key: cord-1052772-zjipq51g authors: Carvalho, F. M.; Lemos, L. N.; Ciapina, L. P.; Moreira, R. G.; Gerber, A.; Guimaraes, A. P.; Fereguetti, T.; Zambelli, V. A. d. A.; Avila, R.; de Almeida, T. B.; Lima, J. S.; Barroso, S. P. C.; Teixeira, M. M.; Souza, R. P.; Cardoso, C. C.; Aguiar, R. S.; Vasconcelos, A. T. R. title: Prevalence of bacterial pathogens and potential role in COVID-19 severity in patients admitted to intensive care units in Brazil date: 2020-12-24 journal: nan DOI: 10.1101/2020.12.22.20248501 sha: 59f38d99c266512ecb94f4794891b867caa94e66 doc_id: 1052772 cord_uid: zjipq51g Secondary bacterial and fungal infections are associated with respiratory viral infections and invasive mechanical ventilation. In Coronavirus disease 2019 (COVID-19), lung injury by SARS-CoV-2 and impaired immune response can provide a favorable environment for microorganism growth and colonization in hospitalized individuals. Recent studies suggest that secondary bacterial pneumonia is a risk factor associated with COVID-19. In Brazil, knowledge about microbiota present in COVID-19 patients is incipient. This work describes the microbiota of 21 COVID-19 patients admitted to intensive care units from two Brazilian centers. We identified respiratory, nosocomial and bacterial pathogens as prevalent microorganisms. Other bacterial opportunistic and commensal species are also represented. Virulence factors of these pathogenic species, metabolic pathways used to evade and modulate immunological processes and the interconnection between bacterial presence and virulence in COVID-19 progression are discussed. acidovorans is usually not pathogenic, but catheter-related bacteremia and cases of pneumonia with lung cavity formation has been observed (23, 24). As expected, an elevated occurrence of the bacterial respiratory pathogen Streptococcus pneumoniae was found independent of the comorbidity, while in patients without comorbidity the occurrence of respiratory and nosocomial pathogens was drastically reduced, with exception of Candida albicans and Candida tropicalis ( Figure 2 and Table 2 ). In contrast, Acinetobacter baumannii was significantly increased in patients with higher SARS-CoV2 infection period (RJ2 and RJ3 samples). No significant difference or direct association was observed between pathogen abundance and comorbidity type. However, the microbiota here identified reinforce the severity of infection under weakened health conditions. Additionally, a large diversity of the oropharynx commensal species was identified, such as Rothia mucilaginosa, Parvimonas micra, Actinomyces odontolyticus, and Streptococcus anginosus (Table 2) . R. mucilaginosa has been associated to pneumonia in patients with chronic obstructive pulmonary disease (COPD) (25). Furthermore, an association of co-infection by A. odontolyticus and P. micra or Streptococcus spp. to lung abscess and acute respiratory failure was reported (26, 27) . Although respiratory disease caused by these commensals is a rare event, their presence in individuals with compromised immune responses should not be neglected. The bacterial pathogens identified in this study, particularly Acinetobacter baumannii, Streptococcus pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, may promote virulence and evade the host immune response by biofilm production, induction of hemolysins, pneumolysin, phospholipases, iron acquisition factors, cytokines, adhesins, and complement system resistance, besides other virulence factors (28 -30). In pulmonary and catheter-associated infections, biofilm formation can be favored by microbial interaction in endotracheal tubes and mechanical-ventilation apparatus, resulting in a diverse microbial complex and enhanced antimicrobial resistance (31, 32) . Studies demonstrated that A. baumannii, Klebsiella pneumoniae, and Enterococcus faecium are also frequently recovered from endotracheal tubes (33-35). In addition, in clinical isolates with persistent infections, P. aeruginosa, S. aureus and C. albicans are the predominant biofilm-producers and species with more biofilm formation capacity are frequently observed to be multidrug-resistant (36-38). For some of these microorganisms, the increase of biofilm, production of bacterial virulence factors and antibiotic resistance are related to enrichment of macrophage secretory products in culture and oxygen-limiting conditions (39, 40) . In S. aureus, for example, biofilm is related to accumulation of activated macrophages exhibiting anti-inflammatory and pro-fibrotic properties (41) . Moreover, it was observed that, when in biofilm, bacterial species are more resistant to the immune response, evading neutrophil mediated phagocytosis (42) . Hemolysin, an important virulence factor of S. aureus, E. coli, and Enterococcus faecalis (43, 44) , is a protein that causes erythrocyte lyse and disrupts the cell membrane, contributing to lung injury and pneumonia. In Streptococcus pneumoniae, when pulmonary surfactant is deficient, there is an expression of hemolysin, which is correlated with lung epithelial cell injury and induction of interleukin (IL)-8 (45) . Similar to hemolysin, pneumolysin is a pore-forming toxin, and most of its isoforms exhibit hemolytic activity. This protein is crucial for virulence and chronic bacteraemia of Streptococcus pneumoniae, and together with polysaccharide capsule proteins and adhesins reduce the S. pneumoniae entrapment in the pulmonary mucus, permitting adherence and inflammatory responses and activating complement and apoptosis (46) . For Candida species identified in this work, phospholipase and biofilm are described as the major virulence factors (47, 48) . Phospholipases possess an important role in lung diseases, promoting change in membrane composition, stimulating chemokines and cytokines, and altering gas exchange and as a lung surfactant (49). Hemolysin, phospholipase and biofilm are some of the many pathogenicity factors in Acinetobacter baumannii. Successful fitness of this bacteria, however, is a consequence of a large virulence repertoire, also composed by outer membrane proteins, secretion systems, surface adhesins, glycoconjugates and iron-chelating activities, which can explain its multi-resistance to antibiotics in nosocomial infections (28). Biofilm-based infections are significantly less susceptible to antimicrobial agents and their treatment is extremely difficult. According to Sanchez et al., 2013 (37) , the investigation of biofilm forming capacity in patients with diverse infective sources revealed that Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli are strong biofilm producers, at levels greater than or equal to Staphylococcus epidermidis, a positive biofilm producing strain. The greatest number of biofilm producing strains corresponded to P. aeruginosa and S. aureus species. Interestingly, the metabolic profile obtained in our study correlated to virulence factors of the species described, particularly biofilm (Tables 3-6 ). Within Central Carbohydrate Metabolism (Table 3) , the TCA_cycle was the predominant pathway (18%), with abundance of dihydrolipoamide dehydrogenase enzyme (Table 4 ). According to Lu and authors (2019) (50), alteration in the metabolites from the TCA cycle, amino acid metabolism, and glycerolipid pathways were observed during biofilm production. Furthermore, characterization of dihydrolipoamide dehydrogenase from Streptococcus pneumoniae showed that this enzyme plays an important role in pneumococcal infection, being necessary for the survival and capsular polysaccharide production of pneumococci within the host (51). 10 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 24, 2020. ; https://doi.org/10.1101/2020.12.22.20248501 doi: medRxiv preprint Besides enzymes associated with biofilm formation, we identified adhesins, permeases for antibiotic export, resistance and toxins, and superantigens belong to the Virulence, Disease and Defense functional category ( Table 5 ). The most abundant adhesins are fibronectin/fibrinogenbinding proteins, although high numbers of sortases, related to attachment of specific proteins to the cell wall, were also found. Toxins and superantigens are composed of the streptolysin S locus, which produces the beta-haemolytic phenotype. Streptolysin S causes impaired phagocytic clearance and promotes epithelial cell cytotoxicity, enhancing the effects of M protein and streptolysin O (52). In the Membrane Transport category, protein secretion system Types I, V, and VI are highlighted, with abundance of exoprotein involved in heme utilization, cytolysin activator and adhesin ( Table 6 ). The scenario highlights the high occurrence of potentially virulent pathogens, which can contribute to severity of lung infection in Coronavirus Disease 2019 patients admitted to intensive care units. Moreover, the prevalence of respiratory pathogens observed in COVID-19 patients analyzed can be orchestrated mainly by the complex and distinct immune events in response to lung damage, as well as by the ecological model proposed by Dickson and collaborators (2014, 2015) (26, 25), which indicate that several lung diseases can alter the growth of local microbiota, leading to an increase in bacterial abundance. A cellular model to explain the bacterial species described in our study correlated to processes that aggravate COVID-19 is presented ( Figure 3 ). Impairment in neutrophil function, macrophage depletion and excessive inflammation has been reported as an important factor in the progression of lower respiratory tract pathologies (53). It was demonstrated that alveolar macrophage depletion could facilitate the bacterial infection by the establishment of a niche for secondary Streptococcus pneumoniae infection, altering cellular innate immunity and resulting in lethal pneumonia (54). Additionally, neutrophilia plays an important role in bacterial pulmonary diseases, including those caused by Streptococcus pneumoniae, Klebsiella 11 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 24, 2020. infection (59, 60) . Increase of cell-free hemoglobin (Hb) and HO-1 expression in lung tissue and bronchoalveolar lavage fluid has been correlated to COPD and acute respiratory distress syndrome (ARDS) severity (61) . Simon and authors (2009)(62) demonstrated an ACE-like activity of Hb in the conversion of angiotensin I to its active metabolites induced by ferrous-and ferryl-Hb, with a possible effect on vasoconstriction. Moreover, intratracheal administration of heme in mice led to alveolar-capillary barrier dysfunction and increased alveolar permeability, contributing to acute lung and ARDS (63) . Curiously, heme uptake by pathogens plays an important role during bacterial infection. These microorganisms induce erythrocyte hemolysis caused by pore-forming toxins, such as hemolysin and phospholipases. Free-heme and iron acquired from erythrocyte disruption is required for invasion, growth and successful pathogen colonization in the host (64) . In addition, Dutra and collaborators (2014) (65) showed that heme promotes the processing of caspase-1, 12 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 24, 2020. ; https://doi.org/10.1101/2020.12.22.20248501 doi: medRxiv preprint inflammasome activation and IL-1β secretion by macrophages that participate in the immune response induced by hemolysis, contributing to inflammation and pathogenesis. Strategies for interfering in inflammasome activation by bacterial pathogens have evolved (66) , and together with SARS-Cov2, may contribute to a hyperactivated inflammatory response (67) . 16 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 24, 2020. 18 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 36 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 24, 2020. ; https://doi.org/10.1101/2020.12.22.20248501 doi: medRxiv preprint Bacterial pathogens evade the host immune response by biofilm production, induction of hemolysins, iron acquisition factors, adhesins, and other virulence factors. The SARS-CoV-2 and bacterial pathogens promote erythrocyte lysis, causing heme(Hb)/iron(Fe) liberation. Heme uptake is required for bacterial host colonization and increased production of virulence factors. Bacterial virulence factors, as well as free-heme, activates the inflammasome, contributing to neutrophil inflammation, cytokine elevation and pathogenesis processes. This figure was generated using the BioRender (available at https://biorender.com/). 38 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 24, 2020. ; https://doi.org/10.1101/2020.12.22.20248501 doi: medRxiv preprint Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia Regional differences in the lung The role of the microbiome in exacerbations of chronic lung diseases Mechanisms of neutrophil accumulation in the lungs against bacteria The frequency of influenza and bacterial coinfection: a systematic review and meta-analysis. Influenza Other Respir Viruses Risk of ruling out severe acute respiratory syndrome by ruling in another diagnosis: variable incidence of atypical bacteria coinfection based on diagnostic assays Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms Deciphering mechanisms of staphylococcal biofilm evasion of host immunity Dynamic interactions of neutrophils and biofilms The hemolysins of Staphylococcus aureus Interaction of Escherichia coli hemolysin with biological membranes. A study using cysteine scanning mutagenesis High incidence of hemolysin production by Enterococcus (Streptococcus) faecalis strains associated with human parenteral infections Group B streptococcal betahemolysin/cytolysin promotes invasion of human lung epithelial cells and the release of interleukin-8 The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease Virulence factors of Candida albicans Heme scavenging reduces pulmonary endoplasmic reticulum stress, fibrosis, and emphysema Heme oxygenase-1 in lung disease Desaturation and heme elevation during COVID-19 infection: A potential prognostic factor of heme oxygenase-1 Targeting the Heme-Heme Oxygenase System to Prevent Severe Complications Following Lung heme oxygenase-1 is elevated in acute respiratory distress syndrome Evaluation of angiotensin converting enzyme (ACE)-like activity of acellular hemoglobin Cell-free hemoglobin: a novel mediator of acute lung injury Heme Uptake and Utilization by Gram-Negative Bacterial Pathogens Hemolysis-induced lethality involves inflammasome activation by heme Inflammasome activation and regulation: toward a better understanding of complex mechanisms The Inflammasome in Times of COVID-19 Differential gene expression in Streptococcus pneumoniae in response to various iron sources Interdependence between iron acquisition and biofilm formation in Pseudomonas aeruginosa Molecular mechanisms of Staphylococcus aureus iron acquisition Iron-source preference of 26 24 99 137 9 50 5 11 285 116 107 26 63 1 59 4 Table 3 . Functional pathway of Central Carbohydrate Metabolism, related to virulence factors reported for the species identified in this work.