Isolation of Clostridioides difficile from the atmospheric fine particulate matter

Clostridioides difficile is a spore−forming, obligate anaerobic bacillus commonly found in soil and the intestinal tracts of animals. It is a major cause of antibiotic−associated diarrhea (AAD) in humans. Our previous study suggested a possible environmental dissemination. To investigate whether atmospheric aerosols contribute to its spread, atmospheric aerosol samples were collected from ten geographic locations in East Asia, and the presence of C. difficile genomic sequences was assessed using 16S rRNA amplicon sequencing. Additionally, atmospheric PM2.5 samples were collected from two sites in Japan, and analyzed by 16S rRNA amplicon sequencing and selective culturing. C. difficile genomic sequences were detected in aerosols from 8 sampling sites, with relative abundances ranging from 0.03% to 9.88% of total prokaryotic reads. From PM2.5 samples, seven C. difficile strains were isolated, and classified into five genotypes. Three genotypes matched low−frequency clinical or environmental strains in Japan (0.1–0.6%), while two were previously unreported. Phylogenetic analysis showed that four genotypes were closely related to strains from North America, Europe, Africa, and Southeast Asia, respectively. These findings are consistent with the possibility that C. difficile may disperse globally via atmospheric aerosols. Successful colonization of C. difficile in the deposited environments likely depends on ecological adaptability.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-026-04888-x.

Highlights

Seven C. difficile strains were isolated from atmospheric aerosols in Japan.

Aerosol-derived strains showed close genetic ties to strains from other continents.

Aerosols may serve as a pathway for the global spread of C. difficile.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-026-04888-x.

Introduction

Clostridioides difficile is a Gram−positive, spore−forming, obligate anaerobic bacillus that inhabits not only the human gastrointestinal tract but also animals, soil and aquatic environments, which serve as reservoirs of strains capable of causing human infections. Humans acquire C. difficile through exposure to these reservoirs [1]. It is a major cause of antibiotic−associated diarrhea, known as C. difficile infection (CDI), and is recognized globally as a leading cause of healthcare−associated infections [2–4]. Although effective treatments for CDI are available, strains with reduced susceptibility to antimicrobial agents have emerged, highlighting the urgent need for the development of new therapeutic options [5, 6].

Molecular epidemiological studies have shown that C. difficile strains with identical genotypes can be found across geographically distant regions, even across oceans [7]. Human travel, as well as the global trade of food and animals, are considered key factors contributing to this wide distribution [8–10]. Migratory birds have also been reported as potential vectors of C. difficile [11]. For example, the hypervirulent ribotype 027 strain is believed to have spread from North America to Europe and Asia [12]. Transmission analyses suggest that this dispersal may be attributable to human carriage, as its spread corresponds with patterns of human mobility flow [13].

We previously conducted a molecular epidemiological study of C. difficile in Japan using polymerase chain reaction (PCR)−based open reading frame (ORF) typing (POT), a genotyping method with high genetic resolution [14]. Analysis of 982 clinical isolates collected from 14 medical institutions across the country revealed a remarkably diverse set of genotypes. While some strains were regionally restricted (indigenous strains), others were consistently isolated across Japan (widely distributed strains). The presence of indigenous strains likely reflects adaptation to the diverse environmental conditions across the country. However, the reason for the widespread distribution of identical genotypes nationwide remains unclear.

Notably, previous studies have reported the detection of various bacteria in atmospheric aerosols from around the world [15–22]. Among them, the genomes of Bacillus species—spore−forming bacteria commonly found in soil—have frequently been identified in aerosols [23]. Spores of Bacillus originating from desert regions are known to travel long distances through the atmosphere [24]. DNA from members of the genus Clostridium has also been detected in atmospheric aerosols [25–29]. Based on this background, we hypothesized that atmospheric aerosols may contribute to the environmental dissemination of C. difficile and sought to test this possibility.

Materials and methods

Sample collection and amplicon sequencing analysis of atmospheric aerosols

Between 2013 and 2018, atmospheric aerosols samples were collected using air pumps on the polycarbonate filters with a pore size of 0.2 µm at ten sampling sites across East Asia (Fig. 1). Sampling sites included Asian dust source regions such as the Gobi Desert (Züünkhangai, Mongolia, N = 22) and the Taklamakan Desert (Dunhuang, China, N = 13), as well as transport and deposition regions such as Lanzhou (China, N = 18), Beijing (China, N = 11), Yongin (South Korea, N = 43), Yonago (Japan, N = 88), Kanazawa (Japan, N = 61), and Tsukuba (Japan, N = 12). The altitude above sea level and the height above ground for all sampling sites are listed in Table 1. Aerosol sampling in Tsukuba at an altitude of 500 m was performed using a helicopter. In addition, aerial sampling was conducted over Suzu on the Noto Peninsula, Japan, at an altitude of 2,000 m using a research aircraft and helicopters (N = 33). In the Mt. Tateyama Mountain Range in Toyama Prefecture, Japan, dust particles preserved in snow layers were collected. In this region, snow accumulates annually from the ground, reaching a thickness of approximately 6–10 m during winter. The accumulated snow was sampled layer by layer in April, under the assumption that aerosol particles deposited during the winter season were entrapped within the snowpack. The detailed sampling procedure has been described previously [17] (N = 69). For amplicon sequencing analysis, DNA extraction and the sample processing procedures have been described in detail in previous studies [17, new 30, 31]. The 16S rRNA gene was amplified by PCR, and bacterial species were identified via next−generation sequencing (NGS) as described previously [17, 31].

Fig. 1.

Atmospheric aerosol sampling sites and detection frequency of C. difficile genomes based on amplicon sequencing analysis. Sampling locations for atmospheric aerosols are indicated by circles on the map. The relative detection frequency of C. difficile genomes, expressed as a percentage of total prokaryotic DNA reads, is shown for each site. Circle size is proportional to the detection frequency, with larger circles indicating higher values. White circles denote sites where C. difficile genomes were not detected. The land is colored in a gradient from green to brown to white according to elevation, with green representing lower altitudes. The sea is colored in blue according to depth, with lighter blues indicating shallower waters. The map was created based on the data from the Geospatial Information Authority of Japan

Table 1.

Amplicon sequencing analysis of atmospheric aerosols for spore-forming soil bacteria

Place	Collection date	Elevation (m)a	Height (m)b	Detection of C. difficilec	Amplicon sequencing analysisd
					Clostridia (%)	C. difficile (%)	Bacillus (%)
Gobi Desert, Mongolia	2014, 2018	1,500	2	3/22 (13.6%)	0.34	0.03	2.80
Taklamakan Desert, China	Mar-Sept 2015	1,200	20	1/13 (7.1%)	17.30	0.05	1.86
Lanzhou, China	Apr 2016	1,600	20	2/18 (11.1%)	1.28	0.06	0.55
Beijing, China	Mar-Apr 2015	50	20	1/11 (9.1%)	13.50	0.66	2.92
Yongin, South Korea	Mar-Jun 2015	130	20	2/43 (4.7%)	10.90	2.22	2.54
Yonago, Japan	Mar-Jun 2015	9	20	4/88 (4.5%)	9.89	0.17	2.66
Kanazawa, Japan	2017–2018	20	20	0/61 (0%)	8.44	0.00	1.12
Suzu, Japan	2014–2018	6	2,000	0/33 (0%)	9.18	0.00	2.93
Mt. Tateyama, Japan	Apr 2013	2,450	1e	2/69 (2.9%)	5.86	0.78	9.90
Tsukuba, Japan	Aug 2018	30	2–500	1/12 (8.3%)	9.88	9.88	12.76
Average	-	-	-	2/37 (5.4%)	8.66	1.39	4.00

^aAbove the sea level

^bSample collection height from the ground level

^cThe number of samples positive for C. difficile genomic reads in the amplicon sequencing analysis

^dThe ratio of Clostridia, C. difficile, or Bacillus genomic reads to the bacterial reads in the amplicon sequencing analysis. Average data of C. difficile-positive samples are shown. When C. difficile was not detected, average data of all the samples are shown

^eAerosol samples recovered from accumulated snow on Mt. Tateyama

Between 2020 and 2023, a total of 61 PM_2.5 particulate matter samples were collected using the cyclone method—48 from Yokohama and 13 from Fukuoka, Japan. The PM2.5 sampling method has been described previously [17, 32, 33]. PM2.5 was collected continuously over sampling periods of approximately one month (as shown in Table 2), and the material collected during each period was pooled and analyzed as a single composite sample. The shotgun metagenomics sequencing analysis of the prokaryotic species contained in the PM2.5 samples was performed as follows. DNA was extracted using the Quick−DNA Fecal/Soil Microbe DNA Miniprep Kit (Zymo Research, Irvine, CA). Library preparation was carried out using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina and the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs, NEB, Ipswich, MA). Libraries were quantified using the Agilent TapeStation 4200 system with High Sensitivity D1000 ScreenTape (Agilent Technologies, Santa Clara, CA, USA). Final library concentrations were measured using the KAPA Library Quantification Kit for Illumina sequencing platforms (Kapa Biosystems, Wilmington, MA, USA). Sequencing was performed on the Illumina NextSeq 1000 platform using NextSeq 1000/2000 P1 Reagents (300 cycles) at the NODAI Genome Research Center, Tokyo University of Agriculture. Sequence data were uploaded to the Galaxy platform [34] and processed using the fastp data preprocessing tool [35]. Taxonomic assignment was conducted using Kraken2 with a confidence threshold of 0.5 [36]. Estimation of class−level relative abundance was performed using Bracken [37].

Table 2.

Characterization of C. difficile strains isolated from PM_2.5 collected in Yokohama, Japan

ID	Collection date	Amount of PM2.5 tested (mg)	POT genotype	Toxinotypea	Sequence type	Ribogype	Clade	Isolation of the same POT genotype strain in Japanb		Metagenomic analysis (%)c
								Medical institution (N = 1066)	Environment (N = 263)	Clostridia (%)	Bacilli (%)
JP-PM2.5–18	Nov 17, 2020 ~ Dec 08, 2020	11.1	604–292	A-, B-, CDT-	ST109	073	4	0%	0%	nt	nt
JP-PM2.5–19	Dec 10, 2020 ~ Jan 06, 2021	10.1	604–292	A-, B-, CDT-	ST109	073	4	0%	0%	nt	nt
JP-PM2.5–22	Mar 10, 2021 ~ Apr 14, 2021	10.6	572–257	A-, B +, CDT-	ST37	017	4	0.1%	0%	0.3%	8.2%
JP-PM2.5–30-1	Sep 13, 2022 ~ Nov 28, 2022	9.7	673–292	A-, B-, CDT-	ST1	027	2	0%	0%	0.5%	7.8%
JP-PM2.5–30-2	Sep 13, 2022 ~ Nov 28, 2022	9.9	405–308	A-, B-, CDT-	ST15	010	1	0.5%	1.5%	0.5%	7.8%
JP-PM2.5–31	Dec 01, 2022 ~ Feb 20, 2023	11.5	405–308	A-, B-, CDT-	ST15	010	1	0.5%	1.5%	0.7%	16.2%
JP-PM2.5–32	Feb 21, 2023 ~ Mar 16, 2023	10.2	485–183	A +, B +, CDT-	ST2	014/020	1	0.6%	0.8%	0.4%	4.5%

^aBased on the POT genotype. A, tcdA; B, tcdB; CDT, cdtA

^bThe percentage of isolates with the same POT genotype identified within each group

^cThe ratio of Clostridia and Bacilli genomic reads to the bacterial reads in the amplicon sequencing analysis. nt, not tested

Isolation of C. difficile strains from atmospheric aerosol particles

Approximately 10 mg of each PM2.5 sample was added to 2 mL of selective enrichment broth for C. difficile (Product ID 717598–3, Kanto Chemical, Tokyo, Japan) and incubated at 37 °C for 2–7 days. When the broth turned yellow, indicating potential anaerobic bacterial growth, 50 µl of the culture was plated onto CCMA selective agar for C. difficile (Nissui, Tokyo, Japan) and incubated anaerobically at 37 °C for 2 days. Anaerobic conditions were achieved using AnaeroPack® (Mitsubishi Gas Chemical, Tokyo, Japan). Colonies suspected to be C. difficile were harvested, and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Tokyo, Japan). The POT genotype was determined using a POT kit (Kanto Chemical). Three independent colonies were examined and confirmed to yield the identical genotypes. Because sequence type (ST) and PCR ribotype (RT) are more widely used for international comparison, STs and RTs were inferred from POT types based on previously reported correlations among these genotyping schemes. For whole−genome sequencing, the isolates were cultured in brain heart infusion (BHI) broth, and genomic DNA was extracted from the culture medium using the DNeasy PowerSoil Pro Kit (QIAGEN). Sequencing was performed on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) using a 300 bp paired−end strategy with the QIAseq FX DNA Library Kit (QIAGEN). Raw reads were assembled using SPAdes v4.0.0 [38]. Multilocus sequence typing (MLST) gene sequences were extracted from the assembled genomes using the PubMLST database (https://pubmlst.org). The production of toxins A and B was verified by C. Diff QUIK CHEK Complete kit (TECHLAB, Blacksburg, VA). Genomic data for the bacterial isolates were registered under BioProject ID PRJDB20345.

Molecular epidemiological analysis of the atmospheric aerosol−derived C. difficile strains

For comparison of atmospheric aerosol−derived C. difficile strains with Japanese isolates, a total of 1,066 clinical isolates (310 POT types) and 263 environmental isolates (118 POT types) collected in Japan were included in the analysis. The clinical isolates comprised 982 strains previously reported from 14 medical institutions [14] and 84 isolates reported from medical institutions in Okinawa [39] and Hiroshima [40], all of which were collected between 2005 and 2021 (Supplementary Data S1). The environmental isolates were obtained from river water collected between 2022 and 2024 at 10 midstream to downstream sites across Japan (Fig. 2). Several liters of river water were filtered using 0.22 µm pore size filters (500 mL Bottle Top Vacuum Filter, 33.2 cm2 PES membrane, Corning, Tokyo, Japan). The filters were treated with 70% ethanol and placed onto CCMA agar plate, which were incubated anaerobically at 37 °C for 2 days to allow colony formation. DNA was extracted from all colonies that emerged and subjected to POT genotyping (Supplementary Data S2).

Fig. 2.

Sampling sites of atmospheric PM_2.5 and locations of medical facilities and rivers where C. difficile strains with matching POT types were isolated. Sample collection sites for PM2.5 particles (Yokohama and Fukuoka) are indicated with green dropper icons. Locations of medical facilities are marked with cross icons, and river water sampling sites are indicated by water drop icons. The POT genotypes of the C. difficile strains identical to those isolated from atmospheric PM_2.5 are indicated at each corresponding site

For comparison of atmospheric aerosol−derived C. difficile strains with international isolates, 108 strains whose genomic sequences were retrieved from PubMLST (48 strains) and NCBI (60 strains) were analyzed, all of which were of clinical origin. Phylogenetic analysis was performed using MEGA software (version 11) based on the sequences of seven housekeeping genes (adk, atpA, dxr, glyA, recA, sodA, tpi) adopted in the MLST, including four strains isolated from PM2.5. The neighbor−joining method was used to construct the phylogenetic tree. The details of the strains used in this study was provided in a separate file (Supplementary Data S3).

Antimicrobial resistance gene analysis

Antimicrobial resistance (AMR) genes were analyzed in the assembles of four atmospheric aerosol–derived C. difficile isolates for which whole−genome sequencing data were available. AMR gene detection was performed using ABRicate (https://github.com/tseemann/abricate), with the NCBI AMRFinderPlus database [41] and the Comprehensive Antibiotic Resistance Database [42] as reference databases.

Results

Amplicon sequencing analysis of atmospheric aerosols

Amplicon sequencing was performed on atmospheric aerosol samples collected from 10 sites across East Asia (Fig. 1) to characterize their bacterial genomic content (Table 1). The relative abundance of sequences classified as Clostridia among total bacterial reads averaged 8.7% (range: 0.34%–17.3%, N = 10). Within this group, genomic sequences of C. difficile were detected in atmospheric aerosol samples of 8 out of the 10 sites (80%) with a mean relative read abundance of 1.4% (range: 0.03%–9.88%, N = 8), corresponding to an average of 15.4% of all Clostridia reads (range: 0.3%–100%, N = 8). However, not all the samples of the 8 collection sites were positive for C. difficile DNA (5.4%, range: 2.9%−11.1%). As a reference, sequences from Bacillus, a Gram−positive, aerobic, spore−forming bacteria widely distributed in soil, were also analyzed. Bacillus sequences were detected at a significantly higher average relative abundance (4.0%, range: 0.55%–12.76%, N = 10) than C. difficile (1.4%, range: 0.03%–9.88%, N = 8; P = 0.009, paired two−sided Student’s t−test). These results suggest that C. difficile is transported through atmospheric aerosols at a relatively high abundance. Notably, while the sample from Taklamakan Desert exhibited a high proportion of Clostridia, the relative abundance of C. difficile was low. In contrast, the Tsukuba sample showed high proportions of both Clostridia and Bacillus, with C. difficile comprising 100% of the Clostridia−classified reads. It was suggested that C. difficile could be present in atmospheric aerosols; however, the data should be interpreted with caution, as amplicon sequencing analysis is not always reliable for bacterial identification at the species level.

Isolation of C. difficile from atmospheric aerosol particles

Because DNA detected in atmospheric aerosols may originate from non−viable cells, the potential role of aerosols in the environmental dissemination of C. difficile cannot be assessed without isolating viable bacteria. Moreover, genotypic analysis requires cultured isolates. To address this, we attempted to isolate C. difficile from atmospheric aerosol particles using selective media. A total of 61 atmospheric PM2.5 samples collected between 2020 and 2023 in Yokohama and Fukuoka, Japan (Fig. 2, green dropper icons), were examined. Seven atmospheric PM2.5 samples yielded C. difficile colonies on the CCMA selective agar (Table 2). The details of the PM2.5 samples and the characteristics of the isolated C. difficle strains were summarized in Table 2. From JP−PM2.5–30, two separate aliquots yielded different C. difficile genotypes. The seven isolates represented five distinct genotypes. The same genotype was isolated from the samples collected on consecutive collection dates (JP−PM2.5–18 and −19, and JP−PM2.5–30−2 and −31, respectively). Among 15 PM2.5 samples examined by the shotgun metagenomic analysis, the proportions of Clostridia genome occupied 0–0.77% among the genomic reads; however, the association was not apparent between this detection frequency of Clostridia genome and the bacterial isolation (data not shown).

The PM_2.5−derived strains were compared with Japanese isolates using POT genotype, owing to its high genetic resolution and the availability of extensive comparative data across Japan. The five POT genotypes identified from atmospheric aerosol−derived C. difficile isolates were compared to 1,066 clinical and 263 environmental isolates collected in Japan. Among the clinical isolates, three POT genotypes matched those found in the PM2.5−derived strains. The isolation frequencies of strains with POT 485–183, POT 405–308, and POT 572–257 were 0.6%, 0.5%, and 0.1%, respectively. These genotypes ranked 32nd, 37th, and 115th out of 310 total POT genotypes, indicating that they were relatively rare. Among the environmental isolates, two genotypes overlapped with the PM2.5−derived strains: POT 405–308 at 1.5% and POT 485–183 at 0.8%. Out of 118 POT genotypes identified among environmental strains, these ranked 11th and 26th, respectively, suggesting that their detection frequencies were also relatively low in Japan. Their sequence types and ribotypes were inferred from the inter−genotype relationships and summarized in Table 2.

With respect to geographic distribution, POT 405–308 was previously identified in clinical isolates from medical institutions in Osaka, Nagoya, and Tokyo, as well as in environmental isolates recovered from river water in Miyagi, Tokyo, and Fukuoka—all of which are geographically distant from the aerosol collection site. Similarly, POT 485–183 was found in clinical isolates from Tokyo, Nagoya, Gifu, Osaka, and Fukuoka, and in environmental isolates from Fukuoka. POT 572–257 was identified in clinical isolates from Osaka. These data indicate that the three PM_2.5−derived strains are geographically distributed across Japan (Fig. 2 and Table 2). The other two genotypes identified in atmospheric aerosol−derived C. difficile isolates, POT 604–292 and POT 673–292, have not been previously detected in Japan.

C. difficile produces three toxins involved in the pathogenesis of CDI: toxin A, toxin B, and binary toxin. The presence of the corresponding toxin genes (tcdA, tcdB, and cdtA) in the atmospheric aerosol−derived isolates was assessed by PCR. Among the seven isolates, two tested positive for toxin genes (Table 2). Immunochromatographic testing confirmed the production of toxin A/B proteins in these toxin gene–positive strains. The proportion of tcdB−positive strains was then analyzed on clinical, environmental, and the PM2.5−derived isolates. The tcdB positivity rates were 81.2% (866/1066), 41.8% (110/263), and 28.6% (2/7), respectively. The toxin positivity rate in the atmospheric aerosol−derived isolates was significantly lower than that in the clinical isolates (P < 0.0035, Fisher’s exact test) but not in the environmental isolates.

The PM2.5−derived isolates were genetically compared to isolates registered from the various parts of the world based on MLST (Fig. 3). These isolates were analyzed alongside 108 strains registered in the public database. JP−PM2.5–32 (ST2) clustered with strains registered from multiple regions worldwide, including Germany, Romania, the USA, Canada, New Zealand, and South Korea (Fig. 3, Cluster A). JP−PM_2.5–31 (ST15) was most closely related to European strains, including one from Great Britain and others from Germany (Fig. 3, Cluster B). JP−PM2.5–18 (ST109) was most closely related to a strain registered from Ghana in 2018 (Fig. 3, Cluster C). JP−PM2.5–22 (ST37) clustered with strains from various Asian countries, including China, India, and Thailand (Fig. 3, Cluster D). None of the PM_2.5−derived strains clustered with the group of strains previously registered from Japan (Cluster J), which is consistent with the POT genotyping results, suggesting that these genotypes are rarely detected in Japan. Similar data was obtained by a phylogenetic analysis of the isolates based on whole−genome single−nucleotide polymorphisms (SNPs, Supplementary Data S4).

Fig. 3.

Molecular epidemiological analysis of the four C. difficile strains isolated from atmospheric aerosol particles. Phylogenetic analysis was performed using the neighbor−joining method on four C. difficile strains isolated from atmospheric PM_2.5 samples (JP−PM2.5–18, −22, −31, and −32; highlighted with red circles). Clusters A–D represent the genetic clusters to which the aerosol−derived strains belong. Insets provide magnified views of each cluster. A cluster primarily composed of strains previously reported from Japan is labeled as Cluster J. Clades and the major sequence types (STs) are shown on the outer ring with a color code for STs shown in the bottom right. Numbers at the branch points represent bootstrap values based on 1,000 replicates. The scale bar indicates the number of nucleotide substitutions per site. Dashed line indicates the genetic distance of 0.02. For space considerations, strain names are abbreviated using a three−letter country code, year of isolation or database registration, and the ST. Detailed strain information is provided in Supplementary Data S3. The phylogenetic tree was constructed using MEGA software (version 11)

Antimicrobial resistance (AMR)–associated genes were analyzed in the four atmospheric aerosol–derived Clostridioides difficile isolates for which whole−genome sequencing data were available. Distinct AMR gene profiles were identified among the isolates. The isolate JP−PM_2.5–18 harbored aac(6')−Ie−aph(2")−Ia, ant(6)−Ia, blaAHM−1, blaCDD−2, cdeA, cplR, nimB, tetA(P), and tetB(P). JP−PM_2.5–22 and JP−PM_2.5–31 each carried blaAHM−1, blaCDD−2, cdeA, cplR, and nimB. JP−PM_2.5–32 harbored blaAHM−1, blaCDD−1, cdeA, cplR, and nimB.

Discussion

This study represents the first report of successful isolation of C. difficile from atmospheric aerosols, followed by genotypic comparison with environmental and clinical isolates using a typing method with higher discriminatory power than conventional MLST and ribotyping. Atmospheric aerosols can circulate globally and travel around the Earth in approximately two weeks via large−scale atmospheric circulation system [43]. For example, aerosols originating from the Sahara Desert are known to cross the Atlantic Ocean and Mediterranean Sea, reaching the Americas and Europe [44]. Similarly, Asian dust (also known as yellow sand), which arises from large−scale soil aerosolization events in deserts such as the Gobi and Taklamakan Deserts, is frequently transported by westerly winds to Southeast Asia and Japan, and in some cases even reaches North America [44].

If soil bacterial cells are aerosolized by dust storms, they could potentially be transported over long distances through the atmosphere [20, 44, 45]. Our findings are consistent with the possibility of the global dissemination of C. difficile through atmospheric aerosol. C. difficile is an obligate anaerobe and typically loses viability within 24 h under aerobic conditions [46, 47]. In aerosols, bacteria are often found attached to other particles, forming aggregates of multiple cells [48]. It is therefore likely that C. difficile persists in the atmosphere in the form of spores adhered to fine particles.

Because PM2.5 was continuously collected at a constant air flow rate by the sampler over a known period, the volume of air corresponding to a given mass of PM2.5 could be estimated from the sampling duration and flow rate. A C. difficile strain was isolated from approximately 10 mg of PM2.5; therefore, the volume of air required to collect this amount of PM2.5 was calculated. Based on this estimate, the atmospheric density of C. difficile spores was approximately one per 1,600 m3. This low concentration suggests that direct uptake of C. difficle from air by humans is unlikely. However, the detection of toxigenic, potentially pathogenic C. difficile strains—comparable to those found in clinical settings [49, 50]—supports the hypothesis that atmospheric aerosols may contribute to the expansion of C. difficile’s ecological niche, potentially impacting human health indirectly.

Considering the potential geographic origins of the C. difficile strains isolated from the atmospheric aerosols in Japan, two possibilities can be proposed: short−distance transport from nearby regions (Japanese origin) and long−distance transport from distant regions (overseas origin). Genotype analysis revealed that the aerosol−derived isolates either belonged to genotypes not previously identified in Japan or to genotypes detected only infrequently. Phylogenetic analysis further showed that these strains were more closely related to isolates from Europe, China, or North America than to those previously detected in Japan, supporting the possibility of long−distance, overseas transport. Notably, large−scale dust storms capable of aerosolizing soil bacteria are limited to geographic regions and specific meteorological conditions worldwide [19, 51, 52]. The observation that the same genotype was found in two consecutive aerosol samples strongly suggests a scenario in which aerosolized soil particles originating from distant locations dispersed gradually through the atmosphere before depositing at the sampling site. Such dispersal events of soil bacteria likely occurred repeatedly throughout history. One possible explanation is that indigenous C. difficile strains in Japan may have adapted to local environmental conditions, thereby limiting the immediate establishment of aerosol−delivered foreign strains. This ecological filtering effect could be partly attributed to the high genomic plasticity of C. difficile, which allows adaptation to diverse environments [53].

Asian dust regularly travels across national borders, reaching China, Korea, Vietnam, and Thailand. Notably, a significant percentage of the PM2.5 that reaches Japan originates from mainland China [54, 55]. The findings of our molecular epidemiological analysis are consistent with the hypothesis that the strain detected in Yokohama, characterized by genotype POT 572–257/ST37, may have originated from Central Asia. In North America, dust storms that aerosolize surface soil from farmland occur annually [56]. Similar events, although less frequent, have also been reported in Europe [57]. Previous studies have shown that C. difficile is more frequently detected in fertilized agricultural soils [58]. Therefore, it is plausible that soil bacteria, including C. difficile spores, became aerosolized during such events and were subsequently transported across long distances, ultimately reaching Japan where they were detected in atmospheric samples.

Although multiple AMR−associated genes were detected in the aerosol−derived C. difficile isolates, their phenotypic relevance should be interpreted cautiously. Aminoglycosides and β−lactam antibiotics are intrinsically ineffective against C. difficile. Accordingly, the presence of aminoglycoside resistance genes and blaCDD is unlikely to confer additional phenotypic resistance beyond intrinsic levels. In contrast, blaAHM has been reported less frequently in global genomic surveys of C. difficile [59], suggesting that this gene may be associated with specific lineages or ecological niches, including environmental reservoirs. Other AMR−associated genes detected in this study, such as cdeA, cplR, and nimB, are generally considered to confer low−level, inducible, or conditional resistance and may not result in clinically relevant resistance. The tetracycline resistance genes tetA(P) and tetB(P) were detected in one aerosol−derived isolate. Although these determinants are generally considered less potent than tet(M)—the most frequently reported tetracycline resistance gene in C. difficile [60]—they have been described in clinical isolates, including strains exhibiting reduced susceptibility to tetracyclines [61–63]. Together, these findings suggest that atmospheric dissemination of C. difficile may contribute not only to the geographic spread of specific lineages but also to the environmental circulation of AMR−associated genetic elements. Further phenotypic analyses using larger numbers of aerosol−derived isolates will be required to clarify the functional and clinical relevance of these genes.

One of the limitations of this study is the technical challenge of isolating C. difficile from the atmospheric aerosol samples. The collected PM2.5 particles may contain cytotoxic substances that are harmful to vegetative cells. Therefore, even if spores germinated, the resulting vegetative forms may have died upon exposure to these toxic components. Therefore, isolates may not represent the full airborne population. Future studies should assess whether all viable C. difficile present in atmospheric aerosols can be successfully cultured. It is also possible that the atmospheric isolates may form stress−resistant spores. The second limitation involves the resolution of the molecular epidemiological analysis. The C. difficile genomes currently registered in public databases mostly consist of clinical isolates. In addition, the geographic origins of these registered strains are unevenly distributed. Genotypic analysis would be more informative if it included environmental C. difficile isolates from a broader and more geographically diverse set of sources. Finally, the number of aerosol−derived isolates was much smaller than that of clinical isolates; therefore, any conclusions drawn from comparisons between these two groups should be interpreted with caution. The biological significance of our findings should be confirmed in future studies by studying using a higher number of atmospheric aerosol−derived C. difficile isolates.

Conclusions

To our knowledge, this study provides the first report of C. difficile being isolated from atmospheric aerosols, and presents a comparative genotype analysis with isolates from environmental and clinical sources. To more accurately determine the origins of aerosol−derived C. difficile, comprehensive and geographically broad environmental surveillance is warranted. Furthermore, profiling the pathogenic potential and antimicrobial resistance of these strains will be critical for assessing their public health implications.

Abbreviations

AAD

Antibiotic−associated diarrhea

AMR

Antimicrobial resistance

BHI

Brain heart infusion

CDI

C. difficile infection

MLST

Multilocus sequence typing

NGS

Next−generation sequencing

ORF

Open reading frame

PCR

Polymerase chain reaction

PM2.5

Particulate matter ≤2.5 µm

POT

PCR−based ORF typing

RT

Ribotype

ST

Sequence type

Authors’ contributions

Investigation: Miyako Ishibashi, Shinya Yonogi, Momoka Tani, Chihiro Hiura, Daisuke Hosokawa, Nami Matsuda, Yusaku Sagisaka, Mayuko Yagi, Yuh Shiwa, Tomoaki Okuda, and Teruya Maki. Formal Analysis: Miyako Ishibashi, Shinya Yonogi, Momoka Tani, Chihiro Hiura, Daisuke Hosokawa, Nami Matsuda, Yusaku Sagisaka, Yuh Shiwa, Tomoaki Okuda, Teruya Maki, and Jun Komano. Resouces: Tomohiro Hasei, Fumito Maruyama, Daisuke Tanaka, Tomoaki Okuda, Teruya Maki. Methodology: Yuh Shiwa, Fumito Maruyama, Tomoaki Okuda, Teruya Maki, and Jun Komano. Data Curation: Shinya Yonogi, Yuh Shiwa, Tomoaki Okuda, Teruya Maki, and Jun Komano. Conceptualization and Writing – Original Draft: Jun Komano. Writing – Review & Editing: All the authors.

Funding

^None.

Data availability

Genomic data for the aerosol-derived *C. difficile* isolates were registered under BioProject ID PRJDB20345. Genomic data for the other *C. difficile* strains used in the phylogenetic analysis are publicly available from either PubMLST or NCBI as listed in Supplementary Data S3.

Declarations

Ethics approval and consent to participate

Not applicable. This study was conducted in accordance with the Declaration of Helsinki. Ethics approval and informed consent were waived for this study in accordance with the Ethical Guidelines for Medical and Biological Research Involving Human Subjects issued by the Japanese Ministry of Education, Culture, Sports, Science and Technology; the Ministry of Health, Labour and Welfare; and the Ministry of Economy, Trade and Industry, as advised by the Research Ethics Committee of Osaka Medical and Pharmaceutical University, Faculty of Pharmacy.

Consent for publication

Not applicable

Competing interests

The authors declare no competing interests.