Distribution of SARS-CoV-2 PCR Cycle Threshold Values Provide Practical Insight Into Overall and Target-Specific Sensitivity Among Symptomatic Patients.

OBJECTIVES: We examined the distribution of reverse transcription polymerase chain reaction (RT-PCR) cycle threshold (CT) values obtained from symptomatic patients being evaluated for coronavirus disease 2019 (COVID-19) to determine the proportion of specimens containing a viral load near the assay limit of detection (LoD) to gain practical insight to the risk of false-negative results. We also examined the relationship between CT value and patient age to determine any age-dependent difference in viral load or test sensitivity. METHODS: We collected CT values obtained from the cobas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assay corresponding to 1,213 combined nasopharyngeal-oropharyngeal specimens obtained from symptomatic individuals that were reported as positive or presumptive positive for SARS-CoV-2. CT values were stratified by SARS-CoV target and patient age group. RESULTS: In total, 93.3% to 98.4% of specimens demonstrated CT values greater than 3× the assay LoD, at which point false-negative results would not be expected. The mean of CT values between age groups was statistically equivalent with the exception of patients in age group 80 to 89 years, which demonstrated slightly lower CTs. CONCLUSIONS: Based on the distribution of observed CT values, including the small proportion of specimens with values near the assay LoD, there is a low risk of false-negative RT-PCR results in combined nasopharyngeal-oropharyngeal specimens obtained from symptomatic individuals.

Structure and Dimerization of IreB, a Negative Regulator of Cephalosporin Resistance in Enterococcus faecalis.

Enterococcus faecalis, a leading cause of hospital-acquired infections, exhibits intrinsic resistance to most cephalosporins, which are antibiotics in the beta-lactam family that target cell-wall biosynthesis. A comprehensive understanding of the underlying genetic and biochemical mechanisms of cephalosporin resistance in E. faecalis is lacking. We previously determined that a transmembrane serine/threonine kinase (IreK) and its cognate phosphatase (IreP) reciprocally regulate cephalosporin resistance in E. faecalis, dependent on the kinase activity of IreK. Other than IreK itself, thus far the only known substrate for reversible phosphorylation by IreK and IreP is IreB, a small protein of unknown function that is well conserved in low-GC Gram-positive bacteria. We previously showed that IreB acts as a negative regulator of cephalosporin resistance in E. faecalis. However, the biochemical mechanism by which IreB modulates cephalosporin resistance remains unknown. As a next step toward an understanding of the mechanism by which IreB regulates resistance, we initiated a structure-function study on IreB. The NMR solution structure of IreB was determined, revealing that IreB adopts a unique fold and forms a dimer in vitro. Dimerization of IreB was confirmed in vivo. Substitutions at the dimer interface impaired IreB function and stability in vivo, indicating that dimerization is functionally important for the biological activity of IreB. Hence, these studies provide new insights into the structure and function of a widely conserved protein of unknown function that is an important regulator of antimicrobial resistance in E. faecalis.

Requirement of the CroRS Two-Component System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium.

Enterococci are serious opportunistic pathogens that are resistant to many cell wall-targeting antibiotics. The CroRS two-component signaling system responds to antibiotic-mediated cell wall stress and is critical for resistance to cell wall-targeting antibiotics in Enterococcus faecalis Here, we identify and characterize an orthologous two-component system found in Enterococcus faecium that is functionally equivalent to the CroRS system of E. faecalis Deletion of croRS in E. faecium resulted in marked susceptibility to cell wall-targeting agents including cephalosporins and bacitracin, as well as moderate susceptibility to ampicillin and vancomycin. As in E. faecalis, exposure to bacitracin and vancomycin stimulates signaling through the CroRS system in E. faecium Moreover, the CroRS system is critical in E. faecium for enhanced beta-lactam resistance mediated by overexpression of Pbp5. Expression of a Pbp5 variant that confers enhanced beta-lactam resistance cannot overcome the requirement for CroRS function. Thus, the CroRS system is a conserved signaling system that responds to cell wall stress to promote intrinsic resistance to important cell wall-targeting antibiotics in clinically relevant enterococci.

Thymidylate Limitation Potentiates Cephalosporin Activity toward Enterococci via an Exopolysaccharide-Based Mechanism.

Multidrug resistant enterococci are major causes of nosocomial infections. Prior therapy with cephalosporins increases the risk of developing an enterococcal infection due to the intrinsic resistance of enterococci to these antibiotics. While progress has been made toward understanding the genetic and biochemical mechanisms of cephalosporin resistance, available data indicate that as-yet-unidentified resistance factors must exist. Here, we describe results of a screen to identify small molecules capable of sensitizing enterococci to broad-spectrum cephalosporins. We found that both Enterococcus faecalis and Enterococcus faecium were sensitized to broad and expanded-spectrum cephalosporins when thymidylate production was impaired, whether by direct inhibition of thymidylate synthase, or by limiting production of cofactors required for its activity. Cephalosporin potentiation is the result of altered exopolysaccharide production due to reduced dTDP-glucose synthesis. Hence, exopolysaccharide production is a previously undescribed contributor to the intrinsic cephalosporin resistance of enterococci and serves as a new target for antienterococcal therapeutics.

SpyA is a membrane-bound ADP-ribosyltransferase of Streptococcus pyogenes which modifies a streptococcal peptide, SpyB.

All sequenced genomes of Streptococcus pyogenes (Group A Streptococcus, GAS) encode a protein, SpyA, with homology to C3-like ADP-ribosyltransferase toxins. SpyA is a novel virulence factor which plays a role in pathogenesis in a mouse model of soft-tissue infection. In this study we demonstrate that SpyA is a surface-exposed membrane protein which is anchored to the streptococcal membrane by an N-terminal transmembrane sequence. We identified a small gene upstream of spyA, designated spyB, which encodes a peptide of 35 amino acids, and is co-transcribed with spyA. Expression of spyBA is strongly influenced by translational coupling: mutational inactivation of spyB translation completely abolishes translation of spyA. spyB expression increases with increasing cell density and reaches its maximum at late exponential growth phase. The SpyB N-terminus is predicted to fold into an amphipathic α-helix, a structural motif that targets a protein to the cytoplasmic membrane. Consistent with the prediction, we found that a SpyB fusion with peptide affinity tags is located in the streptococcal membrane. An ADP-ribosylation assay with recombinant SpyA demonstrated that SpyA modifies SpyB. Thus, our study suggests that ADP-ribosylation of SpyB may be an important function of SpyA.

Reciprocal regulation of cephalosporin resistance in Enterococcus faecalis.

UNLABELLED: Antibiotic-resistant enterococci are major causes of hospital-acquired infections and therefore represent a serious public health problem. One well-known risk factor for the acquisition of hospital-acquired enterococcal infections is prior therapy with broad-spectrum cephalosporin antibiotics. Enterococci can proliferate in patients undergoing cephalosporin therapy due to intrinsic cephalosporin resistance, a characteristic of the genus Enterococcus. However, the molecular basis for cephalosporin resistance in E. faecalis has yet to be adequately elucidated. Previously we determined that a putative Ser/Thr kinase, IreK (formerly PrkC), is required for intrinsic cephalosporin resistance in E. faecalis. Here we show that kinase activity is required for cephalosporin resistance and, further, that resistance in E. faecalis is reciprocally regulated by IreK and IreP, a PP2C-type protein phosphatase encoded immediately upstream of IreK. Mutants of two divergent lineages of E. faecalis lacking IreP exhibit remarkable hyperresistance to cephalosporins but not to antibiotics targeting other cellular processes. Further genetic analyses indicate that hyperresistance of the IreP mutant is mediated by the IreK kinase. Additionally, competition experiments reveal that hyperresistant ΔireP mutants exhibit a substantial fitness defect in the absence of antibiotics, providing an evolutionary rationale for the use of a complex signaling system to control intrinsic cephalosporin resistance. These results support a model in which IreK and IreP act antagonistically via protein phosphorylation and dephosphorylation as part of a signal transduction circuit to regulate cellular adaptation to cephalosporin-induced stress. IMPORTANCE: As a major cause of hospital-acquired infections, antibiotic-resistant enterococci represent a serious public health problem. Enterococci are well-known to exhibit intrinsic resistance to broad-spectrum cephalosporin antibiotics, a trait that enables them to proliferate in patients undergoing cephalosporin therapy, thereby predisposing these patients to acquisition of an enterococcal infection. Thus, inhibition of enterococcal cephalosporin resistance could represent an effective new strategy to prevent the emergence of hospital-acquired enterococcal infections. At this time, however, the molecular basis for cephalosporin resistance in E. faecalis is poorly understood. Our results begin to unravel the details of a new phosphorylation-dependent signal transduction system that controls cephalosporin resistance in enterococci. Deeper understanding of the mechanism underlying cephalosporin resistance in E. faecalis may enable the development of new therapeutics designed to reduce the incidence of hospital-acquired enterococcal infections.

SpyA, a C3-like ADP-ribosyltransferase, contributes to virulence in a mouse subcutaneous model of Streptococcus pyogenes infection.

Streptococcus pyogenes is an important human pathogen with an expansive repertoire of verified and putative virulence factors. Here we demonstrate that a mutant deficient in the production of the streptococcal ADP-ribosyltransferase SpyA generates lesions of reduced size in a subcutaneous mouse infection model. At early stages of infection, when the difference in lesion size is first established, inflamed tissue isolated from lesions of mice infected with spyA mutant bacteria has higher levels of mRNA encoding the chemokines CXCL1 and CCL2 than does tissue isolated from mice infected with wild-type bacteria. In addition, at these early times, the mRNA levels for the gene encoding the intermediate filament vimentin are higher in the mutant-infected tissue. As wound resolution progresses, mRNA levels of the gene encoding matrix metallopeptidase 2 are lower in mutant-infected tissue. Furthermore, we demonstrate that the spyA mutant is internalized more efficiently than wild-type bacteria by HeLa cells. We conclude that SpyA contributes to streptococcal pathogenesis in the mouse subcutaneous infection model. Our observations suggest that the presence of SpyA delays wound healing in this model.

Molecular characterization of conditionally immortalized cell lines derived from mouse early embryonic inner ear.

Inner ear sensory hair cells (HCs), supporting cells (SCs), and sensory neurons (SNs) are hypothesized to develop from common progenitors in the early embryonic otocyst. Because little is known about the molecular signals that control this lineage specification, we derived a model system of early otic development: conditionally immortalized otocyst (IMO) cell lines from the embryonic day 9.5 Immortomouse. This age is the earliest stage at which the otocyst can easily be separated from surrounding mesenchymal, nervous system, and epithelial cells. At 9.5 days post coitum, there are still pluripotent cells in the otocyst, allowing for the eventual identification of both SN and HC precursors--and possibly an elusive inner ear stem cell. Cell lines derived from primitive precursor cells can also be used as blank canvases for transfections of genes that can affect lineage decisions as the cells differentiate. It is important, therefore, to characterize the "baseline state" of these cell lines in as much detail as possible. We characterized seven representative "precursor-like" IMO cell populations and the uncloned IMO cells, before cell sorting, at the molecular level by polymerase chain reaction (PCR) and immunocytochemistry (IHC), and one line (IMO-2B1) in detail by real-time quantitative PCR and IHC. Many of the phenotypic markers characteristic of differentiated HCs or SCs were detected in IMO-2B1 proliferating cells, as well as during differentiation for up to 30 days in culture. These IMO cell lines represent a unique model system for studying early stages of inner ear development and determining the consequences of affecting key molecular events in their differentiation.

Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence.

Genome sequences are available for many bacterial strains, but there has been little progress in using these data to understand the molecular basis of pathogen emergence and differences in strain virulence. Serotype M3 strains of group A Streptococcus (GAS) are a common cause of severe invasive infections with unusually high rates of morbidity and mortality. To gain insight into the molecular basis of this high-virulence phenotype, we sequenced the genome of strain MGAS315, an organism isolated from a patient with streptococcal toxic shock syndrome. The genome is composed of 1,900,521 bp, and it shares approximately 1.7 Mb of related genetic material with genomes of serotype M1 and M18 strains. Phage-like elements account for the great majority of variation in gene content relative to the sequenced M1 and M18 strains. Recombination produces chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. Strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A(2) (designated Sla). Infected humans had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are made in vivo. SpeK and SSA were pyrogenic and toxic for rabbits. Serotype M3 strains with the phage-encoded speK and sla genes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phage-mediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS.

In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence.

Recently, we observed that Staphylococcus aureus strains newly isolated from patients had twofold-higher aconitase activity than a strain passaged extensively in vitro, leading us to hypothesize that aconitase specific activity decreases over time during in vitro passage. To test this hypothesis, a strain recovered from a patient with toxic shock syndrome was serially passaged for 6 weeks, and the aconitase activity was measured. Aconitase specific activity decreased 38% (P < 0.001) by the sixth week in culture. During serial passage, S. aureus existed as a heterogeneous population with two colony types that had pronounced (wild type) or negligible zones of beta-hemolytic activity. The cell density-sensing accessory gene regulatory (agr) system regulates beta-hemolytic activity. Surprisingly, the percentage of colonies with a wild-type beta-hemolytic phenotype correlated strongly with aconitase specific activity (rho = 0.96), suggesting a common cause of the decreased aconitase specific activity and the variation in percentage of beta-hemolytic colonies. The loss of the beta-hemolytic phenotype also coincided with the occurrence of mutations in the agrC coding region or the intergenic region between agrC and agrA in the derivative strains. Our results demonstrate that in vitro growth is sufficient to result in mutations within the agr operon. Additionally, our results demonstrate that S. aureus undergoes significant phenotypic and genotypic changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.