β-Lactamases are bacterial enzymes that inactivate β-lactam antibiotics and, as such, are the most prevalent cause of antibiotic resistance in Gram-negative bacteria. The ever-increasing production and worldwide dissemination of bacterial strains producing carbapenemases is currently a global health concern. These enzymes catalyze the hydrolysis of carbapenems - the β-lactam antibiotics with the broadest spectrum of activity that are often considered as drugs of last resort. The incidence of carbapenem-resistant pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii and carbapenemase or extended spectrum beta-lactamase (ESBL)-producing Enterobacterales, which are frequent in clinical settings, is worrisome since, in some cases, no therapies are available. These include all metallo-β-lactamases (VIM, IMP, NDM, SMP, and L1), and serine-carbapenemases of classes A (KPC, SME, IMI, and GES), and of classes D (OXA-23, OXA-24/40, OXA-48 and OXA-58). Consequently, the early diagnosis of bacterial strains harboring carbapenemases is a pivotal task in clinical microbiology in order to track antibiotic bacterial resistance and to improve the worldwide management of infectious diseases. Recent research efforts on the development of chromogenic and fluorescent chemical sensors for the specific and sensitive detection and quantification of β-lactamase production in multidrug-resistant pathogens are summarized herein. Studies to circumvent the main limitations of the phenotypic and molecular methods are discussed. Recently reported chromogenic and fluorogenic cephalosporin- and carbapenem-based β-lactamase substrates will be reviewed as alternative options to the currently available nitrocefin and related compounds, a chromogenic cephalosporin-based reagent widely used in clinical microbiology laboratories. The scope of these new chemical sensors, along with the synthetic approaches to synthesize them, is also summarized.

Ceramidases (CDases) are important in controlling skin barrier integrity by regulating ceramide composition and affording downstream signal molecules. While the functions of epidermal CDases are known, roles of neutral CDases secreted by skin-residing microbes are undefined. Here, we developed a one-step fluorogenic substrate, S-B, for specific detection of bacterial CDase activity and inhibitor screening. We identified a non-hydrolyzable substrate mimic, C6, as the best hit. Based on C6, we designed a photoaffinity probe, JX-1, which efficiently detects bacterial CDases. Using JX-1, we identified endogenous low-abundance PaCDase in a P. aeruginosa monoculture and in a mixed skin bacteria culture. Harnessing both S-B and JX-1, we found that CDase activity positively correlates with the relative abundance of P. aeruginosa and is negatively associated with wound area reduction in clinical diabetic foot ulcer patient samples. Overall, our study demonstrates that bacterial CDases are important regulators of skin ceramides and potentially play a role in wound healing.

β-glucosidases hydrolyse glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides via catalytic mechanisms. It is very well known that the β-glucosidase enzyme is used in biorefineries for cellulose degradation, where β-glucosidases is the rate-limiting enzyme for the final glucose production from cellobiose. The β-glucosidase enzyme is used as a catalyst in other industrial sectors, including pharmaceuticals, breweries, dairy, and food processing. With the aid of β-glucosidase enzymes, cyanogenic glycosides and plant glycosides are transformed into sugar moiety and aglycones. These aglycone compounds are employed as aromatic compounds in the food processing and brewing industries. They are also used as medications and dietary supplements based on their pharmacological qualities. Applications of aglycones and the microbiological sources of β-glucosidase in aglycone production have been discussed in this review.

The enzyme-linked immunospot (ELISpot) is a highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single-cell level. The secreted molecules are detected by using a detection antibody system similar to that used in the enzyme-linked immunosorbent assay (ELISA). The ELISpot assay is carried out in a 96-well plate and an automated ELISpot reader is used for analysis. The assay is easy to perform, robust and allows rapid analysis of a large number of samples and is not limited to measurement of cytokines; it is suitable for almost any secreted protein where single-cell analysis is of interest.

The ability to detect, identify and quantify bacteria is crucial in clinical diagnostics, environmental testing, food security settings and in microbiology research. Recently, the threat of multidrug-resistant bacterial pathogens pushed the global scientific community to develop fast, reliable, specific and affordable methods to detect bacterial species. The use of synthetically modified enzyme substrates is a convenient approach to detect bacteria in a specific, economic and rapid manner. The method is based on the use of specific enzyme substrates for a given bacterial marker enzyme, conjugated to a signalogenic moiety. Following enzymatic reaction, the signalophor is released from the synthetic substrate, generating a specific and measurable signal. Several types of signalophors have been described and are defined by the type of signal they generate, such as chromogenic, fluorogenic, luminogenic, electrogenic and redox. Signalophors are further subdivided into groups based on their solubility in water, which is key in defining their application on solid or liquid media for bacterial culturing. This comprehensive review describes synthetic enzyme substrates and their applications for bacterial detection, showing their mechanism of action and their synthetic routes.

The preparation and evaluation of chromogenic substrates for detecting bacterial glycosidase enzymes is reported. These substrates are monoglycoside derivatives of the metal chelators catechol, 2,3-dihydroxynaphthalene (DHN) and 6,7-dibromo-2,3-dihydroxynaphthalene (6,7-dibromo-DHN). When hydrolysed by appropriate bacterial enzymes these substrates produced coloured chelates in the presence of ammonium iron(III) citrate, thus enabling bacterial detection. A β-d-riboside of DHN and a β-d-glucuronide derivative of 6,7-dibromo-DHN were particularly effective for the detection of S. aureus and E. coli respectively.

Rapid, sensitive, and selective detection and identification of pathogenic bacteria is required in terms of food security. In this study, exogenous VOCs liberated by Salmonella strains have been identified and quantified via head space-solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) in milk samples. The specific enzymes targeted for detection and/or differentiation of Salmonella were C8 esterase, α-galactosidase and pyrrolidonyl peptidase using the following enzyme substrates: 2-chlorophenyl octanoate, phenyl α-d-galactopyranoside and L-pyrrollidonyl fluoroanilide, respectively. Detection of the exogenous VOCs, 2-chlorophenol, phenol and 3-fluoraniline was possible with typical limits of detection of 0.014, 0.045 and 0.005 μg/mL, respectively and correlation coefficients >0.99. The developed methodology was able to detect and identify Salmonella species within a 5 h incubation at 37 °C by the detection of the liberated VOCs. It was found that the milk samples tested were Salmonella free.

A series of carboxy-substituted 2-(nitroaryl)benzothiazole derivatives and carboxy-substituted 2-(nitroaryl)benzoxazole derivatives were prepared and evaluated as potential nitroreductase substrates for the purpose of detecting clinically important microorganisms. Several of the substrates produced highly fluorescent colonies with the majority of a panel of 10 Gram-negative bacteria and also with two of a panel of 8 Gram-positive bacteria.

The rapid detection of Listeria monocytogenes contamination in food is essential to prevent food-borne illness in humans. The aim of this study was to differentiate non-contaminated milk from milk contaminated with L. monocytogenes using enzyme substrates coupled with the analysis of volatile organic compounds (VOCs). The method is based on the activity of β-glucosidase and hippuricase enzymes and the detection of a specific VOC i.e. 2-nitrophenol and 3-fluoroaniline, respectively. VOCs were extracted, separated and detected by headspace-solid phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME GC-MS). This approach required the inclusion of the selective agent\'s cycloheximide, nalidixic acid and acriflavine HCl in the growth medium to inhibit interfering bacteria. The VOCs were liberated by L. monocytogenes provided that samples contained at least 1-1.5×10(2) CFU ml(-1) of milk prior to overnight incubation. This approach shows potential for future development as a rapid method for the detection of L. monocytogenes contaminated milk.