RESEARCH ARTICLE

Characterization of Lung and Oral Microbiomes in Lung Cancer

Patients Using Culturomics and 16S rRNA Gene Sequencing
Yifan Sun,a Yuejiao Liu,a Jianjie Li,b Yafang Tan,a Tongtong An,b Minglei Zhuo,b Zhiyuan Pan,a Menglei Ma,b Bo Jia,b
Hongwei Zhang,b Ziping Wang,b Ruifu Yang,a Yujing Bia

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
a

b Department of Thoracic Oncology, Peking University Cancer Hospital, Beijing, China

Yifan Sun and Yuejiao Liu contributed equally to this work. Author order was determined by the corresponding author after negotiation.

ABSTRACT Recently, microbiota dysbiosis in lung cancer has attracted immense atten-
tion. Studies on lung microbes are mostly based on sequencing, which has left the poten-
tially functional bacteria with extremely low abundance uncovered. In this study, we char-
acterized and compared the lung and oral cavity microbiotas using culturomics and 16S
rRNA gene sequencing. Of the 198 bacteria identified at the species level from bronchoal-
veolar lavage fluid (BALF) samples, Firmicutes was predominant (39.90%). Twenty bacterial
species isolated from BALF samples were present in at least half of the patients and were
also highly abundant in oral samples. Of all isolated strains, Streptococcus and Veillonella
were highly dominant. The abundance of Prevotella and Veillonella decreased from the
oral cavity to the lung, whereas that of Pseudomonas increased. Linear discriminant analy-
sis effect size demonstrated that Prevotella was more abundant in the healthy samples
than in the cancerous ones, which is in accordance with the isolation of Prevotella oralis
only from the healthy group using culturomics. Moreover, Gemella sanguinis and
Streptococcus intermedius were isolated only from the non-small-cell lung cancer (NSCLC)
group, and 16S rRNA gene sequencing showed that they were higher in the NSCLC than
in the small-cell lung cancer group. Furthermore, while Bacillus and Castellaniella were
enriched in lung adenocarcinoma, Brucella was enriched in lung squamous cell carcinoma.
Overall, alterations were observed in the microbial community of patients with lung can-
cer, whose diversity might be site and pathology dependent. Using culturomics and 16S
rRNA gene amplicon sequencing, this study has provided insights into pulmonary and
oral microbiota alterations in patients with lung cancer.
IMPORTANCE The relationship between lung microbiota and cancer has been explored
based on DNA sequencing; however, culture-dependent approaches are indispensable Editor Diyan Li, Chengdu University
for further studies on the lung microbiota. In this study, we applied a comprehensive Ad Hoc Peer Reviewers Raunak Das, Vellore
Institute of Technology University; Hassan
approach combining culturomics and 16S rRNA gene amplicon sequencing to detect Zafar, Stredoevropsky technologicky institut;
members of the microbiotas in saliva and BALF samples from patients with unilateral lo- Pierre Le Bars, University of Nantes;
bar masses. We found alterations in the microbial community of patients with lung can- Pablo Millones Gómez, Universidad Señor
de Sipán
cer, whose diversity might be site and pathology dependent. These features may be
Copyright © 2023 Sun et al. This is an open-
potential bacterial biomarkers and new targets for lung cancer diagnosis and treatment. access article distributed under the terms of
In addition, a lung and oral microbial biobank from lung cancer patients was estab- the Creative Commons Attribution 4.0
International license.
lished, which represents a useful resource for studies of host-microbe interactions.
Address correspondence to Ziping Wang,
KEYWORDS microbiota, lung cancer, BALF, oral bacteria, culturomics, 16S rRNA gene, wangzp2007@126.com, Ruifu Yang,
13801034560@163.com, or Yujing Bi,
DNA sequencing, lung infection, oral microbiota byj7801@sina.com.
The authors declare no conflict of interest.
Received 19 January 2023

L ung cancer, the most common type of cancer worldwide, is closely associated with
chronic inflammation (1). Inflammation caused by microbial infections might contribute
to cancer development and progression (2). Polymorphic microbiomes have recently been
Accepted 3 April 2023
Published 24 April 2023

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Lung and Oral Microbiomes of Lung Cancer Patients Microbiology Spectrum

added as one of the four new “hallmarks” of cancer (3). Increasing evidence shows that the
lung microbiome is involved in cancer pathogenesis.
Healthy lungs, conventionally considered sterile, are now known to harbor a diverse
microbiota (4). To date, several culture-independent analyses have reported the association
between microbial population diversity and lung cancer (5, 6). Lung tissue samples from
lung cancer patients demonstrated an increase in a diversity and the phylum Bacteroidetes
compared with lung tissue samples from emphysema patients (7). A study found that the
genus Streptococcus was more abundant in the bronchoalveolar lavage fluid (BALF) sam-
ples from patients with lung cancer than in those of healthy controls (8). Few studies have
shown the association between lung bacteria and the histological subtypes of lung can-
cers; the genera Veillonella, Megasphaera, Enterobacter, Morganella, and Klebsiella were sig-
nificantly higher in lung adenocarcinoma (ADC) than in lung squamous cell carcinoma
(SCC) (9, 10). As the oral cavity is the entry point to the respiratory tract, its microbiome
may contribute to lung cancer (11). Tsay et al. reported that lower airway dysbiosis induced
by microaspiration of oral commensals promotes an interleukin-17-driven inflammatory
phenotype, which exacerbates lung tumorigenesis (12). Sphingomonas and Blastomonas
were shown to be more abundant in the oral microbiomes of lung cancer patients (13).
However, the possible variations in the oral and lung microbiotas of lung cancer patients
and the differences in the microbial diversity of their saliva and BALF samples remain
unclear.
While numerous DNA sequencing-based investigations have explored the relation-
ship between the lung microbiota and cancer, they have several inherent drawbacks,
such as depth bias and a high detection threshold (14). Therefore, culture-dependent
approaches are indispensable for further studies of the lung microbiota. Culturomics,
in which multiple culture conditions are combined for rapid identification, provides
new perspectives on host-bacterium relationships (15). However, the use of culturo-
mics for the culture and identification of bacteria in BALF has rarely been reported.
In this study, we applied a comprehensive approach combining culturomics and
16S rRNA gene amplicon sequencing to the saliva and BALF samples from 25 patients
with unilateral lobar masses. To the best of our knowledge, this is the first study report-
ing the bacterial diversity and richness in the oral and BALF microbiotas of patients
with lung cancer, using culturomics and 16S rRNA gene sequencing.

RESULTS
Clinical information. We recruited 25 patients with unilateral lobar masses from
Beijing Peking University Cancer Hospital between January 2021 and May 2022, of
which 23 were newly diagnosed with lung cancer via histological confirmation but 2
were not. There were 16 non-small-cell lung cancer (NSCLC) patients, including 8 with
ADC, 7 with SCC, and one with nonspecified NSCLC, and 7 small-cell lung cancer
(SCLC) patients. None had previously received any anticancer therapy, radiation ther-
apy, or antibiotic treatment. Of the 23 patients with lung cancer, 14 were men and 9
were women. Furthermore, 18 were smokers. There were 17 and 6 patients with and
without distant metastasis, respectively (Table 1).
Characteristics of bacteria isolated from the lung and oral cavity via culturo-
mics. Figure 1 shows the culturomics workflow. Briefly, total 45 samples were collected
from 15 lung cancer patients, and four culture conditions were tested for each sample. We
obtained 12,379 colonies from the BALF samples (from both cancerous [C] and healthy [H]
sites), and 198 bacteria were identified at the species level using matrix-assisted laser de-
sorption ionization–time of flight (MALDI-TOF) or 16S rRNA gene sequencing. The identi-
fied species belonged to six phyla, including Firmicutes (39.90%), Proteobacteria (27.78%),
Actinobacteria (19.19%), Bacteroidetes (9.60%), Fusobacteria (2.02%), and Synergistetes
(1.52%) (Fig. 2A and B). Comparison with the previously established repertoire of microor-
ganisms isolated from the human gut, urine, vagina, and oral/respiratory tract revealed
that approximately 1/4 of the species isolated in this study had been isolated previously
from these four sites (47/198 [23.7%]) (Fig. 2C).
We obtained 5,671 colonies from the oral samples and identified 156 bacterial species

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TABLE 1 Clinical characteristics of patients

Value for patient type

Variable NSCLC SCLC Noncancer

No. 16 7 2
Age [mean (SD)] 65 (7.0) 67 (12.8) 69 (8.2)

Gender [no. (%)]

Male 9 (56) 5 (72) 2
Female 7 (44) 2 (14) 0

Smoking [no. (%)]

Current or former smoker 12 (75) 6 (86) 1
Nonsmoker 6 (25) 1 (14) 1

Pathological diagnosis [no. (%)]

Adenocarcinoma 8 (50)
Squamous cell carcinoma 7 (44)
Unidentified 1 (6)

Distant metastasis [no. (%)]a

M0 4 (25) 2 (29)
M1 12 (75) 5 (71)
aM0, without metastasis; M1, with metastasis.

(see Fig. S1 in the supplemental material). At the phylum level, the bacterial diversity of the
oral sample was similar to that of BALF, consisting predominantly of Firmicutes and
Proteobacteria (.66%). In addition to the previously known bacteria, we isolated 15 poten-
tial new species (Table S1).
Comparison of microbiotas in lung and oral cavity. We compared the microbiota
composition between the C and H lung sites and the oral cavity (O) using 16S rRNA gene
amplicon sequencing. There was a noticeable difference in the microbiota composition
between the lung and oral cavity. In the lung samples, we observed a moderate difference
between the C and H samples at the phylum level (Fig. 3A). The three dominant phyla
were Proteobacteria, Firmicutes, and Bacteroidetes. At the genus level, Pseudomonas
(Proteobacteria), Streptococcus (Firmicutes), Veillonella (Firmicutes), and Prevotella_7 (Bacteroi-
detes) were the most common ones in the BALF samples. Contrastingly, Prevotella_7
(Bacteroidetes), Neisseria (Proteobacteria), Streptococcus (Firmicutes), Veillonella (Firmicutes),
and Haemophilus (Proteobacteria) were the most common ones in the oral samples (Fig.
S2A). We did not observe any significant difference in the richness or diversity of the micro-
bial community (a diversity) between the BALF samples from C and H sites, as measured
using the Shannon index (P = 0.527) and Chao1 index (P = 0.428). Moreover, there was no
difference in the overall microbiota (b diversity) between the C and H groups, as measured
using the Bray-Curtis distances (P = 0.39) (Fig. S3). Nevertheless, the oral sample was signifi-
cantly different from both the C and H lung samples in a and b diversity (Fig. 3B and C).
Metastats analysis at the genus level further revealed unique anatomy-related microbial

FIG 1 Summary of culturomics methods and workflow.

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FIG 2 Bacteria identified from the BALF samples. (A) Phylogenetic tree and (B) proportion of 198 bacterial species isolated from the BALF samples listed
according to phylum. (C) UpSet plot showing the shared cultured species among human oral/respiratory, gut, urine, and vagina samples.

features, such as higher abundance of Prevotella in oral samples (Fig. 3D). Pseudomonas
was the only genus concentrated in BALF samples (Fig. 3D; Fig. S2B).
Furthermore, we compared the bacteria isolated via culturomics among different
groups. Unlike the sequencing data, there was no obvious difference in the bacterial
proportion at the phylum level among C, H, and O samples, with Firmicutes being dom-
inant in all three sites (Fig. 3E). More than half of the species (89) were isolated in all
three sites, and 34, 32, and 39 unique species were isolated from the C, H, and O
groups, respectively (Fig. 3F). In addition, we analyzed the prevalence of bacteria in the
lungs and oral cavity. Based on the culturomics results, we defined the strains isolated
from over 50% of the patients as prevalent strains. Twenty prevalent strains were iden-
tified from the BALF samples, which belonged to 12 genera (Table 2). Streptococcus
was the major genus, and Streptococcus oralis, Veillonella atypica, Parvimonas micra,
and Actinomyces odontolyticus were found in almost all BALF samples. These 20 species
were also cultured at a high frequency from oral samples, which indicated that the pul-
monary microbiota might originate from the oral cavity.
Differences in the microbiota composition between cancerous and healthy
lungs. To compare the relative contributions of different taxa, we used linear discrimi-
nant analysis (LDA) effect size (LEfSe) to detect taxa with differential abundances
between the two groups We identified 14 different taxa at various levels with signifi-
cantly different abundances between the two groups, of which four were differentially
abundant at the genus level. Prevotella and Prevotella 7 were more abundant in the H
group, whereas Carnobacterium and Brucella were more abundant in the C group
(Fig. 4A).
Moreover, we compared the differences between the H and C groups of bacteria
obtained via culturomics. We drew a heat map of the proportion of each bacterium in
the total sample. Streptococcus oralis, Veillonella atypica, and Parvimonas micra were
cultured at a high frequency without significant differences. A significantly higher fre-
quency of Prevotella oralis was found in the H group (P = 0.019) (Fig. 4B), which agreed
with the sequencing results.

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FIG 3 Variations in microbial composition in different anatomical sites. (A) Taxonomic composition at the phylum level in BALF and oral samples based on 16S
rRNA gene amplicon sequencing (top 30). (B) Shannon diversity index of BALF and oral samples. P values were calculated using the Wilcoxon test. *, P , 0.05. (C)
PCoA of samples from three different anatomical site in patients with lung cancer. ANOSIM was performed to statistically evaluate significant difference. (D)
Notched box plots illustrating the differences of the significantly 2 genus relative abundances in three different anatomy sites. *, P , 0.05. (E) Proportions of
bacterial species isolated from C, H, and O samples listed according to phylum. (F) Venn diagram of the culturable bacterial species from the three anatomical
sites. C, H, and O, samples from the cancerous site and contralateral healthy controls from lungs and the oral site, respectively, of patients with lung cancer.

Different pathological strains reshape different pulmonary microbiomes. We

examined the differences in the lung microbiotas of NSCLC and SCLC groups with compa-
rable basic information, including age, sex, smoking status, and distant metastasis (Table
S2). Bacteria with an abundance of .4% were considered dominant. Pseudomonas (32%),
Streptococcus (5%), and Bacillus (4%) were the dominant genera in the 32 NSCLC lung sam-
ples, while in SCLC samples, the dominant genera were Pseudomonas (31%), Veillonella
(7%), and Prevotella 7 (6%) (Fig. 5A). The a diversity of NSCLC was not significantly different
from that of SCLC, as measured using the Shannon index (P = 0.931) and Chao1 index
(P = 0.72), or b diversity, as measured using the Bray-Curtis distances (P = 0.489) (Fig. S4A
and B). The culturomics study revealed that 77 and 33 species were isolated only from
NSCLC and SCLC lung samples, respectively (Fig. 5B). Only bacterial species isolated from
at least half of the patients in the group were compared. Later, we analyzed the bacteria
isolated from only one group with an isolation rate of $50%. Gemella sanguinis, Pseudo-
ramibacter alactolyticus, Bifidobacterium dentium, and Streptococcus intermedius were the
four species isolated with a relatively high frequency only from the NSCLC group.
Prevotella pallens was the only species isolated at a relatively high frequency from the SCLC
lung samples. Subsequently, we compared the relative abundances of these five bacteria
at the genus level using 16S rRNA gene amplicon sequencing. Although there was no sig-
nificant difference between the two groups, their abundance tended to be higher in the
site from which they were isolated (Fig. 5C).
In the subtype analysis of patients with NSCLC, Pseudomonas (26%) and Bacillus (8%)
were dominant in the 16 ADC samples. In contrast, in the 14 SCC samples, the dominant
genera were Pseudomonas (38%) and Streptococcus (8%) (Fig. 5D). Shannon index (P = 0.27)
and Chao1 index (P = 0.402) were not significantly different between the ADC and SCC
groups. However, b diversity showed that the microbiota constitution of the ADC lung sam-
ples was significantly different from that of the SCC samples (P = 0.021) (Fig. S4C and D). In
the LEfSe analysis of ADC and SCC, 11 taxa displayed contrasting correlations between the
NSCLC subtypes. A differential abundance analysis at the genus level between ADC and SCC

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TABLE 2 Summary of the proportion of the top 20 bacteria in BALF samples and the related
proportions in the oral cavity
Frequency of detection (%) in
sample typea

Genus Species C H O
Streptococcus Streptococcus salivarius 60.00 60.00 73.33
Streptococcus pseudopneumoniae 53.33 40.00 33.33
Streptococcus parasanguinis 93.33 86.67 100.00
Streptococcus oralis 93.33 93.33 100.00
Streptococcus mitis 93.33 86.67 100.00
Streptococcus gordonii 53.33 53.33 80.00
Streptococcus constellatus 66.67 73.33 93.33
Streptococcus anginosus 66.67 66.67 100.00

Veillonella Veillonella parvula 60.00 60.00 73.33

Veillonella atypica 66.67 80.00 66.67

Solobacterium Solobacterium moorei 66.67 60.00 73.33

Slackia Slackia exigua 60.00 73.33 60.00
Parvimonas Parvimonas micra 80.00 80.00 93.33
Mogibacterium Mogibacterium diversum 53.33 46.67 53.33
Granulicatella Granulicatella adiacens 66.67 53.33 46.67
Gemella Gemella morbillorum 66.67 53.33 66.67
Fusobacterium Fusobacterium nucleatum 40.00 66.67 80.00
Dialister Dialister invisus 73.33 53.33 73.33
Anaeroglobus Anaeroglobus geminatus 53.33 60.00 73.33
Actinomyces Actinomyces odontolyticus 80.00 80.00 66.67
aC,
H, and O, samples from the cancerous lung sites and contralateral healthy control and the oral site,
respectively, of patients with lung cancer.

showed enrichment of Bacillus and Castellaniella in patients with ADC, whereas those with
SCC had a higher abundance of Brucella (Fig. 5E).

DISCUSSION
The relationship between the microbiota and cancer is being extensively investigated
(16). Accumulating evidence indicates that the gut microbiota contributes to carcinogenesis
and response to immunotherapy (16, 17). While many studies have focused on analyzing
the influence of the gut microbiota, its composition differs substantially from that of the
lung microbiota (18, 19). Although microaspiration commonly occurs in healthy subjects, it
is more frequent in those with chronic inflammatory airway diseases (20, 21). The entry of
more oral microbes into the lungs is associated with the increased release of lung proinflam-
matory cytokines and a proinflammatory phenotype characterized by elevated Th-17 lym-
phocytes (22, 23). Lung microbiota dysbiosis might modulate the risk of malignancy at mul-
tiple levels, including chronic inflammation and activation of oncogenes (24, 25). Although
the lung microbiome has been implicated in lung cancer in several specific ways, its exact
role in carcinogenesis has not yet been elucidated. One of the possible reasons for this is
that current studies are mostly based on sequencing levels, which results in a lack of strain
materials for further study. Here, we utilized both culturomics and 16S rRNA gene amplicon
sequencing to evaluate and compare the structure and diversity characteristics of oral and
pulmonary microbiotas associated with lung cancer.
Culturomics can identify bacteria at strain level, and successfully isolating living bacteria
is crucial for carrying out subsequent experimental work (14, 26). A previous study summa-
rizing a list of microbes isolated from the human lower respiratory tract showed that the
lung microbiome was dominated by the phyla Pseudomonadota, Firmicutes, Bacteroidota,
and Actinomycetota, which was consistent with our study (27). Here, we cultured 198 iden-
tified bacterial species from human BALF and 156 from oral samples from lung cancer
patients, including 15 potentially new taxa. A separate article describing the novel bacteria,
new.4 and new.10, belonging to a novel genus, which we named “Curtanaerobium” (28).
The present study enabled us to expand the human respiratory and oral repertoire. We

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FIG 4 Differentially abundant taxonomy between cancer and paired healthy lung. (A) LEfSe was used to identify the bacterial microbiota that significantly
differed between the cancerous and healthy lungs. Only taxa meeting a significant LDA threshold value of .3.5 and having a P value of ,0.05 are shown.
(B) Heat map analysis of lung microbiotas at C and H sites of patients with lung cancer based on culturomics (n = 15). Each row represents an individual
species with the higher isolated rate in the two groups. Top 25 bacterial species were recovered from either group. Transition from blue to red represents
the frequency of culturomics recovery from 0 to 1. Statistical comparisons were made using Fisher’s exact test with FDR correction. *, P , 0.05.

also found 20 prevalent strains in both BALF and oral samples from patients with lung can-
cer, which indicates that the pulmonary microbiota might originate from the oral cavity.
Culturomics might reduce the number of these unclassed or no-rank operational taxonomic
units (OTUs) by increasing the number of pure-cultured microbial species. In this study,
Pseudoramibacter alactolyticus was recovered with a relatively high frequency only from the
NSCLC group but was not detected in 16S rRNA gene amplicon sequencing.
Parvimonas micra is an opportunistic pathogen that is frequently associated with several
human infections and also causes purulent infections in multiple organs (29, 30). P. micra is
highly abundant in patients with colorectal cancer and promotes colorectal tumorigenesis
by inducing colonocyte proliferation and altering the Th17 immune response (31, 32). Our
previous results showed that P. micra promoted colorectal cancer progression by upregulat-
ing miR-218-5p expression and ultimately activating the Ras/ERK/c-Fos signaling pathway
(33). However, its correlation with lung cancer has not yet been reported. In this study, P.
micra was cultured at a high frequency in patients with lung cancer, which provides valuable
information for further studies.
We analyzed the microbiota composition at the genus level via taxonomic analysis,
and the results showed that the representative flora differed based on the sampling site.
We found that Streptococcus, Veillonella, and Prevotella were enriched in the oral samples,
whereas Pseudomonas was enriched in the BALF samples. Streptococcus and Veillonella
which were considered oral commensals were reported to increase in lower airways of
lung cancer patients (34). Pseudomonas species, commonly found in the respiratory tract,
are involved in the pathogenesis of lung diseases, such as chronic obstructive pulmonary
disease and cystic fibrosis (35, 36). A study demonstrated that transplanted lungs are sus-
ceptible to the growth of multiple Pseudomonas species (37). Pseudomonas aeruginosa
possesses numerous virulence factors that attack the respiratory epithelial cells, such as
the type III secretion system (38). P. aeruginosa possesses pili and flagella that are neces-
sary for motility and respiratory infection, as they enable attachment to the respiratory epi-
thelium via respiratory mucins and the glycolipid asialo-GM1 (39). A study reported that P.
aeruginosa was significantly more abundant in NSCLC patients with brain metastasis than
in those without metastasis (40). Likewise, most of the patients in our study had distant

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FIG 5 Characterization of lung microbiotas in different lung cancer subtypes. (A) Taxonomic composition at the genus level in NSCLC and SCLC lung samples.
(B) Venn diagram of the culturable bacterial species isolated only from NSCLC and only from SCLC lung samples. (C) Differences in the relative abundance of
the genera corresponding to the species isolated from NSCLC and SCLC samples. Differential microbial taxa were identified using a paired t test, and the
P values were adjusted for multiple comparison using the FDR. (D) Taxonomic composition at genus level in ADC and SCC lung samples. (E) LEfSe was used to
identify the bacterial microbiotas that significantly differed between NSCLC and SCLC. Only taxa meeting a significant LDA threshold value of .4 and having a
P value of ,0.05 are shown.

metastasis, and Pseudomonas was the most prevalent genus in the BALF samples. These
findings, including ours, suggest that Pseudomonas frequently colonizes the respiratory
tracts of patients with lung cancer and might promote distant metastasis.
Based on 16S rRNA gene sequencing, we found that Brucella melitensis was more
enriched in the cancerous lung than in the healthy lung. The genus Brucella includes
several significant human and veterinary pathogens, which predominantly maintain an
intracellular lifestyle in their mammalian hosts. These pathogens exhibit numerous
pathogen-associated molecular patterns that can be recognized by innate receptors in
airway epithelial cells (41). Their type IV secretion system secretes effector molecules
into the host cell cytoplasm, which direct the intracellular trafficking of the brucellae
and modulate host immune responses (42). Thus, we speculated that Brucella melitensis
is a possible lung cancer biomarker.
Gemella sanguinis, Pseudoramibacter alactolyticus, and Streptococcus intermedius are
involved in human inflammatory diseases, such as endocarditis (43), periodontal infections
(44), and brain abscesses (45). Germ-free mice or mice treated with antibiotics had signifi-
cantly lower incidence of lung cancer than specific-pathogen-free mice (46, 47). Our results
showed that these three species were isolated only from the lung microbiotas of patients
with NSCLC and not from those with SCLC. This observation was consistent with the results
of 16S rRNA gene amplicon sequencing, which indicated that specific bacteria might medi-
ate the development of NSCLC by inducing chronic inflammation.
When bacteria were categorized based on the lung cancer subtypes, Bacillus and
Castellaniella were significantly enriched in the BALF samples from patients with ADC,
whereas Brucella was enriched in the SCC group. Another study reported the enrich-
ment of Acinetobacter in BALF samples from patients with ADC, whereas Bacillus and
Brucella showed no significant differences between the ADC and SCC groups (10).
These conflicting associations of microbiomes with different pathological subtypes
could be attributed to the significant variations across individuals and sampling
methods.

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The lung microbiome is similar to other microbiomes in other parts of the human body,
and there are significant individual differences. Although we screened some differential
microorganisms via culture or sequencing, the sample size was small, and more clinical
samples are needed for verification. The resolution of 16S rRNA gene sequencing is limited,
since only bacterial identification at the genus level was relatively accurate, limiting our
comparison between the two methods to genus information and ignoring the species in-
formation from the culturomics method. Metagenomic sequencing combined with cul-
turomics should be preferable for the parallel comparison at the species level. Moreover,
the causal relationship between the screened microorganisms and the occurrence and de-
velopment of lung cancer remains uncertain, and follow-up studies are also required.
In conclusion, we examined the pneumonic and oral microbiotas of lung cancer
patients using culturomics and 16S rRNA gene sequencing. We found alterations in the
microbial community of patients with lung cancer, whose diversity might be site and
pathology dependent. Using culturomics, we found that Streptococcus and Veillonella
were highly dominant in both pneumonic and oral samples from patients with lung
cancer, which suggests the possible deleterious effects of airway microbial dysbiosis
originating from the oral cavity. We showed that Prevotella oralis was isolated only
from the H group and that Gemella sanguinis was isolated only from the NSCLC group,
which was consistent with the findings of 16S rRNA gene sequencing. This study pro-
vides basic data on the microbiota diversity in pneumonic and oral samples from
patients with lung cancer. These organisms might serve as potential bacterial bio-
markers and new targets for lung cancer diagnosis and treatment, and the causative
relationships can be explored using the isolated strains.

MATERIALS AND METHODS

Patient recruitment and sample collection. This study was approved by the Institutional Review
Board of Peking University School of Oncology, China, and informed consent was obtained from all par-
ticipants. The BALF samples were collected as previously described (48). The saliva samples were col-
lected before the patients underwent bronchoscopy examination. All the participants were instructed to
not eat and drink for 1 h prior to saliva sample collection. Twenty-five patients with unilateral lobar
masses who consented to undergo bronchoscopic examination at Peking University Cancer Hospital
were enrolled. All patients underwent transbronchoendoscopy to avoid contaminating the upper respi-
ratory tract or oral microbiota, and paired BALF samples (one each from the cancerous site [C] and the
contralateral healthy lung [H]) were collected before the operation. None of the participants had
recently suffered from oral disease. Before the bronchoscopy, oral samples from 21 patients were col-
lected. The sample was divided into two parts: one part (including 45 samples from 15 lung cancer
patients) was used for culturing bacteria, and the other part (all of the 71 samples) was used for 16S
rRNA gene amplicon sequencing. Fresh samples were collected in a sterile tube, placed on ice, and
transported to the laboratory within 1 h. Culturomics was performed immediately after sample collec-
tion in the laboratory, and the aliquots were stored at 280°C before high-throughput sequencing.
Culturomics. (i) The process of culturomics. Culturomics is a high-throughput method that multi-
plies culture conditions to detect high bacterial diversity and pure bacterial cultures. This analysis
involved preculturing under different conditions: aerobically with 5% sheep blood, aerobically with 5%
rumen fluid, anaerobically with 5% sheep blood, and anaerobically with 5% rumen fluid. Sample dilu-
tion, strain isolation, and identification were performed as previously described (49). On days 1, 3, 6, 9,
15, and 30, the enriched culture samples were extracted from the bottles using a syringe. Subsequently,
100-m L doubling dilutions were spread onto Columbia agar supplemented with 5% sheep blood at 37°C
under aerobic conditions for 24 h or anaerobic conditions for 72 h. Colonies were picked and identified
using MALDI-TOF mass spectrometry (MS) systems (Autof MS1000). Colonies that could not be identified
using the MALDI-TOF MS database V1.1.12 (score , 9) were subjected to 16S rRNA gene sequencing
with the primers 27F (59-AGAGTTTGATCMTGGCTCAG-39) and 1492R (59-GGTTACCTTGTTACGACTT-39).
The sequencing results were analyzed using the NCBI BLAST algorithm for homologous sequence
searches with type strains. If the 16S rRNA gene sequence is ,98.65% similar to the closest type strain,
the isolate might be a new species (50).
(ii) Classification of cultivated species. We classified all isolates into four categories: oral/respira-
tory (this study and reference 27), gut (51), urine (52, 53), and vagina (54, 55). We also performed litera-
ture searches on PubMed to compare our results with the published data to confirm the classification.
DNA extraction, 16S rRNA gene amplification, and sequencing. DNA was extracted from each
sample using a HiPure bacterial DNA kit (Mageon, China) according to the manufacturer’s instructions.
The V3-V4 region of the 16S rRNA gene was amplified using specific primers (341F, 59-CCTAYGGGR
BGCASCAG-39; 806R, 59-GGACTACNNGGGTATCTAAT-39). Sequencing libraries were generated using a
TruSeq DNA PCR-free sample preparation kit (Illumina, USA) following the manufacturer’s recommenda-
tions, and index codes were added. The library quality was assessed with the Qubit 2.0 fluorometer

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Lung and Oral Microbiomes of Lung Cancer Patients Microbiology Spectrum

(Thermo Scientific) and Agilent Bioanalyzer 2100 system. The sequencing was performed with on an
Illumina NovaSeq 6000 sequencing platform (Illumina, San Diego, CA, USA), and 250-bp paired-end
reads were generated.
Sequence data analysis. Raw reads were filtered to remove adaptors, low-quality reads, and ambig-
uous bases. Clean data were extracted from the raw data using USEARCH 8.0. The OTUs were classified
based on 97% similarity after chimeric sequences were removed using UPARSE (version 7.0.1001 [http://
drive5.com/uparse/]), and the representative sequence from each OTU cluster was obtained. The a di-
versity was assessed using the nonparametric Shannon and Simpson indices; the b diversity was calcu-
lated using Bray-Curtis distances in QIIME and visualized by principal-coordinate analysis (PCoA). LEfSe
was used to detect taxa with differential abundance among groups. Metastats analysis was employed to
detect the differences in the microbiota composition at the genus level.
Statistical analyses. Statistical analyses were performed using the R software (v3.4.10) and SPSS
20.0. Independent t and chi-square tests were used to analyze the demographic and clinical data.
Fisher’s exact test with false-discovery-rate (FDR) correction was used to compare frequency of isolated
species between groups. Sample diversity metrics were assessed based on the nonparametric Shannon
index and Chao1 index. PCoA plots were generated to visualize the separation of samples based on pair-
wise distances, and analysis of similarity (ANOSIM) was performed to evaluate the statistically significant
difference in PCoA. P values of ,0.05 were considered statistically significant.
Ethics approval and consent to participate. All procedures performed in studies involving human
participants or animals were approved by the Institutional Review Board of the Peking University School
of Oncology (no. 2018KT89). All patients gave their written consent before inclusion in the study.
Data availability. All the data generated or analyzed in this study are included in this article (or its
supplemental material). The read sequences obtained from Illumina NovaSeq were submitted to the
NCBI Sequence Read Archive (SRA) under accession number PRJNA904049 (BioProject ID).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 0.6 MB.

ACKNOWLEDGMENTS
We gratefully acknowledge all of the participants and the medical staff who helped
collect the samples in this study.
We declare no competing interests.
This research was supported by the National Key Research and Development
Program of China (2021YFC2301000), and the National Natural Science Foundation of
China (31970863 and 81790632).
Y.B. designed research and project outline. R.Y. and Z.W. directed the research. Y.S., J.L.,
Y.L., T.A., M.Z., M.M., B.J., and H.W. performed isolation, deposition, and identification. Y.T.
and Z.P. performed genome analysis. Y.S. and Y.B. drafted the manuscript. All authors read
and approved the final manuscript.

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