The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Enterococcus spp., and Klebsiella spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥10³ CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application.

Macrophages use diverse strategies to restrict intracellular pathogens, including either depriving the bacteria of (micro)nutrients such as transition metals or intoxicating them via metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum, a close relative of Mycobacterium tuberculosis (Mtb), and its hosts. We use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn²⁺ during M. marinum infection. We show that M. marinum senses toxic levels of Zn²⁺ and responds by upregulating one of its isoforms of the Zn²⁺ efflux transporter CtpC. Deletion of ctpC (MMAR_1271) leads to growth inhibition in broth supplemented with Zn²⁺ as well as reduced intracellular growth. Both phenotypes were fully rescued by constitutive ectopic expression of the Mtb CtpC orthologue demonstrating that MMAR_1271 is the functional CtpC Zn²⁺ efflux transporter in M. marinum. Infection leads to the accumulation of Zn²⁺ inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn²⁺ efflux pumps ZntA and ZntB. In cells lacking ZntA, there is further attenuation of M. marinum growth, presumably due to a compensatory efflux of Zn²⁺ into the MCV, carried out by ZntB, the main Zn²⁺ transporter in endosomes and phagosomes. Counterintuitively, bacterial growth is also impaired in zntB KO cells, in which MCVs appear to accumulate less Zn²⁺ than in wild-type cells, suggesting restriction by other Zn²⁺-mediated mechanisms. Absence of CtpC further epistatically attenuates the intracellular proliferation of M. marinum in zntA and zntB KO cells, confirming that mycobacteria face noxious levels of Zn²⁺. IMPORTANCE Microelements are essential for the function of the innate immune system. A deficiency in zinc or copper results in an increased susceptibility to bacterial infections. Zn²⁺ serves as an important catalytic and structural cofactor for a variety of enzymes including transcription factors and enzymes involved in cell signaling. But Zn²⁺ is toxic at high concentrations and represents a cell-autonomous immunity strategy that ensures killing of intracellular bacteria in a process called zinc poisoning. The cytosolic and lumenal Zn²⁺ concentrations result from the balance of import into the cytosol via ZIP influx transporters and efflux via ZnT transporters. Here, we show that Zn²⁺ poisoning is involved in restricting Mycobacterium marinum infections. Our study extends observations during Mycobacterium tuberculosis infection and explores for the first time how the interplay of ZnT transporters affects mycobacterial infection by impacting Zn²⁺ homeostasis.

Introduction: Streptococcus agalactiae, also known as group B streptococci (GBS), is associated with invasive infections in neonates. Identification of GBS vaginal colonization in pregnant women before delivery is essential for treatment with antibiotics to prevent intrapartum vertical transmission to the newborn. This study was designed to evaluate applicability of two rapid real‐time PCRs in comparison to standard culture identification.Material and methods: We compared the Xpert GBS assay, hereafter referred to as Xpert, and GenomEra GBS PCR, hereafter referred to as GenomEra. The standard culture identification consisted of two different agar plates as well as an enrichment broth.Results: We analyzed vaginal samples of 260 pregnant women; 42 samples were tested GBS‐positive by using standard culture as a gold standard, 30 by Xpert, and 37 by GenomEra. Xpert and GenomEra assays performed with sensitivities of 71.4% and 88.1% as well as specificities of 98.6% and 99.1%, respectively. Twelve vaginal samples were false‐negative by Xpert and five samples by GenomEra. Interestingly, three negative Xpert results of standard culture‐positive samples exhibited high Ct‐values indicating the presence of GBS. If higher Ct‐values are taken into consideration, the sensitivity of Xpert increases up to 78.6%. Moreover, only three Xpert PCRs had to be repeated, whereas two Genomera were invalid even after repetition and further 15 GenomEra PCRs were repeated because of borderline results or inhibition of the PCR test.Conclusions: In this study, GenomEra assay performed with a higher sensitivity than the Xpert PCR. On the other hand, the Xpert assay needs less hands‐on‐time for a sample preparation and requires approximately four‐fold less repetitions as compared to the GenomEra assay. This robust performance of the Xpert assay make it applicable as a rapid intrapartum point‐of‐care test, although a higher sensitivity would be desirable. Therefore, culture in the 35–37 week of gestation remains the gold standard to detect vaginal colonization.