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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., Author summary COVID-19 caused by SARS-CoV-2 induces immune-paralysis characterized by misdirected host responses and altered levels of inflammatory mediators. Additionally, bacterial superinfections can further exacerbate the disease. Here, we report an in-depth functional characterization of the effector response and phenotypic properties of neutrophils and monocytes derived from critically ill COVID-19 patients towards bacterial superinfection. We show that elevated levels of specific cytokine clusters, positively correlate with the development of bacterial superinfections in these patients. Neutrophils and monocytes of critically ill COVID-19 patients showed impaired bactericidal capacity, which was mediated by elevated inflammatory mediators in the plasma. The observed impaired bactericidal capacity in critically ill COVID-19 patients was due to reduced classical effector functions of neutrophils and monocytes. Interestingly, lower levels of overall inflammatory mediators as well as reduction in length of ICU stay and ventilation-days in critically ill COVID-19 patients treated with dexamethasone was observed. These data suggest that the exhaustion and paralysis of the cellular innate immunity against bacterial challenge in critically ill COVID-19 patients is driven by a misdirected host-response, characterized by a hyperinflammatory environment including dysregulated cytokine levels. These results further emphasize the utility and importance of dampening these inflammatory mediators via broad anti-inflammatory therapy in COVID-19 patients, which improves the antibacterial effector functions of neutrophils and monocytes.

SummaryBackgroundDifficult-to-treat infections caused by antibiotic susceptible strains have been linked with the occurrence of persisters. Persisters are a subpopulation of dormant bacteria that tolerate antibiotic exposure despite lacking genetic resistance. They can be identified phenotypically upon plating on nutrient agar because of their altered growth dynamics, resulting in colony size heterogeneity. The occurrence of within-patient bacterial phenotypic heterogeneity in various infections and clinical determinants of persister formation remain unknown.MethodsWe plated bacteria derived from 132 patient-samples of difficult-to-treat infections directly on nutrient-rich agar and monitored colony growth by time-lapse imaging. Of these, we retained 36 Staphylococcus aureus mono-cultures for further analysis. We investigated clinical factors potentially associated with increased colony growth-delay with regression analyses. Additionally, we corroborated the clinical findings using in vitro grown static biofilms, exposed to distinct antibiotics.ResultsThe extent of phenotypic heterogeneity of patient-derived S. aureus varied substantially between patients. Increased heterogeneity coincided with increased median growth-delay. Multivariable regression showed that rifampicin treatment was significantly associated with increased median growth-delay. S. aureus grown in biofilms and exposed to high concentrations of rifampicin or a combination of rifampicin with either clindamycin or levofloxacin exhibited prolonged growth-delay, correlating with a strain-dependent increase in antibiotic tolerance.ConclusionsUpon direct cultivation on nutrient-rich agar, S. aureus from difficult-to-treat infections commonly exhibited colony size heterogeneity. This was due to heterogeneous delays in growth resumption, with delays larger than two days in the most extreme cases. Since bacteria in a dormant state are tolerant to antibiotics, the observation of large growth-delays might have direct clinical implications. Future studies are needed to assess the potential of bacterial phenotypic heterogeneity quantification for staphylococcal infections prognosis.

Antibiotic tolerant Staphylococcus aureus pose a great challenge to clinicians as well as to microbiological laboratories and are one reason for treatment failure. Antibiotic tolerant strains survive transient antibiotic exposure despite being fully susceptible in vitro. Thus, fast and reliable methods to detect tolerance in the routine microbiology laboratory are urgently required.We therefore evaluated the feasibility of the replica plating tolerance isolation system (REPTIS) to detect antibiotic tolerance in S. aureus isolates derived directly from patients suffering from different types of infections and investigated possible connections to clinical presentations and patient characteristics.One hundred twenty-five S. aureus isolates were included. Replica plating of the original resistance testing plate was used to assess regrowth in the zones of inhibition, indicating antibiotic tolerance. Bacterial regrowth was assessed after 24 and 48 hours of incubation and an overall regrowth score (ORS) was assigned. Regrowth scores were compared to the clinical presentation.Bacterial regrowth was high for most antibiotics targeting protein synthesis and relatively low for antibiotics targeting other cellular functions such as DNA-replication, transcription and cell wall synthesis, with the exception of rifampicin. Isolates with a blaZ penicillinase had lower regrowth in penicillin and ampicillin. Low ORSs were more prevalent among isolates recovered from patients with immunosuppression or methicillin-resistant S. aureus (MRSA) isolates.In conclusion, REPTIS is useful to detect antibiotic tolerance in clinical microbiological routine diagnostics. Rapid detection of antibiotic tolerance offers a new diagnostic readout that might allow more tailored treatments in the future.

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.

Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells., Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections.

The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time. Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus. Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections., Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureus.

Treatment failure and recurrent infections can occur even when patients are treated with antibiotics to which bacteria are susceptible. These treatment failures could be due to a sub-population of bacteria persisting through the treatment. In this study, we tested the hypothesis that such bacterial persisters manifest in clinical samples as small colony variants (SCVs). We worked with the bacterial pathogen Staphylococcus aureus, where SCVs are frequently observed in clinics. The small size of SCV-colonies is often considered to be the result of mutations that reduce the bacterial growth rate. Alternatively, it is possible that the small colony size could result from a long lag time of bacteria in plated samples. We used automated plate imaging and single-cell microscopy to precisely quantify growth kinetics of bacteria sampled from patient and mice abscesses, and from low-pH in vitro cultures mimicking the host. Under these conditions, the small colony size was the consequence of a long lag time and was associated with tolerance towards antibiotics. We found that bacteria with a long lag time emerged de novo during the growth phase in the murine abscess as well as in the low-pH in vitro cultures, and that their proportion increased during stationary phase. Antibiotic exposure further increased the proportion of bacteria with a long lag time. Thus, the persisters found in low-pH host compartments could be a cause of treatment failure, and antibiotic treatment potentially aggravates the problem. These insights call for antibiotic treatment strategies that explicitly address the problem of persister formation.Significance StatementBacterial pathogens often contain subpopulations that are multidrug tolerant and metabolically inactive. These subpopulations are known as persisters and are hypothesised to be linked to treatment failure and recurrent infections even in bacteria that are not genetically resistant to antibiotics. Identifying persisters in clinical samples remains a challenge due to their metabolicaly active state. Here, we used time-lapse plate imaging and single cell microscopy to link the presence of persisters to the nonstable small colony variant (SCV) phenotype that is observed in some Staphylococcus aureus infections. These findings may help to identify treatment regimens that prevent or address the emergence of persistence and thus to reduce relapses.

Background The cell adhesion and tumor suppressor protein E-cadherin is an important factor in the establishment and maintenance of epithelial integrity. E-cadherin is a single transmembrane protein, which consists of an intracellular domain (IC), a transmembrane domain (TD), and five extracellular domains (EC). EC domains form homophilic interactions in cis and trans that require calcium binding to the linker region between the EC domains. In our previous studies, we identified the serine protease high temperature requirement A (HtrA) from the human pathogen and class-I carcinogen Helicobacter pylori (H. pylori) as a bacterial E-cadherin-cleaving protease that targets the linker region of the EC domains, thereby disrupting gastric epithelial integrity. However, it remains unclear how calcium binding to the E-cadherin linker regions affects HtrA-mediated cleavage. Results Investigating the influence of calcium on the HtrA-mediated cleavage of recombinant E-cadherin (rCdh1) in vitro, we tested different concentrations of calcium ions and the calcium chelator ethylenediaminetetraacetic acid (EDTA). Calcium efficiently reduced HtrA-mediated E-cadherin fragmentation. Conversely, the addition of EDTA strongly increased cleavage, resulting in a ladder of defined E-cadherin fragments. However, calcium ions did not affect HtrA oligomerization and protease activity as monitored by degradation of the universal protease substrate casein. Finally, addition of ethyleneglycol-bis-tetraacetic acid (EGTA) slightly enhanced E-cadherin cleavage during H. pylori infection of gastric epithelial cells. Conclusions Our results suggest that calcium blocks HtrA-mediated cleavage by interfering with the accessibility of calcium-binding regions between the individual EC domains, which have been identified as cleavage sites of HtrA., Gut Pathogens, 8, ISSN:1757-4749

Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications.