Identification of the Bacterial Pathogens in Children with Otitis Media: A Study in the Northwestern Portuguese District of Braga

Understanding the bacterial etiology of otitis media (OM) is important when designing and evaluating the best course of treatment. This study analyzed middle ear fluid (MEF) and nasopharynx (NP) samples collected from 49 children with OM undergoing myringotomy in the northwestern Portuguese district of Braga. A correlation between species in the NP and MEF was observed following pathogen detection by culture and quantitative polymerase chain reaction (qPCR) methods. Bacterial identification using culturing methods showed that Moraxella catarrhalis was the most representative in NP and MEF, followed by Streptococcus pneumoniae. However, qPCR of MEF showed a higher prevalence (61%) of Haemophilus influenzae. S. pneumoniae was not the most frequently identified species, but it still remains one of the leading causes of OM in this region despite 93.9% of the children being vaccinated with the pneumococcal conjugate vaccine. Furthermore, 46% of the samples analyzed by qPCR identified more than two bacterial species. M. catarrhalis and S. pneumoniae were the most frequent combination identified in NP and MEF samples by culturing methods. Additionally, a few NP and MEF samples simultaneously presented the three main otopathogens. These results point out that polymicrobial infections play an important role in OM. Further studies characterizing the serotypes of the strains isolated, their resistance profile, and their biofilm forming ability would help in the development of more targeted strategies against otitis media.

226. Multidrug Resistant Polymicrobial Gram-negative Bacteremia in Hematologic Cancer Patients with Febrile Neutropenia at the Uganda Cancer Institute

Background Bloodstream infections (BSI) are associated with significant mortality in hematologic cancer patients with febrile neutropenia. Poor clinical outcomes are associated with presence of multidrug resistant (MDR) organisms and polymicrobial infections. We sought to determine antimicrobial resistance and outcomes of polymicrobial bloodstream infections in hematologic cancer patients with febrile neutropenic episodes (FNEs) at the Uganda Cancer Institute. Methods Blood drawn from participants during an FNE (fever ≥ 37.5°C and neutrophil count ≤ 1000 cells/µL) was cultured in the BACTEC 9120 blood culture system. Bacteria from positive cultures were identified biochemically. Antimicrobial susceptibility testing was performed with the disc diffusion method. Participants were followed for 30 days from first FNE onset for death from any cause. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (95%). Results Six hundred and twenty-nine participants were followed for FNE. Two hundred and twenty-eight FNEs in 159 participants were observed. Of 181 FNEs with blood cultures completed, 65 (36%) had pathogenic organism isolated. A total of 74 Gram negative and 18 Gram positive bacteria were isolated. Forty-eight (74%) FNEs had monomicrobial (MBSI) and 17 (26%) had polymicrobial (PBSI) bloodstream infections. Gram negative - Gram negative (10 out of 17, 59%) was the most frequent PBSI combination (Fig 1). Up to 75% (12 out of 16) of Gram-negative PBSI were MDR. The most common organism isolated was E. coli (38% of isolates). Participants with PBSI had higher early mortality rates at 7 days compared to MBSI and negative cultures (44%, 22%, and 16% for PBSI, MBSI, and negative respectively; HR (95% CI): 3.63 (1.49, 8.86) for PBSI v. negative/MBSI cultures). Similarly, PBSI was associated with higher mortality within 30 days of FNE onset (63%, 52%, and 38% for PBSI, MBSI, and negative respectively; HR (95% CI): 2.17 (1.09, 4.32) for PBSI v. negative/MBSI) (Fig 2). Figure 1. Bar graph showing combinations for polymicrobial bloodstream infections (PBSI). GNGN: Gram-negative – Gram-negative; GNGP: Gram-negative – Gram-positive; GNO: Gram-negative – Other (fungi); GPGP: Gram-positive – Gram-positive Figure 2. Kaplan-Meier failure curves of participants with negative cultures, monomicrobial infections and polymicrobial infections Conclusion PBSI episodes were more likely to be multidrug resistant and are associated with higher mortality. Empirical therapy for patients with PBSI should consider multidrug resistant Gram-negative bacteria Disclosures All Authors: No reported disclosures

1152. Microbiology of Pediatric Neck Infections Based on Age and Anatomic Location

Background Studies of pediatric neck infections demonstrate an increase in methicillin resistant Staphylococcus aureus (MRSA), and predominance of Staphylococcus aureus (S. aureus) in infants, and commonly polymicrobial infections. Thus, some providers treat acute neck infections with empiric broad spectrum antibiotics, often with two drugs. Our institution often uses clindamycin plus ampicillin-sulbactam as empiric therapy for hospitalized children with acute neck infection. We aimed to identify the microbiology of acute neck abscesses at our institution to determine if stratifying by age and abscess location would allow for single agent therapy. Table 1. Causative organism based on anatomic location of neck infection. Methods Diagnosis codes identified patients hospitalized with acute neck infections. Cases with underlying malignancy, cervicofacial malformations, or lymphatic malformations were excluded. Patients with surgical cultures were categorized into two groups based on anatomic location of infection: medial (retropharyngeal, parapharyngeal, and peritonsillar), lateral (other locations), or both. Within each group, causative pathogen(s) were explored and further categorized by age (infants: < 1 year old; non-infants: ≥1 year old). Results 412 patients were hospitalized for acute neck infection of which 132 had surgical cultures. 110 had growth of one or more pathogens (20 infants, 90 non-infants). 53 infections were located medially, 54 laterally, and 3 had both locations involved. S. aureus was most commonly identified, with lateral infections accounting for the majority (Table 1). 40/44 S. aureus isolates were susceptible to clindamycin. Among medial infections, Streptococcus Anginosus and Group A Streptococcus were most common followed by S. aureus (Table 1). 17/20 (85%) positive cultures in infants grew S. aureus with 8/17 (47%) MRSA. No polymicrobial infections were identified in infants. Among non-infants, 0/39 lateral infections had polymicrobial growth but 23/50 (46%) of medial infections did. Conclusion Local epidemiology based on anatomic location and patient age suggests a single agent (clindamycin for lateral and penicillin with beta-lactamase inhibitor for medial) may be reasonable for non-infants with uncomplicated neck infections. For infants, coverage of MRSA, regardless of anatomic location, is advisable. Disclosures All Authors: No reported disclosures

Model Systems to Study the Chronic, Polymicrobial Infections in Cystic Fibrosis: Current Approaches and Exploring Future Directions

A recent workshop titled “Developing Models to Study Polymicrobial Infections,” sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 35+ investigators over two virtual sessions.

Clinical Relevance of the Microbiome in Odontogenic Abscesses

Odontogenic abscesses are usually caused by bacteria of the oral microbiome. However, the diagnostic culture of these bacteria is often prone to errors and sometimes fails completely due to the fastidiousness of the relevant bacterial species. The question arises whether additional pathogen diagnostics using molecular methods provide additional benefits for diagnostics and therapy. Experimental 16S rRNA gene analysis with next-generation sequencing (NGS) and bioinformatics was used to identify the microbiome of the pus in patients with severe odontogenic infections and was compared to the result of standard diagnostic culture. The pus microbiome was determined in 48 hospitalized patients with a severe odontogenic abscess in addition to standard cultural pathogen detection. Cultural detection was possible in 41 (85.42%) of 48 patients, while a pus-microbiome could be determined in all cases. The microbiomes showed polymicrobial infections in 46 (95.83%) cases, while the picture of a mono-infection occurred only twice (4.17%). In most cases, a predominantly anaerobic spectrum with an abundance of bacteria was found in the pus-microbiome, while culture detected mainly Streptococcus, Staphylococcus, and Prevotella spp. The determination of the microbiome of odontogenic abscesses clearly shows a higher number of bacteria and a significantly higher proportion of anaerobes than classical cultural methods. The 16S rRNA gene analysis detects considerably more bacteria than conventional cultural methods, even in culture-negative samples. Molecular methods should be implemented as standards in medical microbiology diagnostics, particularly for the detection of polymicrobial infections with a predominance of anaerobic bacteria.

Single-Cell Imaging Reveals That Staphylococcus aureus Is Highly Competitive Against Pseudomonas aeruginosa on Surfaces

Pseudomonas aeruginosa and Staphylococcus aureus frequently occur together in polymicrobial infections, and their interactions can complicate disease progression and treatment options. While interactions between P. aeruginosa and S. aureus have been extensively described using planktonic batch cultures, little is known about whether and how individual cells interact with each other on solid substrates. This is important because both species frequently colonize surfaces to form aggregates and biofilms in infections. Here, we performed single-cell time-lapse fluorescence microscopy, combined with automated image analysis, to describe interactions between P. aeruginosa PAO1 with three different S. aureus strains (Cowan I, 6850, JE2) during microcolony growth on agarose surfaces. While P. aeruginosa is usually considered the dominant species, we found that the competitive balance tips in favor of S. aureus on surfaces. We observed that all S. aureus strains accelerated the onset of microcolony growth in competition with P. aeruginosa and significantly compromised P. aeruginosa growth prior to physical contact. Upon direct contact, JE2 was the most competitive S. aureus strain, simply usurping P. aeruginosa microcolonies, while 6850 was the weakest competitor itself suppressed by P. aeruginosa. Moreover, P. aeruginosa reacted to the assault of S. aureus by showing increased directional growth and expedited expression of quorum sensing regulators controlling the synthesis of competitive traits. Altogether, our results reveal that quantitative single-cell live imaging has the potential to uncover microbial behaviors that cannot be predicted from batch culture studies, and thereby contribute to our understanding of interactions between pathogens that co-colonize host-associated surfaces during polymicrobial infections.

Ventilator-Associated Pneumonia in COVID-19 Patients: A Retrospective Cohort Study

Introduction: Aim of this study is to analyse the characteristics of ventilator-associated pneumonia (VAP) inpatients infected by severe acute respiratory syndrome-coronavirus 2 (SARS-Cov2). Materials and Methods: A retrospective study was conducted, including coronavirus infectious disease 2019 (COVID-19) patients who developed VAP from March to May 2020 (VAP COVID-19). They were compared to non-COVID-19 patients who developed VAP from January 2011 to December 2019 (VAP NO COVID-19) and COVID-19 patients who did not develop VAP (NO VAP COVID-19). Results: Overall, 42 patients were included in the VAP COVID-19group, 37 in the NO VAP COVID-19 group, and 188 in the VAP NO COVID-19 group. VAP COVID-19 had significantly higher rates of shock (71% vs. 48%, p = 0.009), death in ICU (52% vs. 30%, p = 0.011), VAP recurrence (28% vs. 4%, p < 0.0001), positive blood culture (26% vs. 13%, p = 0.038), and polymicrobial culture (28% vs. 13%, p = 0.011) than VAP NO COVID-19. At the multivariate analysis, death in patients with VAP was associated with shock (p = 0.032) and SARS-Cov2 (p = 0.008) infection. Conclusions: VAP in COVID-19 patients is associated with shock, bloodstream, and polymicrobial infections.

Rapid and strain-specific resistance evolution of Staphylococcus aureus against inhibitory molecules secreted by Pseudomonas aeruginosa

Pseudomonas aeruginosa and Staphylococcus aureus frequently occur together in polymicrobial infections, and there is evidence that their interactions negatively affect disease outcome in patients. At the molecular level, interactions between the two bacterial taxa are well-described, with P. aeruginosa usually being the dominant species suppressing S. aureus through a variety of inhibitory molecules. However, in polymicrobial infections, the two species interact over prolonged periods of time, and S. aureus might evolve resistance against inhibitory molecules deployed by P. aeruginosa. Here, we used experimental evolution to test this hypothesis by exposing three different S. aureus strains (Cowan I, 6850, JE2) to the growth-inhibitory supernatant of P. aeruginosa PAO1 over 30 days. We found that all three S. aureus strains rapidly evolved resistance against inhibitory molecules and show that (i) adaptations were strain-specific; (ii) resistance evolution affected the expression of virulence traits; and (iii) mutations in membrane transporters were the most frequent evolutionary targets. Our work indicates that adaptations of S. aureus to co-infecting pathogens could increase virulence and decrease antibiotic susceptibility, because both virulence traits and membrane transporters involved in drug resistance were under selection. Thus, pathogen co-evolution could exacerbate infections and compromise treatment options.