Deciphering the potential of plasma cell-free metagenomic next-generation sequencing using the Karius test.

PURPOSE OF REVIEW: Plasma cell-free metagenomic next-generation sequencing (cf-mNGS) is increasingly employed for the diagnosis of infection, but a consensus for optimal use has not been established. This minireview focuses on the commercially available Karius Test and is aimed at local leaders seeking to understand the complexities of cf-mNGS to make informed test utilization policies and better interpret results. RECENT FINDINGS: Recent retrospective studies have reported how the Karius Test was applied at their institutions and identified areas of potential patient benefit. In addition, substantive studies have reported how this test performs in specific indications, particularly invasive fungal disease, endovascular infection and lower respiratory infection. SUMMARY: Successfully integrating plasma cf-mNGS requires careful assessment of performance in the specific applications and patient populations in which it is used. Individual institutions must independently evaluate implementation strategies and determine where diagnostic yields outweigh the potential pitfalls.

A Novel Phenotypic Method To Screen for Plasmid-Mediated Colistin Resistance among Enterobacteriales.

Plasmid-mediated colistin resistance (PMCR), a consequence of the mcr genes, is a significant public health concern given its potential to easily spread among clinical pathogens. Recently, it was discovered that MCR enzymes require zinc for activity. Thus, we modified the colistin broth-disk elution (CBDE) test to screen for plasmid-mediated colistin resistance (PMCR) genes based on any reduction of colistin MIC in the presence of EDTA. Eighty-five isolates of the order Enterobacteriales (12 mcr positive) were tested by CBDE ± EDTA. The sensitivity and specificity of the EDTA-CBDE method to detect PMCR compared to the molecular genotype results were 100% and 95.8%, respectively. Isolates positive by the EDTA-CBDE test should be further evaluated to confirm the presence of mcr genes.

Two-Site Evaluation of the Colistin Broth Disk Elution Test To Determine Colistin In Vitro Activity against Gram-Negative Bacilli.

Limited methods for colistin MIC determination are available to clinical microbiology laboratories. The purpose of this study was to evaluate the accuracy of the colistin broth disk elution (CBDE) test compared to that of broth microdilution (BMD) for identifying colistin MICs. CBDE was compared to colistin BMD using a collection of Gram-negative bacilli tested at two U.S. microbiology laboratories. The isolates tested included 121 retrospective clinical isolates, 45 prospective clinical isolates, and 6 mcr-1-positive Escherichia coli isolates. CBDE was performed with four 10-ml cation-adjusted Mueller-Hinton broth tubes per isolate, to which 0, 1, 2, and 4 colistin 10-µg disks were added, generating final concentrations in the tubes of 0 (growth control), 1, 2, and 4 µg/ml, respectively. MICs were evaluated visually and interpreted using Clinical and Laboratory Standards Institute breakpoints. Site 2 also compared CBDE to the reference broth macrodilution (BMAD) method (n = 110 isolates). Overall, CBDE yielded a categorical agreement (CA) and essential agreement (EA) of 98% and 99%, respectively, compared to the results of colistin BMD. Very major errors occurred for mcr-1-producing strains, with MICs fluctuating from 2 to 4 µg/ml on repeat testing. The results for all other isolates were in CA with those of BMD. CBDE versus BMAD had an EA of 100% and a CA of 100%. Compared to currently used techniques, CBDE is an easy and practical method to perform colistin MIC testing. Some mcr-1-producing isolates yielded MICs of 2 µg/ml by CBDE and 4 µg/ml by BMD. As such, the results for isolates with colistin MICs of 2 µg/ml by CBDE should be confirmed by the reference BMD method, and isolates with MICs of ≥2 µg/ml should be evaluated for the presence of mcr genes.

Non-classical transpeptidases yield insight into new antibacterials.

Bacterial survival requires an intact peptidoglycan layer, a three-dimensional exoskeleton that encapsulates the cytoplasmic membrane. Historically, the final steps of peptidoglycan synthesis are known to be carried out by D,D-transpeptidases, enzymes that are inhibited by the ß-lactams, which constitute >50% of all antibacterials in clinical use. Here, we show that the carbapenem subclass of ß-lactams are distinctly effective not only because they inhibit D,D-transpeptidases and are poor substrates for ß-lactamases, but primarily because they also inhibit non-classical transpeptidases, namely the L,D-transpeptidases, which generate the majority of linkages in the peptidoglycan of mycobacteria. We have characterized the molecular mechanisms responsible for inhibition of L,D-transpeptidases of Mycobacterium tuberculosis and a range of bacteria including ESKAPE pathogens, and used this information to design, synthesize and test simplified carbapenems with potent antibacterial activity.

Mutation in an Unannotated Protein Confers Carbapenem Resistance in Mycobacterium tuberculosis.

ß-Lactams are the most widely used antibacterials. Among ß-lactams, carbapenems are considered the last line of defense against recalcitrant infections. As recent developments have prompted consideration of carbapenems for treatment of drug-resistant tuberculosis, it is only a matter of time before Mycobacterium tuberculosis strains resistant to these drugs will emerge. In the present study, we investigated the genetic basis that confers such resistance. To our surprise, instead of mutations in the known ß-lactam targets, a single nucleotide polymorphism in the Rv2421c-Rv2422 intergenic region was common among M. tuberculosis mutants selected with meropenem or biapenem. We present data supporting the hypothesis that this locus harbors a previously unidentified gene that encodes a protein. This protein binds to ß-lactams, slowly hydrolyzes the chromogenic ß-lactam nitrocefin, and is inhibited by select penicillins and carbapenems and the ß-lactamase inhibitor clavulanate. The mutation results in a W62R substitution that reduces the protein's nitrocefin-hydrolyzing activity and binding affinities for carbapenems.

An evolved oxazolidinone with selective potency against Mycobacterium tuberculosis and gram positive bacteria.

Innovation of new antibacterials that are effective against strains that have developed resistance to existing drugs would strengthen our ability to treat and subsequently control spread of pathogenic bacteria. Increasing incidence of infections with drug resistant bacteria has become a common occurrence in recent times. We have developed an evolved oxazolidinone, T145, which inhibits growth of Enterococcus faecalis, Staphylococcus aureus and Mycobacterium tuberculosis (Mtb) with sub µg/ml potencies that are potentially therapeutically valuable. The oxazolidinone is bactericidal against Mtb but bacteriostatic against E. faecalis and S. aureus. In addition to therapeutically valuable potency and bactericidal activity against Mtb, T145 minimizes selection of spontaneous resistant mutants, a trait that prolongs longevity of a drug in clinical use.