [19].

Specifi city test
In order to exclude false-positive results due to the  [20]) were assayed using the miniaturized assay.

On fi eld application
The developed protocol was applied to the analysis of seawater samples. Surface samples were collected from selected sites of Messina shoreline differently impacted by urban wastes (Tremestieri, Pagliarisi, Annunziata, Tennis), fi ltered through a 0.45 μm pore size mixed cellulose esters fi ltering membrane (Millipore Corporation, Bedford, USA) and 10 times concentrated by resuspension in sterile physiological saline, in order to obtain a signal detectable by the microplate reader. The -glucuronidase activity rates were reported as the maximal velocity of enzymatic reaction (V max ), which was calculated through the Lineweaver Burke transformation of each substrate concentration plotted versus the corresponding velocity of hydrolysis of IBDG substrate. V max values were multiplied by the initial concentration factor and reported per 100 ml of sample.

Plate counts
Contextually to the enzymatic assay, standard plate counts were carried out by culture method on m-FC agar medium [21], in order to compare and relate the measured -glucuronidase activity rates with E. coli abundance. Blue colonies grown on m-FC agar were confi rmed to be E. coli after addition of James indole reagent (Biomérieux, Marcy l'Etoile, France).

Specifi city test
No positive results with the strains assayed in the specifi city test were obtained, suggesting that the substrate IBGD was specifi cally metabolised by E. coli and that the enzymatic assay was specifi c for the detection of this microorganism. The -glucuronidase activity rates, reported as V max values, correlated signifi cantly with plate counts, which showed concentrations of 5.52x10 2 , 6.0x10 2 , 3.24x10 2 and 5.40x10 2 CFU 100 ml -1 at the stations Tremestieri, Pagliarisi, Annunziata and Tennis, respectively ( Figure 3).

Discussion
This study can be considered as a further contribute in the fi eld of rapid analytical methods for environmental quality monitoring. In this context, the set-up of laboratory procedures allowing us to detect and quantify in short times (within a few hours) E. coli abundance, can play a strategic role. The developed protocol was applied to the analysis of natural samples collected from coastal sites more or less affected by fecal pollution, in comparison with the plate count method, for the quantitative determination of E. coli in seawater. The assay also included a preliminary step of concentration, which was necessary to overcome possible diffi culties encountered in detecting E. coli in the case that this bacterium was present in low abundances in natural seawater.
Previous experiments, performed through the incubation of a concentrated sample with the fl uorogenic 4-metilumbellyferil--d-glucuronide (MUG) substrate, showed the suitability of the enzymatic method as a rapid tool for the screening of marine environments according to their pollution degree [7]. Following the same methodological approach (i.e. addition of multiple concentrations of the substrate), but modifi ed through the use of the chromogenic IBDG substrate and automatic readings of the hydrolysis product, a new experimental protocol for the estimation of -glucuronidase present in natural seawater samples was developed.
Such method has been conceived with the fi nal objective of miniaturizing the enzymatic assay and automating their OD measurements. Using the proposed procedure, it is possible to monitor indirectly the presence and distribution of E. coli in seawater. Compared to other technologies developed for the rapid detection of bacteria in recreational waters, the developed protocol offers also the advantages of working with low volumes of sample, disposable microplates, and simple instrumentation, therefore it is cheaper than other commercial diagnostic kits for E. coli detection based on molecular approaches [22][23].
The microplate reader can run a fully automated analysis of the substrate hydrolytic reaction and the -glucuronidase activity rates can be available in short times, typically within a few hours. The frequency of automatic readings can be regulated according to the operator needs, but normally an incubation period of 2 hours is required to obtain a detectable signal. The use of IBDG in a liquid mixture also involves a signifi cant decrease in the response time compared with its incorporation into a solid medium, for which at least 18 hours of incubation are normally needed to appreciate bacterial growth. Another important aspect is the high incubation temperature (44.5°C) set up in the assay, which increases the specifi city of the reaction for E. coli. All these features make our assay particularly suitable to match with the requirements (speed and specifi city of detection) for early warning of bacterial pollution in coastal monitoring.

Conclusion
The results obtained from the application of the developed enzymatic assay to naturally polluted samples are encouraging.