Pleural space infection microbiology as assessed using a clinical sequencing-based assay: Fusobacterium nucleatum group, Streptococcus intermedius, and other oral normal microbiota are the most common bacteria identified in community-acquired pleural space infections

ABSTRACT

The definition of the microbiology of pleural space infection has been challenging due to the poor yield of conventional culture. Here, the results of a 16S ribosomal RNA gene PCR/sequencing assay performed on pleural fluid in routine clinical practice between August 2020 and January 2023 were evaluated. Amplified 16S rRNA gene DNA was submitted to Sanger sequencing and/or next-generation sequencing or results were reported as negative, depending on PCR crossing threshold value. In all, 496 pleural fluids were tested at Mayo Clinic Laboratories, with 227 positive results, including 57 from Mayo Clinic patients. Among the 57 Mayo Clinic patients, pleural space infection was community acquired in 48 (84%); Fusobacterium nucleatum group and/or Streptococcus intermedius were detected in 31/57 (54%) cases [including 28/48 (58%) community-acquired cases], with additional facultative and/or anaerobic species also found in various combinations in 17/31 (55%). Results of this study suggest that the most frequent microorganism profile involved in community-acquired pleural space infection may be a combination of F. nucleatum group and/or S. intermedius, with or without other normal microbiota.

IMPORTANCE

We describe here the most frequent microorganisms detected in community-acquired pleural space infection using a clinically performed sequencing-based assay. We found that the most common detection was the Fusobacterium nucleatum group and/or Streptococcus intermedius, with or without other normal microbiota. We propose the term e-FuSion (effusion with Fusobacterium nucleatum group, Streptococcus intermedius, and other oral normal microbiota) for this entity.

INTRODUCTION

Pleural space infection has significant morbidity and appears to be increasing worldwide (1, 2). In the United States, a 38% relative increase in pleural space infection-related hospitalizations was reported between 2007 and 2016 (3). In France, there were 7.2 cases per 100,000 habitants reported in 2013, and 7.8 cases per 100,000 inhabitants reported in 2017 (4), and in England there was an increase from 6.4 to 8.4 per 100,000 hospital admissions between 2008 and 2017 (5).

The yield of pleural fluid cultures is low in pleural space infections (6), whether due to the fastidious nature of the microorganisms involved or the administration of antimicrobial therapy (7). Identifying the causative microorganism(s) is important to guide antimicrobial therapy; without that knowledge, pleural space infections are empirically treated. Knowledge of the microbiology of pleural space infection can inform a transition from empiric to tailored treatment, potentially shortening hospital stays, reducing complications from unneeded antibiotic use, and/or decreasing healthcare costs (8). A limited number of studies have evaluated the etiology of pleural space infection using 16S ribosomal RNA (rRNA) gene PCR followed by sequencing (9–11).

This study aimed to describe the microbiologic and clinical findings of pleural fluids testing positive by a 16S rRNA gene PCR assay followed by Sanger or next-generation sequencing performed in routine clinical practice.

MATERIALS AND METHODS

Study design and study population

This retrospective study analyzed pleural fluids tested at Mayo Clinic (Rochester MN) by 16S rRNA gene PCR/sequencing between August 2020 and January 2023. Specimens other than pleural fluid were not analyzed. Clinical characteristics were only available for Mayo Clinic subjects and not for specimens submitted for testing through Mayo Clinic Laboratories. The laboratory and imaging findings of the former cohort were ascertained through an electronic medical record review. Specimens submitted for testing through Mayo Clinic Laboratories were from 24 US states and Mexico.

Pleural space infection was defined as the presence of bacteria or pus in the pleural cavity (12). A pleural fluid specimen testing positive by 16S rRNA gene PCR/sequencing only was considered an infection if results were not interpreted as representing a contaminant (or contaminants) by the treating medical team and provided the team considered the subject to be infected. Community-acquired infection was defined as infection contracted outside the hospital or diagnosed within 48 hours of admission; hospital-acquired infection was defined as infection acquired during hospitalization, diagnosed more than 48 hours after admission.

16S rRNA gene PCR/sequencing

100 µL of each pleural fluid was transferred to a lysis tube containing 20 µL of 0.1 mm silica/zirconium beads, 170 µL proteinase K buffer, and 20 µL proteinase K. Lysis tubes were incubated in a thermomixer at 300 rpm for 1 hour at 60°C, and then a thermomixer at 2,000 rpm for 5 minutes at 100°C. The tube was then centrifuged and cooled for 5 minutes. The homogenized specimen preparation (270 µL) was transferred to pre-warmed lysis buffer and extracted on an eMAG instrument (bioMérieux, France) generating 50 µL of purified eluate. 5 µL of the extract was used to amplify the V1 to V3 region of the 16S rRNA gene on a LightCycler 480II (Roche Diagnostics, Risch-Rotkreuz, Switzerland) using dual priming oligonucleotides, as previously described (13). A crossing threshold (Ct) value was determined for each sample. Samples with Ct values of <32 cycles were submitted to Sanger sequencing. Samples with a Ct between 32 and 34 cycles were submitted to next-generation sequencing (NGS). Samples with Ct values >34 cycles were considered negative and not further analyzed, except if a well-defined melting temperature peak was noted, in which case they were sent to NGS. Samples that did not produce an interpretable result from Sanger sequencing were submitted to NGS. Bidirectional Sanger sequencing was performed on an Applied Biosystems 3500xl instrument (Thermo Fisher Scientific, Waltham, Massachusetts, United States) and NGS on an Illumina MiSeq (Illumina, San Diego, California, United States) with a 500-cycle (2 × 250 paired-end read) v2 nano kit generating 2 million paired-end reads per run. Bioinformatic analysis was performed with RipSeq NGS software (Pathogenomix, Santa Cruz, California, United States). Doctoral-level clinical microbiologists interpreted results; organisms considered potential pathogens were reported and organisms found in the negative control and/or across different samples in the same sequencing run were generally not reported (14).

Conventional microbiologic methods

Conventional cultures included aerobic and anaerobic cultures and any others (e.g., mycobacterial cultures, fungal cultures) clinically ordered and performed at Mayo Clinic. Concordance between conventional cultures and 16S RNA PCR/sequencing was defined as detection of the same microorganism(s) by both techniques. Concordance was not used to calculate the performance characteristics of 16S RNA PCR/sequencing because the yield of conventional cultures was low.

Statistical analysis

Descriptive summaries are reported as median (minimum, maximum) for continuous variables and frequencies and percentages for categorical variables. Analyses were performed using SAS software version 9.4 (SAS Inc. Cary, NC).

RESULTS

Samples

In all, 496 samples were studied (including 143 from Mayo Clinic patients), 269 (including 86 from Mayo Clinic patients) with negative and 227 (including 57 from Mayo Clinic patients) with positive 16S ribosomal RNA gene PCR/sequencing results. Of the 227 positive samples, 184 samples had Ct values <32 cycles, of which 90 yielded results reported based on Sanger sequencing and 94 underwent reflexive NGS, all of which were associated with one or more reported bacteria. In all, 18 pleural fluids had Ct values between 32 and 34 cycles; all of these underwent NGS, with all 18 yielding results reported as positive. In total, 294 pleural fluids yielded Ct values >34 cycles, 38 of which were sent to NGS, with 25 yielding results reported as positive (Fig. 1).

Fig 1.

Sample flowchart. Abbreviations: Ct: cycle threshold; NGS: next-generation sequencing.

Clinical analysis (Mayo Clinic patients)

There were 57 pleural fluids from Mayo Clinic patients with positive 16S rRNA gene PCR/sequencing results. The median patient age was 63 years (range 5–83 years) and 75% were male. Pleural space infection was community acquired in 48/57 (84%). 48/57 (84%) patients had received antibiotics in the 2 weeks preceding specimen collection. The most frequent symptom, dyspnea, was noted in 44 (77%), followed by chest pain in 28 (49%). Twenty-one (37%) had a fever. Hypoxemia was documented in 25 (44%). Median neutrophil count, C-reactive protein, and lactate values were 16.7 /L (range 4.8–50.6/L), 174 mg/L (range 77–340 mg/L), and 1.5 mmol/L (0.6–6.5 mml/L), respectively. Median pleural fluid total cell count, neutrophil percentage, pH, glucose, protein, and lactate dehydrogenase were 8,297/µL (range 104–469,536/μL), 88% (range 36%–100%), 7.1 (range 6.1–7.5), 88 mg/dL (range 36–100 mg/dL), 4 g/dL (range 1.7–6 mg/dL), and 1,399 U/L (range 43–10,410 U/L). Radiological consolidation was observed in 36 (63%), including 28/48 (58%) with community-acquired infection. Pleural fluid was loculated in 33 (58%), with lytic therapy used in 36 (63%). Mortality at 30 days was 12% (none related to pleural space infection); no recurrent infection ocurred with the same microorganism at 30 days.

Demographics (non-Mayo Clinic patients)

There were 168/353 pleural fluids from non-Mayo Clinic patients with positive 16S rRNA gene PCR results; 112/168 (67%) were male and the median patient age was 51 years.

Microbiology (Mayo Clinic patients)

Pleural fluid cultures were positive in 32% (18/57) of the Mayo Clinic patients with positive 16S rRNA gene PCR/sequencing results (and none [0/57] with negative 16S rRNA gene PCR/sequencing results). In those with positive 16S rRNA gene PCR/sequencing results, pleural fluid cultures were concordant with 16S rRNA gene PCR/sequencing in eight cases, partially concordant in five cases, and not concordant in five cases. Sputum cultures were positive in 9/57 cases, and partially concordant with 16S rRNA gene PCR/sequencing in two cases. Blood cultures were positive in 11% (5/57), being concordant with 16S rRNA gene PCR/sequencing in three cases and partially concordant in two cases.

Forty percent (23/57) of detections by 16S rRNA gene PCR/sequencing were polymicrobial. The most common microorganisms detected alone were Staphylococcus aureus (3/57), Streptococcus pyogenes (2/57), Enterobacterales (2/57), Streptococcus mitis group (2/57), Staphylococcus caprae/capitis (2/57), and Cutibacterium acnes (2/57) (Table 1).

TABLE 1.

Microbiology by 16S ribosomal RNA gene PCR/sequencing of non-Fusobacterium nucleatum group, Streptococcus intermedius, and other oral normal microbiota group pleural space infections from Mayo Clinic subjects

Microorganisms	n
Staphylococcus aureus	3
Streptococcus pyogenes	2
Enterobacterales	2
Streptococcus mitis	2
Staphylococcus caprae/capitis	2
Cutibacterium acnes	2
Streptococcus pneumoniae	1
Pseudomonas aeruginosa	1
Staphylococcus epidermidis	1
Fusobacterium necrophorum	1
Legionella londiniensis	1
Aggregatibacter aphrophilus	1
Morococcus/Neisseria species	1
Polymicrobial detectionsa	6

^a Haemophilus influenzae, Enterococcus species, Metamycoplasma salivarum, Neisseria species (not Neisseria gonorrhoeae or Neisseria meningitidis), Parvimonas micra, Porphyromonas species, Anaeroglobus geminates, Bacteroides thetaiotaomicron, Odoribacter splanchnicus, Lawsonella clevelandensis, Leuconostoc lactis, Staphylococcus caprae/capitis, Staphylococcus epidermidis, Bergeyella zoohelcum, Mycobacterium avium complex, Streptococcus mitis group, Streptococcus pyogenes, Cardiobacterium hominis, Citrobacter freundii.

Fusobacterium nucleatum group and/or Streptococcus intermedius were detected in 31/57 (54%) pleural fluids by 16S rRNA gene PCR/sequencing, with additional facultative and/or anaerobic bacterial species in various combinations found in 17/31 (55%) of these cases (Table 2). Considering recent taxonomy changes, the term “F. nucleatum group” is used here to include species closely related to F. nucleatum, including former subspecies. Overall, F. nucleatum group was detected in 17/31 and S. intermedius in 22/31 of these cases, with the two detected together in 8/31 and just one of the two in 23/31. 19/31 (61%) of these 16S rRNA gene PCR-positive samples yielded negative cultures. 7/31 had positive pleural fluid cultures with microorganisms detected by culture also found by sequencing alone or in combination with other bacteria.

TABLE 2.

Microbiology by 16S ribosomal RNA gene PCR/sequencing of Fusobacterium nucleatum group, Streptococcus intermedius, and other oral normal microbiota group pleural space infections from Mayo Clinic subjects

Microorganisms	n
Streptococcus intermedius	12
Fusobacterium nucleatum group	2
S. intermedius and F. nucleatum group	1
S. intermedius, F. nucleatum group and other normal oral microbiotaa	7
S. intermedius and other normal oral microbiotab	2
F. nucleatum group and other normal oral microbiotac	7

^a Parvimonas micra, Gemella bergeri, Eubacterium brachy, Prevotella species, Campylobacter gracilis, Campylobacter rectus, Porphyromonas species, Schaalia species, and Capnocytophaga species.

^b Prevotella intermedia, Porphyromonas species, Fusobacterium gonidiaformans, Gemella bergeri, P. micra, and Prevotella pleuritidis.

^c Bacteroides fragilis, Corynebacterium species, P. micra, Porphyromonas species, Lancefieldella rimae, Prevotella species, Solobacterium moorei, Streptococcus anginosus, Campylobacter rectus, Eubacterium brachy, Schaalia species, Streptococcus constellatus, Dialister pneumosintes, and Fusobacterium periodonticum.

Clinical analysis of F. nucleatum group, S. intermedius, and oral microbiota group

In this group, 28/31 (90%) were community-acquired infections. The most common initial presentations were dyspnea in 29/31 (93%), pleuritic pain in 17/31 (55%), and fever in 9/31 (29%). Radiological consolidation was observed in 18 (58%) and pleural fluid was loculated in 26 (84%). Lytic therapy was administrated to 24 subjects (77%) (Table 3). 30-day mortality was 6% (with no deaths related to pleural space infection).

TABLE 3.

Demographics and clinical characteristics of subjects with Fusobacterium nucleatum group, Streptococcus intermedius, and other oral normal microbiota group pleural space infections from Mayo Clinic subjects

Basal characteristics	n
Age (years)	58
Male sex (%)	23/31 (74%)
Community-acquired infection (%)	28/31 (90%)
Symptoms/signs
Dyspnea (%)	29/31 (93%)
Pleuritic pain (%)	17/31 (55%)
Fever (%)	9/31 (29%)
Microbiologic studies
Positive blood culturea (%)	1/31 (3%)
Positive pleural fluid cultureb (%)	10/31 (32%)
Positive sputum culturec (%)	5/31 (16%)
Radiographic findings
Pulmonary consolidation (%)	18/31 (58%)
Unilateral effusion (%)	2/31 (6%)
Left-sided effusion (%)	15/31(48 %)
Pleural space loculation (%)	26/31 (84%)

^a Positive blood culture: isolation of a microorganism in two or more sets of blood cultures.

^b Positive pleural fluid culture: isolation of a microorganism in pleural fluid culture.

^c Positive sputum culture: isolation of a microorganism in sputum culture.

Microbiology (non-Mayo Clinic patients)

The most common microorganisms detected in pleural fluids by 16S rRNA gene PCR/sequencing were F. nucleatum group (49), Streptococcus anginosus group (42), Prevotella species (42), Parvimonas micra (30), S. intermedius (28), and S. mitis group (15) (Table 4). There were 94 monomicrobial and 74 polymicrobial detections.

TABLE 4.

Microbiology by 16S ribosomal RNA gene PCR/sequencing of pleural space infections of non-Mayo Clinic Laboratories subjects

Microorganisms	n
Fusobacterium nucleatum group	49
Streptococcus anginosus group	42
Prevotella species	42
Parvimonas micra	30
Streptococcus intermedius	28
Streptococcus mitis group	15
Dialister species	12
Enterobacterales	11
Streptococcus pneumoniae	9
Eubacterium species	9
Campylobacter species	9
Staphylococcus aureus	7
Tanerella forsythia	7
Gemella species	6
Filifactor alocis	6
Enterococcus species	5
Schaalia species	5
Veillonella species	4
Capnocytophaga species	4
Pseudomonas aeruginosa	3
Peptoniphilus species	3
Abiotrophia defectiva	3
Alloprevotella species	3
Bacteroidales	3
Eikenella corrodens	3
Granulicatella species	3
Mogibacterium timidum	3
Neisseria species	3
Rothia species	3
Treponema species	3
Corynebacterium species	2
Lancefieldella rimae	2
Nocardia species	2
Acticaseibacillus paracase	2
Streptococcus pyogenes	2
Fusobacterium necrophorum	2
Bulleida extructa	2
Anaeroglobus geminatus	2
Aggregatibacter species	2
Metamycoplasma salivarum	2
Stenotrophomonas maltophilia	2
Solobacterium moorei	2
Haemophilus influenzae	2
Haemophilus parainlfuenzae	2
Streptococcus dysgalactiae	2
Burkholderia cepacia	1
Staphylococcus epidermidis	1
Pasterella multocida	1
Porphyromonas species	1
Kingella kingae	1
Kluyvera species	1
Moraxella catarrhalis	1
Fusobacterium gonidiaformans	1
Moryella indoliges	1
Peptostreptococcus stomatis	1
Pyramidobacter psicolins	1
Paeniclostridium ghonii	1
Synergistales	1
Trueperella bernardiae	1
Actinomyces gerencseriae	1
Clostridium perfringes	1

DISCUSSION

This real-world study provides information about the use of 16S rRNA gene PCR/sequencing of pleural fluids in clinical practice. The most common detection was F. nucleatum group and/or S. intermedius, with or without other normal microbiota, for which we propose the term e-FuSion (effusion with Fusobacterium nucleatum group, Streptococcus intermedius, and other oral normal microbiota), based on the frequent presence of these microorganisms in community-acquired pleural space infections.

In a Norwegian study, F. nucleatum group and S. intermedius were suggested as key pathogens for the establishment of community-acquired pleural infection (10). Here, 77 patients with community-acquired pleural space infections were included, among whom 63 (82%) were infected with S. intermedius and/or F. nucleatum (10). In another retrospective study including 64 patients with pleural space infections, S. intermedius and/or F. nucleatum were also detected (11). Together with our findings, a key role for S. intermedius and/or F. nucleatum in community-acquired pleural space infections is suggested.

Other studies also support a role of F. nucleatum group and S. intermedius in pleural space infections. Gimenez-Miranda et al. analyzed the pleural fluids of 47 patients; 20 were clinically infected, 7 had positive pleural fluid cultures and 14 had positive targeted metagenomic sequencing results. Streptococcus and Staphylococcus species were the most common organisms identified, with S. intermedius/constellatus identified in five subjects. Polymicrobial infections were found in 6 of 20 infected subjects, with anaerobes being the most common microorganisms identified in these cases (9). Shiraishi and collaborators described 19 cases of pleural empyema or parapneumonic effusion analyzed by 16S rRNA gene PCR followed by NGS and reported detection of Streptococcus species (n ꞊ 6, including S. intermedius), Fusobacterium species (n ꞊ 5, including F. nucleatum), Porphyromonas species (n ꞊ 5), and Prevotella species (n ꞊ 4) (15). In a community-acquired pleural space infection cohort in Canada, “Streptococcus milleri group” was the most common pathogen isolated in culture (16). S. intermedius, S. constellatus, and S. mitis combined were also the most common organism types in the UK MIST1 trial cohort (17). In a study performed in Sweden of patients with pneumonia and parapneumonic effusions, viridans group streptococci and anaerobic bacteria were the most common microorganisms identified using 16S rRNA gene PCR/Sanger sequencing (18).

Results of this study show that 16S rRNA gene PCR/sequencing (including NGS) increases the microbiological diagnosis of pleural space infection compared with culture. Our prior real-world study using this approach to test a variety of clinical specimens showed improved microbial detection compared to culture, especially in patients who had received antibiotics before sampling (14). Specifically in pleural infections, a few studies have shown improvement in sensitivity with sequencing-based assays as compared to culture (15, 19, 20). In the study by Shiraishi et al., only 8 (42%) pleural effusions were culture positive, whereas the combination of 16S rRNA gene-specific PCR with NGS identified 14 anaerobes and 7 aerobes/facultative anaerobes (15). Insa et al. reported that in infected samples, 16S rRNA gene PCR was positive in 82%, while conventional culture was positive in 55% (19). Lampejo et al. observed that 28/90 (31%) of culture-negative pleural fluids tested by 16S rRNA gene PCR yielded a positive result; 25 (28 %) had positive 16S rRNA PCR for a “causative” microorganism, with an impact on clinical management in 76% of cases (20). An important limitation of Sanger sequencing is its poor performance in polymicrobial samples, which is especially important for pleural fluids because of the frequency of polymicrobial infections. Using NGS allows detection of polymicrobial infections (21, 22). Given the findings reported here, if either S. intermedius or F. nucleatum group is detected by Sanger sequencing from pleural fluid, either NGS should be reflexively performed or it should be assumed that other microorganisms in the F. nucleatum group, S. intermedius and other oral normal microbiota may be present.

The organisms involved in the entity described are typically oral microbiota; how they seed the pleural space is unclear. Interestingly, the microbiology is similar to that of brain abscess (23). Here, radiological consolidation was observed in 58% of cases, suggesting spread from the lung to the pleural cavity, at least in some cases. Given that an etiologic diagnosis of pneumonia is established in less than half of cases (24), and that oral microbiota such as F. nucleatum group and S. intermedius would not be recovered or reported in routine sputum cultures, findings presented raise the question as to the role of these bacteria as etiologic agents of pneumonia. A hematogenous route has also been suggested (11), from a deep-sited odontogenic source. The initial antibiotic regimen for pleural space infection is usually empiric, with selection based on patient condition, antibiotic resistance patterns, and whether the infection is community or hospital acquired (1). The exact definition of the causative agents of pleural space infection may allow early tailoring of antibiotic therapy.

This study has several limitations. First, it was a retrospective study, with bias inherent to this study design. Second, it was a single-center study, although given that Mayo Clinic Laboratory specimens were analyzed, patients at several institutions were included. Third, among the samples clinically evaluated, there was just one pediatric patient, so the results are not representative of the pleural fluid microbiology in pediatric populations. Finally, antibiotic susceptibility was not assessed. In future studies, a definition of the ideal treatment for pleural space infections caused by F. nucleatum group, S. intermedius, and other oral normal microbiota will be needed.

In conclusion, consistent with previous results, this study shows the benefit of adding 16S rRNA gene PCR/sequencing to conventional cultures in defining the microbiology of pleural space infection. Molecular diagnostics should become standard for the diagnosis of pleural space infection. With this approach, infection caused by F. nucleatum group, S. intermedius, and other oral normal microbiota was found to be the most common type of pleural space infection.

DATA AVAILABILITY

Data are shown in Table S1 in the supplemental material.

ETHICS APPROVAL

The Mayo Clinic Institutional Review Board approved the study.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jcm.00694-24.

Table S1. jcm.00694-24-s0001.docx.

Microbiologic findings from pleural fluid 16S ribosomal RNA gene PCR/sequencing and conventional cultures for Mayo Clinic subjects.

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