Validation of a qualitative real-time PCR assay for the detection of Candida auris in hospital inpatient screening.

Candida auris is a multidrug-resistant opportunistic fungal pathogen capable of causing serious infections and healthcare-associated outbreaks. Screening for colonization with C. auris has become routine and is recommended in many hospitals and healthcare facilities as an infection control and prevention strategy. Subsequently, and since there are currently no FDA-approved tests for this purpose, clinical microbiology laboratories have become responsible for developing protocols to detect C. auris using axial and inguinal screening swabs. In a College of American Pathologists-accredited large academic healthcare center setting, we implemented a laboratory-developed nucleic-acid amplification test for the detection of C. auris DNA. Our test validation evaluated the performance of the DiaSorin C. auris primer set used in a real-time qualitative PCR assay on the LIAISON MDX thermocycler with the Simplexa Universal Disc. The assay was highly sensitive and specific, with a limit of detection of 1-2 CFU/reaction, with no observed cross-reactivity with other Candida spp., bacterial skin commensal organisms or commonly encountered viruses. When run in parallel with a culture-based detection method, the PCR assay was 100% sensitive and specific. The assay was precise, with low variability between replicates within and between runs. Lastly, pre-analytical factors, including swab storage time, temperature, and transport media, were assessed and found to have no significant effect on the detection of C. auris at variable concentrations. Taken together, this study expands the available options for nucleic acid detection of C. auris and characterizes pre-analytical factors for implementation in both high- and low-volume laboratory settings. IMPORTANCE: This study overviews the validation and implementation of a molecular screening tool for the detection of Candida auris in a College of American Pathologist-accredited clinical laboratory. This molecular laboratory-developed test is both highly sensitive and specific and has significant health-system cost-savings associated with significantly reduced turn-around-time compared to traditional standard-of-care culture-based work up. This method and workflow is of interest to support clinical microbiology diagnostics and to help aid in hospital inpatient, and infection prevention control screening.

Phosphorus starvation response and PhoB-independent utilization of organic phosphate sources by Salmonella enterica.

IMPORTANCE: Phosphorus (P) is the fifth most abundant element in living cells. This element is acquired mainly as inorganic phosphate (Pi, PO4 3-). In enteric bacteria, P starvation activates a two-component signal transduction system which is composed of the membrane sensor protein PhoR and its cognate transcription regulator PhoB. PhoB, in turn, promotes the transcription of genes that help maintain Pi homeostasis. Here, we characterize the P starvation response of the bacterium Salmonella enterica. We determine the PhoB-dependent and independent transcriptional changes promoted by P starvation and identify proteins enabling the utilization of a range of organic substrates as sole P sources. We show that transcription and activity of a subset of these proteins are independent of PhoB and Pi availability. These results establish that Salmonella enterica can maintain Pi homeostasis and repress PhoB/PhoR activation even when cells are grown in medium lacking Pi.

An intracellular phosphorus-starvation signal activates the PhoB/PhoR two-component system in Salmonella enterica.

Bacteria acquire P primarily as inorganic orthophosphate (Pi, PO43-). Once internalized, Pi is rapidly assimilated into biomass during the synthesis of ATP. Because Pi is essential, but excessive ATP is toxic, the acquisition of environmental Pi is tightly regulated. In the bacterium Salmonella enterica (Salmonella), growth in Pi-limiting environments activates the membrane sensor histidine kinase PhoR, leading to the phosphorylation of its cognate transcriptional regulator PhoB and subsequent transcription of genes involved in adaptations to low Pi. Pi limitation is thought to promote PhoR kinase activity by altering the conformation of a membrane signaling complex comprised by PhoR, the multicomponent Pi transporter system PstSACB and the regulatory protein PhoU. However, the identity of the low Pi signal and how it controls PhoR activity remain unknown. Here we characterize the PhoB-dependent and independent transcriptional changes elicited by Salmonella in response to P starvation, and identify PhoB-independent genes that are required for the utilization of several organic-P sources. We use this knowledge to identify the cellular compartment where the PhoR signaling complex senses the Pi-limiting signal. We demonstrate that the PhoB and PhoR signal transduction proteins can be maintained in an inactive state even when Salmonella is grown in media lacking Pi. Our results establish that PhoR activity is controlled by an intracellular signal resulting from P insufficiency.

Coordination of Phosphate and Magnesium Metabolism in Bacteria.

The majority of cellular phosphate (PO4-3; Pi) exists as nucleoside triphosphates, mainly adenosine triphosphate (ATP), and ribosomal RNA (rRNA). ATP and rRNA are also the largest cytoplasmic reservoirs of magnesium (Mg2+), the most abundant divalent cation in living cells. The co-occurrence of these ionic species in the cytoplasm is not coincidental. Decades of work in the Pi and Mg2+ starvation responses of two model enteric bacteria, Escherichia coli and Salmonella enterica, have led to the realization that the metabolisms of Pi and Mg2+ are interconnected. Bacteria must acquire these nutrients in a coordinated manner to achieve balanced growth and avoid loss of viability. In this chapter, we will review how bacteria sense and respond to fluctuations in environmental and intracellular Pi and Mg2+ levels. We will also discuss how these two compounds are functionally linked, and how cells elicit physiological responses to maintain their homeostasis.

Limitation of phosphate assimilation maintains cytoplasmic magnesium homeostasis.

Phosphorus (P) is an essential component of core biological molecules. In bacteria, P is acquired mainly as inorganic orthophosphate (Pi) and assimilated into adenosine triphosphate (ATP) in the cytoplasm. Although P is essential, excess cytosolic Pi hinders growth. We now report that bacteria limit Pi uptake to avoid disruption of Mg2+-dependent processes that result, in part, from Mg2+ chelation by ATP. We establish that the MgtC protein inhibits uptake of the ATP precursor Pi when Salmonella enterica serovar Typhimurium experiences cytoplasmic Mg2+ starvation. This response prevents ATP accumulation and overproduction of ribosomal RNA that together ultimately hinder bacterial growth and result in loss of viability. Even when cytoplasmic Mg2+ is not limiting, excessive Pi uptake increases ATP synthesis, depletes free cytoplasmic Mg2+, inhibits protein synthesis, and hinders growth. Our results provide a framework to understand the molecular basis for Pi toxicity. Furthermore, they suggest a regulatory logic that governs P assimilation based on its intimate connection to cytoplasmic Mg2+ homeostasis.

Genetic Modification of Sodalis Species by DNA Transduction.

Bacteriophages (phages) are ubiquitous in nature. These viruses play a number of central roles in microbial ecology and evolution by, for instance, promoting horizontal gene transfer (HGT) among bacterial species. The ability of phages to mediate HGT through transduction has been widely exploited as an experimental tool for the genetic study of bacteria. As such, bacteriophage P1 represents a prototypical generalized transducing phage with a broad host range that has been extensively employed in the genetic manipulation of Escherichia coli and a number of other model bacterial species. Here we demonstrate that P1 is capable of infecting, lysogenizing, and promoting transduction in members of the bacterial genus Sodalis, including the maternally inherited insect endosymbiont Sodalis glossinidius While establishing new tools for the genetic study of these bacterial species, our results suggest that P1 may be used to deliver DNA to many Gram-negative endosymbionts in their insect host, thereby circumventing a culturing requirement to genetically manipulate these organisms.IMPORTANCE A large number of economically important insects maintain intimate associations with maternally inherited endosymbiotic bacteria. Due to the inherent nature of these associations, insect endosymbionts cannot be usually isolated in pure culture or genetically manipulated. Here we use a broad-host-range bacteriophage to deliver exogenous DNA to an insect endosymbiont and a closely related free-living species. Our results suggest that broad-host-range bacteriophages can be used to genetically alter insect endosymbionts in their insect host and, as a result, bypass a culturing requirement to genetically alter these bacteria.

Conjugal DNA Transfer in Sodalis glossinidius, a Maternally Inherited Symbiont of Tsetse Flies.

Stable associations between insects and bacterial species are widespread in nature. This is the case for many economically important insects, such as tsetse flies. Tsetse flies are the vectors of Trypanosoma brucei, the etiological agent of African trypanosomiasis-a zoonotic disease that incurs a high socioeconomic cost in regions of endemicity. Populations of tsetse flies are often infected with the bacterium Sodalis glossinidius Following infection, S. glossinidius establishes a chronic, stable association characterized by vertical (maternal) and horizontal (paternal) modes of transmission. Due to the stable nature of this association, S. glossinidius has been long sought as a means for the implementation of anti-Trypanosoma paratransgenesis in tsetse flies. However, the lack of tools for the genetic modification of S. glossinidius has hindered progress in this area. Here, we establish that S. glossinidius is amenable to DNA uptake by conjugation. We show that conjugation can be used as a DNA delivery method to conduct forward and reverse genetic experiments in this bacterium. This study serves as an important step in the development of genetic tools for S. glossinidius The methods highlighted here should guide the implementation of genetics for the study of the tsetse-Sodalis association and the evaluation of S. glossinidius-based tsetse fly paratransgenesis strategies.IMPORTANCE Tsetse flies are the insect vectors of T. brucei, the causative agent of African sleeping sickness-a zoonotic disease that inflicts a substantial economic cost on a broad region of sub-Saharan Africa. Notably, tsetse flies can be infected with the bacterium S. glossinidius to establish an asymptomatic chronic infection. This infection can be inherited by future generations of tsetse flies, allowing S. glossinidius to spread and persist within populations. To this effect, S. glossinidius has been considered a potential expression platform to create flies which reduce T. brucei stasis and lower overall parasite transmission to humans and animals. However, the efficient genetic manipulation of S. glossinidius has remained a technical challenge due to its complex growth requirements and uncharacterized physiology. Here, we exploit a natural mechanism of DNA transfer among bacteria and develop an efficient technique to genetically manipulate S. glossinidius for future studies in reducing trypanosome transmission.