Manual counting of bacterial colony forming units (CFUs) on agar plates is laborious and error-prone. We therefore implemented a colony counting system with a novel segmentation algorithm to discriminate bacterial colonies from blood and other agar plates. A colony counter hardware was designed and a novel segmentation algorithm was written in MATLAB. In brief, pre-processing with Top-Hat-filtering to obtain a uniform background was followed by the segmentation step, during which the colony images were extracted from the blood agar and individual colonies were separated. A Bayes classifier was then applied to count the final number of bacterial colonies as some of the colonies could still be concatenated to form larger groups. To assess accuracy and performance of the colony counter, we tested automated colony counting of different agar plates with known CFU numbers of S. pneumoniae , P. aeruginosa and M. catarrhalis and showed excellent performance.

25Background: Multiple epidemiological studies identify Dolosigranulum pigrum as a 26 candidate beneficial bacterium based on its positive association with health, including 27 negative associations with nasal/nasopharyngeal colonization by the pathogenic 28 species Staphylococcus aureus and Streptococcus pneumoniae. 29Results: Using a multipronged approach to gain new insights into D. pigrum function, 30 we observed phenotypic interactions and predictions of genomic capacity that support a 31 role for microbe-microbe interactions involving D. pigrum in shaping the composition of 32 human nasal microbiota. We identified in vivo community-level and in vitro phenotypic 33 cooperation by specific nasal Corynebacterium species. Also, D. pigrum inhibited S. 34 aureus growth in vitro. Whereas, robust inhibition of S. pneumoniae required both D. 35 pigrum and a nasal Corynebacterium together, and not either alone. D. pigrum L-lactic- 36 acid production was insufficient to account for these inhibitions. Genomic analysis of 11 37 strains revealed that D. pigrum has a small genome (average 1.86 Mb) and multiple 38 predicted auxotrophies consistent with D. pigrum relying on its human host and 39 cocolonizing bacteria for key nutrients. Further, the accessory genome of D. pigrum 40 3 encoded a diverse repertoire of biosynthetic gene clusters, some of which may have a 41 role in microbe-microbe interactions. 42 Conclusions: These new insights into D. pigrum's functions advance the field from 43 compositional analysis to genomic and phenotypic experimentation on a potentially 44 beneficial bacterial resident of the human upper respiratory tract and lay the foundation 45 for future animal and clinical experiments. 46 47 48 Streptococcus pneumoniae, microbe-microbe interactions, interspecies interactions, 50 upper respiratory tract, nasal, microbiota, comparative genomics 51 4 Background 52 Colonization of the human nasal passages by Staphylococcus aureus or Streptococcus 53pneumoniae is a major risk factor for infection by the colonizing bacterium at a distant 54 body site [1][2][3][4][5]. Interventions that reduce the prevalence of colonization also reduce the 55 risk of infection and transmission, e.g., as in [6, 7]. S. aureus and S. pneumoniae are 56 major human pathogens that cause significant morbidity and mortality worldwide [8][9][10][11]. 57 There are also concerns regarding rising rates of antimicrobial resistance [12] and the 58 potential for long-term effects of antibiotics early in life [13]. Thus, efforts have recently 59 focused on the identification of candidate bacteria that confer colonization resistance 60 against S. aureus [14-21] and S. pneumoniae [22-25], with particular urgency for S. 61 aureus in the absence of an effective vaccine. 62 Dolosigranulum pigrum has emerged in multiple studies of the human upper respiratory 63 tract microbiota, colonizing with or without Corynebacterium species, as potentially 64 beneficial and/or protective against colonization by S. aureus and ...

Objectives While superinfections are associated with unfavourable disease course, their impact on clinical outcomes in critically ill COVID-19 patients remains largely unknown. We aimed to investigate the burden of superinfections in COVID-19 patients. Methods In this prospective single centre cohort study in an intensive care setting patients aged ≥ 18 years with COVID-19 acute respiratory distress syndrome were assessed for concomitant microbial infections by longitudinal analysis of tracheobronchial secretions, bronchoalveolar lavages and blood. Our primary outcome was ventilator-free survival on day 28 in patients with and without clinically relevant superinfection. Further outcomes included the association of superinfection with ICU length of stay, incidence of bacteremia, viral reactivations, and fungal colonization. Results In 45 critically ill COVID-19 patients, we identified 19 patients with superinfections (42.2%) by longitudinal analysis of 433 TBS, 35 BAL and 455 blood samples, respectively. On average, superinfections were detected on day 10 after ICU admission. The most frequently isolated clinically relevant bacteria were Enterobacteriaceae, Streptococcus pneumoniae, and Pseudomonas aeruginosa. Ventilator-free survival was substantially lower in patients with superinfection (subhazard ratio 0.37, 95%-CI 0.15-0.90, p=0.028). Patients with pulmonary superinfections more often had bacteraemia, virus reactivations, yeast colonization, and needed ICU treatment for a significantly longer time. Conclusions The detection of superinfections was frequent and associated with reduced ventilator-free survival. Despite empirical antibiotic therapy, superinfections lead to an extended ICU stay in COVID 19 patients. Longitudinal microbiological sampling in COVID-19 patients could allow targeted antimicrobial therapy, and therefore minimize the use of broad-spectrum and reserve antibiotics.