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.

Nucleic acid tests have been widely used for diagnosis of diseases by detecting the relevant genetic markers that are usually amplified using polymerase chain reaction (PCR). This work reports the use of a plasmonic device as an efficient and low-cost PCR thermocycler to facilitate nucleic acid-based diagnosis. The thermoplasmonic device, consisting of a one-dimensional metal grating, exploited the strong light absorption of plasmonic resonance modes to heat up PCR reagents using a near-infrared laser source. The plasmonic device also integrated a thin-film thermocouple on the metal grating to monitor the sample temperature. The plasmonic thermocycler is capable of performing a PCR amplification cycle in ~2.5 minutes. We successfully demonstrated the multiplex and real-time PCR amplifications of the antibiotic resistance genes using the genomic DNAs extracted from Acinetobacter baumannii, Klebsiella pneumonia, Escherichia coli and Campylobacter.

Lab-on-a-chip multiplex assays allow a rapid identification of multiple parameters in an automated manner. Here we describe a lab-based preparation followed by a rapid and fully automated DNA microarray hybridization and readout in less than 10 min using the Fraunhofer in vitro diagnostics (ivD) platform to enable rapid identification of bacterial species and detection of antibiotic resistance. The use of DNA microarrays allows a fast adaptation of new biomarkers enabling the identification of different genes as well as single-nucleotide-polymorphisms (SNPs) within these genes. In this protocol we describe a DNA microarray developed for identification of Staphylococcus aureus and the mecA resistance gene.

Helicobacter pylori (H. pylori) is a species of bacteria that can colonize the human stomach mucosa. It is closely associated with gastric diseases such as ulcer and inflammation. Recently, some H. pylori strains were found to express resistance to a family of antibiotics known as quinolones due to single-point mutations. Although traditional polymerase chain reaction (PCR) and molecular diagnostic-based approaches can be used to determine the presence and abundance of antibiotic-resistant H. pylori strains, such processes are relatively expensive, labor-intensive, and require bulky and costly equipment. This study therefore reports an advanced diagnostic assay performed on an integrated microfluidic system for rapid detection of antibiotic resistance in H. pylori. The assay features three components: (1) nucleic acid extraction by specific probe-conjugated magnetic beads, (2) amplification of the target deoxyribonucleic acid (DNA) fragments by using single-nucleotide-polymorphism polymerase chain reaction (SNP-PCR), and (3) optical detection of the PCR products. The device integrates several microfluidic components including micro-pumps, normally-closed micro-valves, and reaction chambers such that the entire diagnostic assay can be automatically executed on a single microfluidic system within one hour with detection limits of 10(0), 10(2), and 10(2) bacterial cells for H. pylori detection and two different SNP sites strains. Three PCR-based assays for determining presence of H. pylori infection and two DNA single-point mutation assays aimed at determining whether the infected strains were resistant to quinolone can be performed simultaneously on a single chip, suggesting that this microfluidic system could be a promising tool for rapid diagnosis of the presence of antibiotic-resistant H. pylori strains.

We report on the synthesis of three nitrocefin analogues and their evaluation as substrates for the detection of β-lactamase activity. These compounds are hydrolyzed by all four Ambler classes of β-lactamases. Kinetic parameters were determined with eight different β-lactamases, including VIM-2, NDM-1, KPC-2, and SPM-1. The compounds do not inhibit the growth of clinically important antibiotic-resistant gram-negative bacteria in vitro. These chromogenic compounds have a distinct absorbance spectrum and turn purple when hydrolyzed by β-lactamases. One of these compounds, UW154, is easier to synthesize from commercial starting materials than nitrocefin and should be significantly less expensive to produce.