Limited Adaptation of Staphylococcus aureus during Transition from Colonization to Invasive Infection.

Staphylococcus aureus carriage is a risk factor for invasive infections. Unique genetic elements favoring the transition from colonizing to invasive phenotype have not yet been identified, and phenotypic adaptation traits are understudied. We therefore assessed phenotypic and genotypic profiles of 11 S. aureus isolate pairs sampled from colonized patients simultaneously suffering from invasive S. aureus infections. Ten out of 11 isolate pairs displayed the same spa and multilocus sequence type, suggesting colonization as an origin for the invasive infection. Systematic analysis of colonizing and invasive isolate pairs showed similar adherence, hemolysis, reproductive fitness properties, antibiotic tolerance, and virulence in a Galleria mellonella infection model, as well as minimal genetic differences. Our results provide insights into the similar phenotypes associated with limited adaptation between colonizing and invasive isolates. Disruption of the physical barriers of mucosa or skin was identified in the majority of patients, further emphasizing colonization as a major risk factor for invasive disease. IMPORTANCE S. aureus is a major pathogen of humans, causing a wide range of diseases. The difficulty to develop a vaccine and antibiotic treatment failure warrant the exploration of novel treatment strategies. Asymptomatic colonization of the human nasal passages is a major risk factor for invasive disease, and decolonization procedures have been effective in preventing invasive infections. However, the transition of S. aureus from a benign colonizer of the nasal passages to a major pathogen is not well understood, and both host and bacterial properties have been discussed as being relevant for this behavioral change. We conducted a thorough investigation of patient-derived strain pairs reflecting colonizing and invasive isolates in a given patient. Although we identified limited genetic adaptation in certain strains, as well as slight differences in adherence capacity among colonizing and invasive isolates, our work suggests that barrier breaches are a key event in the disease continuum of S. aureus.

Serine-threonine phosphoregulation by PknB and Stp contributes to quiescence and antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus can cause infections that are often chronic and difficult to treat, even when the bacteria are not antibiotic resistant because most antibiotics act only on metabolically active cells. Subpopulations of persister cells are metabolically quiescent, a state associated with delayed growth, reduced protein synthesis, and increased tolerance to antibiotics. Serine-threonine kinases and phosphatases similar to those found in eukaryotes can fine-tune essential bacterial cellular processes, such as metabolism and stress signaling. We found that acid stress-mimicking conditions that S. aureus experiences in host tissues delayed growth, globally altered the serine and threonine phosphoproteome, and increased threonine phosphorylation of the activation loop of the serine-threonine protein kinase B (PknB). The deletion of stp, which encodes the only annotated functional serine-threonine phosphatase in S. aureus, increased the growth delay and phenotypic heterogeneity under different stress challenges, including growth in acidic conditions, the intracellular milieu of human cells, and abscesses in mice. This growth delay was associated with reduced protein translation and intracellular ATP concentrations and increased antibiotic tolerance. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified targets of serine-threonine phosphorylation that may regulate bacterial growth and metabolism. Together, our findings highlight the importance of phosphoregulation in mediating bacterial quiescence and antibiotic tolerance and suggest that targeting PknB or Stp might offer a future therapeutic strategy to prevent persister formation during S. aureus infections.

Antibiotic prophylaxis before dental procedures to prevent infective endocarditis: a systematic review.

PURPOSE: Infective endocarditis (IE) is a severe bacterial infection. As a measure of prevention, the administration of antibiotic prophylaxis (AP) prior to dental procedures was recommended in the past. However, between 2007 and 2009, guidelines for IE prophylaxis changed all around the word, limiting or supporting the complete cessation of AP. It remains unclear whether AP is effective or not against IE. METHODS: We conducted a systematic review whether the administration of AP in adults before any dental procedure, compared to the non-administration of such drugs, has an effect on the risk of developing IE. We searched for studies in the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE via OVID, and EMBASE. Two different authors filtered articles independently and data extraction was performed based on a pre-defined protocol. RESULTS: The only cohort study meeting our criteria included patients at high-risk of IE. Analysis of the extracted data showed a non-significant decrease in the risk of IE when high-risk patients take AP prior to invasive dental procedures (RR 0.39, p-value 0.11). We did not find other studies including patients at low or moderate risk of IE. Qualitative evaluation of the excluded articles reveals diversity of results and suggests that most of the state-of-the-art articles are underpowered. CONCLUSIONS: Evidence to support or discourage the use of AP prior to dental procedures as a prevention for IE is very low. New high-quality studies are needed, even though such studies would require big settings and might not be immediately feasible.

Hyperinflammatory environment drives dysfunctional myeloid cell effector response to bacterial challenge in COVID-19.

COVID-19 displays diverse disease severities and symptoms including acute systemic inflammation and hypercytokinemia, with subsequent dysregulation of immune cells. Bacterial superinfections in COVID-19 can further complicate the disease course and are associated with increased mortality. However, there is limited understanding of how SARS-CoV-2 pathogenesis and hypercytokinemia impede the innate immune function against bacterial superinfections. We assessed the influence of COVID-19 plasma hypercytokinemia on the functional responses of myeloid immune cells upon bacterial challenges from acute-phase COVID-19 patients and their corresponding recovery-phase. We show that a severe hypercytokinemia status in COVID-19 patients correlates with the development of bacterial superinfections. Neutrophils and monocytes derived from COVID-19 patients in their acute-phase showed an impaired intracellular microbicidal capacity upon bacterial challenges. The impaired microbicidal capacity was reflected by abrogated MPO and reduced NETs production in neutrophils along with reduced ROS production in both neutrophils and monocytes. Moreover, we observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes, in line with suppressed autocrine and paracrine cytokine signaling. This phenotype was characterized by a high expression of CD66b, CXCR4 and low expression of CXCR1, CXCR2 and CD15 in neutrophils and low expression of HLA-DR, CD86 and high expression of CD163 and CD11b in monocytes. Furthermore, the impaired antibacterial effector function was mediated by synergistic effect of the cytokines TNF-α, IFN-γ and IL-4. COVID-19 patients receiving dexamethasone showed a significant reduction of overall inflammatory markers in the plasma as well as exhibited an enhanced immune response towards bacterial challenge ex vivo. Finally, broad anti-inflammatory treatment was associated with a reduction in CRP, IL-6 levels as well as length of ICU stay and ventilation-days in critically ill COVID-19 patients. Our data provides insights into the transient functional dysregulation of myeloid immune cells against subsequent bacterial infections in COVID-19 patients and describe a beneficial role for the use of dexamethasone in these patients.

Editor's Choice - Validation of the Management of Aortic Graft Infection Collaboration (MAGIC) Criteria for the Diagnosis of Vascular Graft/Endograft Infection: Results from the Prospective Vascular Graft Cohort Study.

OBJECTIVE: The timely management of vascular graft/endograft infection (VGEI) is crucial to a favourable outcome, yet can be challenging as there is no validated gold standard diagnostic test. Recently, a new case definition has been proposed by the Management of Aortic Graft Infection Collaboration (MAGIC) to close the diagnostic gap. The aim of this study was to validate the MAGIC criteria as a suggested diagnostic standard for the diagnosis of suspected VGEI in the prospective Vascular Graft Cohort study (VASGRA). METHODS: VASGRA is an open, prospective, observational cohort study. Prospective participants in VASGRA between 2013 and 2019 were included (257 patients; 137 with VGEI). The accuracy of the MAGIC criteria for a diagnosis of VGEI was evaluated retrospectively by calculating the sensitivity and specificity vs. the consensually adjudicated VASGRA infection status. RESULTS: The VASGRA cohort categorised 137 (53.3%) patients as "diseased" and 120 patients as "not diseased"; using the MAGIC criteria, 183/257 (71.2%) patients were considered to be "diseased". Thus, for the MAGIC criteria, a sensitivity of 99% (95% confidence interval [CI] 96-100) and a specificity of 61% (95% CI 52-70) were calculated. Considering suspected VGEI according to the MAGIC criteria as "not diseased" achieved congruent assessments of the VASGRA team and the MAGIC criteria, with a sensitivity of 93% and a specificity of 93%. The accuracy of the MAGIC criteria for the different graft locations were also compared. If the suspected VGEIs were assigned to the "not diseased" group, VGEIs of the thoracic aorta seemed to have a poorer sensitivity (86%; 95% CI 73-95) than the other graft locations. CONCLUSION: The current MAGIC criteria offer good sensitivity and specificity in the context of true infections but a reduced specificity for a possible VGEI.

Quantifying Variation in Bacterial Reproductive Fitness: a High-Throughput Method.

To evaluate changes in reproductive fitness of bacteria, e.g., after acquisition of antimicrobial resistance, a low-cost high-throughput method to analyze bacterial growth on agar is desirable for broad usability. In our bacterial quantitative fitness analysis (BaQFA), arrayed cultures are spotted on agar and photographed sequentially while growing. These time-lapse images are analyzed using a purpose-built open-source software to derive normalized image intensity (NI) values for each culture spot. Subsequently, a Gompertz growth model is fitted to NI values, and fitness is calculated from model parameters. To represent a range of clinically important pathogenic bacteria, we used different strains of Enterococcus faecium, Escherichia coli, and Staphylococcus aureus, with and without antimicrobial resistance. Relative competitive fitness (RCF) was defined as the mean fitness ratio of two strains growing competitively on one plate.BaQFA permitted the accurate construction of growth curves from bacteria grown on semisolid agar plates and fitting of Gompertz models. Normalized image intensity values showed a strong association with the total CFU/ml count per spotted culture (P < 0.001) for all strains of the three species. BaQFA showed relevant reproductive fitness differences between individual strains, suggesting substantially higher fitness of methicillin-resistant S. aureus JE2 than Cowan (RCF, 1.58; P < 0.001). Similarly, the vancomycin-resistant E. faecium ST172b showed higher competitive fitness than susceptible E. faecium ST172 (RCF, 1.59; P < 0.001). Our BaQFA method allows detection of fitness differences between bacterial strains and may help to estimate epidemiological antimicrobial persistence or contribute to the prediction of clinical outcomes in severe infections.IMPORTANCE Reproductive fitness of bacteria is a major factor in the evolution and persistence of antimicrobial resistance and may play an important role in severe infections. With a computational approach to quantify fitness in bacteria growing competitively on agar plates, our high-throughput method has been designed to obtain additional phenotypic data for antimicrobial resistance analysis at a low cost. Furthermore, our bacterial quantitative fitness analysis (BaQFA) enables the investigation of a link between bacterial fitness and clinical outcomes in severe invasive bacterial infections. This may allow future use of our method for patient management and risk stratification of clinical outcomes. Our proposed method uses open-source software and a hardware setup that can utilize consumer electronics. This will enable a wider community of researchers, including those from low-resource countries, where the burden of antimicrobial resistance is highest, to obtain valuable information about emerging bacterial strains.

Molecular reprogramming and phenotype switching in Staphylococcus aureus lead to high antibiotic persistence and affect therapy success.

Staphylococcus aureus causes invasive infections and easily acquires antibiotic resistance. Even antibiotic-susceptible S. aureus can survive antibiotic therapy and persist, requiring prolonged treatment and surgical interventions. These so-called persisters display an arrested-growth phenotype, tolerate high antibiotic concentrations, and are associated with chronic and recurrent infections. To characterize these persisters, we assessed S. aureus recovered directly from a patient suffering from a persistent infection. We show that host-mediated stress, including acidic pH, abscess environment, and antibiotic exposure promoted persister formation in vitro and in vivo. Multiomics analysis identified molecular changes in S. aureus in response to acid stress leading to an overall virulent population. However, further analysis of a persister-enriched population revealed major molecular reprogramming in persisters, including down-regulation of virulence and cell division and up-regulation of ribosomal proteins, nucleotide-, and amino acid-metabolic pathways, suggesting their requirement to fuel and maintain the persister phenotype and highlighting that persisters are not completely metabolically inactive. Additionally, decreased aconitase activity and ATP levels and accumulation of insoluble proteins involved in transcription, translation, and energy production correlated with persistence in S. aureus, underpinning the molecular mechanisms that drive the persister phenotype. Upon regrowth, these persisters regained their virulence potential and metabolically active phenotype, including reduction of insoluble proteins, exhibiting a reversible state, crucial for recurrent infections. We further show that a targeted antipersister combination therapy using retinoid derivatives and antibiotics significantly reduced lag-phase heterogeneity and persisters in a murine infection model. Our results provide molecular insights into persisters and help explain why persistent S. aureus infections are so difficult to treat.

CASPR, an analysis pipeline for single and paired guide RNA CRISPR screens, reveals optimal target selection for long non-coding RNAs.

MOTIVATION: CRISPR-Cas9 loss-of-function (LOF) pooled screening promises to identify which long non-coding RNAs (lncRNAs), amongst the many thousands to have been annotated so far, are capable of mediating cellular functions. The two principal LOF perturbations, CRISPR-inhibition and CRISPR-deletion, employ one and two guide RNAs, respectively. However, no software solution has the versatility to identify hits across both modalities, and the optimal design parameters for such screens remain poorly understood. RESULTS: Here, we present CRISPR Analysis for Single and Paired RNA-guides (CASPR), a user-friendly, end-to-end screen analysis tool. CASPR is compatible with both CRISPRi and CRISPR-del screens, and balances sensitivity and specificity by generating consensus predictions from multiple algorithms. Benchmarking on ground-truth sets of cancer-associated lncRNAs demonstrates CASPR's improved sensitivity with respect to existing methods. Applying CASPR to published screens, we identify two parameters that predict lncRNA hits: expression and annotation quality of the transcription start site. Thus, CASPR is a versatile and complete solution for lncRNA CRISPR screen analysis, and reveals principles for including lncRNAs in screening libraries. AVAILABILITY AND IMPLEMENTATION: https://judithbergada.github.io/CASPR/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.