Microbiome of laboratory‐reared and environmentally collected cockroaches

Cockroaches are insects found in almost all habitats, including unsanitary environments. Understanding their microbial communities is crucial for assessing the potential risks they pose as vectors of pathogens. In this study, we assessed the microbial communities of omnivorous cockroaches collected from external environments and those reared in a clean laboratory for extended periods (5–20 years). Using the iSeq 100 system, we examined the relative abundance of microbial communities at the phylum, family and genus levels. Our results revealed that the predominant taxa in these cockroaches were Proteobacteria, Bacteroidetes and Firmicutes. Interestingly, the bacterial communities of samples from the same cockroach species, regardless of their living conditions, clustered together, indicating species‐specific similarities in microbiomes. The symbiont genus Blattabacterium was consistently present in all samples, delivering nutrients to the host. Pathogen detection at the genus level indicated a higher prevalence of potential pathogens in cockroaches collected from field environments, compared with those from laboratory‐reared cockroaches. These findings underscore the importance of cockroaches as pathogen reservoirs and vectors of opportunistic infections, emphasizing the need for further studies to identify specific microorganisms and confirm their pathogenicity. As cockroaches inhabit human environments, their potential to spread harmful bacteria through defecation warrants attention and underscores the significance of understanding their microbial ecology for public health implications.


Introduction
Cockroaches inhabit unsanitary environments and are distributed in many areas where humans are active, such as hospitals, restaurants and residences in urban areas (Guzman & Vilcinskas 2020).Cockroaches are considered disease carriers and can pose a serious threat to human health (Pai et al. 2004;Schapheer et al. 2018).Concerns regarding the hygiene of cockroaches have been a topic of discussion for years, particularly in relation to issues such as food poisoning and contamination (Leibovitz 1951;Rueger & Olson 1969).Recent advancements in omics technology and molecular diagnostics have allowed researchers to identify pathogenic bacteria (including multidrug-resistant bacteria) and viruses (SARS-CoV-2) in cockroaches (Kalantari et al. 2023;Luckyjane Molewa et al. 2022).Cockroaches can spread these bacteria and viruses by roaming around food and over surfaces, posing a major health risk to humans.
The gut microbiome of this omnivorous insect participates in food digestion, protects against pathogens, and contributes positively to host physiology by providing vitamins and amino acids.However, harmful microbes, such as fungi, pathogenic bacteria and viruses, can be spread to humans through cockroach feces (Crawford et al. 2015;Kalantari et al. 2023;Pérez-Cobas et al. 2015).Cockroaches are considered reservoirs of pathogens because of the intestinal accumulation of these microbes.However, the degree of risk associated with cockroaches causing human infection is poorly understood and is still a matter of debate (Guzman & Vilcinskas 2020).
Blattella germanica and Periplaneta americana are commonly found around people, and Periplaneta japonica is also found in places where food is stored, such as kitchens and bathrooms (Nasirian & Salehzadeh 2019;Vicente et al. 2018).Cockroaches are often exposed to unsanitary environments, which can cause the spread of pathogenic microorganisms.Environmental pollution caused by wastewater discharge from hospitals and factories can become a hotspot for the emergence of "superbug" bacteria, through the acquisition of various antibiotic resistance genes as a result of horizontal gene transfer (Dai et al. 2023;Talat et al. 2023).Moreover, antibiotic resistance is a global public health emergency.The recent detection of antibiotic-resistant bacteria (e.g., Enterococcus spp.and Dysgonomonas spp.) in the cockroach microbiome suggests that new resistance genes may be transferred in the insect gut, which can potentially harm human and animal health (Anacarso et al. 2016;Laborda et al. 2022;Li et al. 2020).However, the detection of putative pathogenic bacteria in cockroaches using microbiome analysis remains lacking.Here, we analyzed the 16S rDNA metabarcoding data from three species of laboratory-reared and wild cockroaches, and our results suggest the potential of cockroaches acting as pathogen carriers.

Study animals
Three cockroach species (B.germanica, P. americana and P. japonica, termed BG, PA and PJ, respectively) were used for the microbiome analysis.Fifteen cockroaches were used in this study, nine of which were collected from various locations in the Republic of Korea and six of which came from populations that have been laboratory reared for 5-28 years (Table 1).Sample names beginning with "C" or "L" represent collected and laboratory-reared specimens, respectively.The wild cockroaches were collected from seven food factories and two public facilities in the Republic of Korea in March-June 2022 using a commercial sticky trap and were immediately frozen at À80°C.Meanwhile, the laboratory-reared cockroaches used in the study were reared for 5-28 years in our laboratory (Table S1).The laboratory-reared cockroaches were reared in plastic cages (27 × 34 × 19 cm, replaced every 4 months), incubated at 25°C and 50% relative humidity, and supplied with sterilized fish-added experimental mouse feed (Purina, St. Louis, MO, USA) and autoclaved tap water.All laboratory-reared cockroaches were anesthetized in CO 2 for 1 min prior to preparation for DNA extraction.2 Illumina sequencing metadata analysis All cockroaches were washed in sterile distilled water and then 70% ethanol before DNA extraction.Each cockroach was broken down using an autoclaved mortar and pestle.Liquid nitrogen was added to break down the tissues and bacterial cell walls, and disrupted samples were used for DNA extraction.
Total DNA was extracted from individual cockroaches using the NucleoSpin ™ DNA Insect Mini Kit (Macherey-Nagel, Düren, Nordrhein-Westfalen, Germany), as previously described (Lee et al. 2023).For microbial community studies, the V4 region of the 16S rRNA gene was amplified via polymerase chain reaction (PCR) using the primers 515F (50-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGG TGCCAGCMGCCGCGGTAA-30) and 806R (50-GTCTCG TGGGCTCGGAGATGTGTATAAGAGACAGGGACTACH VGGGTWTCTAAT-30) (Kim et al. 2021).KAPA HiFi HotStart ReadyMix (Roche Sequencing Solutions, Pleasanton, CA, USA) was used for PCR amplification under the following conditions: one cycle at 95°C for 5 min; followed by 25 cycles of 98°C for 30 s, 55°C for 30 s and 72°C for 30 s; and a final cycle of 72°C for 5 min.AMPure XP (Beckman Coulter, Brea, CA, USA) was used for DNA purification.A limited cycling amplification step (8 cycles) was performed to add multiplexing indices and Illumina sequencing adapters.Mixed amplicons were pooled and sequenced on an Illumina iSeq 100 sequencing system using the Illumina iSeq ™ 100 i1 Reagent v2 Kit (Illumina Inc., San Diego, CA, USA), according to the manufacturer's instructions.
Bacterial microbiome analysis of the 16S rRNA gene sequence data from cockroaches, including taxonomy identification and principal coordinates analysis (PCoA), was performed using EzBioCloud (https://www.ezbiocloud.net), a commercially available bioinformatics cloud platform for microbiome research developed by ChunLab (Seoul, Republic of Korea).Bioinformatics analysis was performed to analyze the clustering, as described previously (Yoon et al. 2017).Briefly, raw reads were quality checked and low-quality (< Q25) reads were filtered using Trimmomatic 0.32 (Bolger et al. 2014).Paired-end sequence data were then merged using PandaSeq (Masella et al. 2012).Primers were then trimmed using the ChunLab in-house program, which applied a similarity cut-off of 0.8.Sequences were denoised using the pre-clustering program Mothur, which merges sequences and extracts unique sequences, allowing up to two differences between sequences (Masella et al. 2012).The EzBioCloud database (https://www.ezbiocloud.net)(Yoon et al. 2017) was used for taxonomic assignment using BLAST 2.2.22, and pairwise alignments were generated for similarity calculations (Altschul et al. 1990;Schloss et al. 2009).The UCHIME algorithm and non-chimeric 16S rRNA database from EzTaxon were used to detect chimeric sequences for reads with a best-hit similarity rate of <97% (Edgar et al. 2011) (a 97% similarity is generally used as the cut-off for species-level identification).Sequence data were then clustered using CD-Hit and UCLUST (Myers & Miller 1988).All subsequent analyses were performed using EzBioCloud.We performed PCoA and permutational multivariate analysis of dispersion (PERMANOVA) (Gower 1966), based on the generalized Bray-Curtis distance.We selected putative pathogens that matched the genus level, as identified in the 16S amplicon, based on the National Center for Biotechnology Information (NCBI) Pathogen Detection Browser database (https://www.ncbi.nlm.nih.gov/pathogens/isolates).

Results and discussion
Figure 1 shows the relative abundance of microbial communities at the phylum, family and genus levels of the two groups of cockroaches analyzed using the iSeq 100 system.The total bacterial community was classified into 25 phyla and 602 genera; in particular, the taxa Proteobacteria, Bacteroidetes and Firmicutes dominated (Fig. 1A; Table S2).Numerous studies have consistently found that the cockroach microbiome is primarily comprised of Proteobacteria, Bacteroidetes and Firmicutes (Berlanga 2015;Dietrich et al. 2014;Domínguez-Santos et al. 2021).These findings not only assist in identifying the core bacterial phylum of this species but also suggest the potential for establishing a symbiotic relationship with its host.These symbiotic relationships have been instrumental in enhancing the limited metabolic networks of most cockroaches, fostering a range of prokaryotic metabolic capabilities, including methanogenesis, chemotrophy, nitrogen assimilation and more (Berlanga 2015;Jahnes & Sabree 2020).
The bacterial communities were clustered in a cockroach species-specific way on the PCoA plot after β-diversity analysis, regardless of whether the cockroaches were reared in the laboratory or caught from the wild (Fig. 2, P < 0.001, PERMANOVA).This suggests that despite the variation in living environments and nutritional status of the cockroaches tested, the composition of the microbiome is similar in the same species of cockroach and that bacterial communities from each cockroach species were distinct from one another.Additionally, only in B. germanica were the laboratory-reared and wild-collected groups found to have a distinct microbial composition (Fig. S1, P < 0.024, PERMANOVA), suggesting environmental factors still affect the microbial community found in cockroaches.
The dominant bacterial taxa in B. germanica were Proteobacteria, Bacteroidetes and Firmicutes.This finding aligns with a prior study on the microbiome of German cockroaches (Guzman & Vilcinskas 2020), which shows that

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the distribution of these taxa remained consistent in most German cockroaches, irrespective of their habitat or dietary preferences.The similarity in bacterial taxa provides insights into the potential influence of host genetics on microbial community structure.These findings contribute to our understanding of the intricate relationship between cockroach hosts and their microbial companions, highlighting the need for further research to uncover the mechanisms that govern these interactions.
Blattabacterium, which belongs to the Blattabacteriaceae family, is a symbiont that can deliver nutrients to the host through amino acid and vitamin/cofactor biosynthesis (Jennings et al. 2020).Blattabacterium was found in all samples and was dominant in most samples (comprising approx.5%-84%; Fig. 1C), except in P. americana (CPA1 = 0.21%, CPA2 = 0.22% and LPA1 = 1.93%).In a prior investigation of the microbiome of P. americana across life stages, it was discovered that Blattabacterium dominated the microbial composition found in the ootheca.However, its abundance significantly diminished in the nymph and adult stages, exhibiting considerable variance among samples (Chen et al. 2023).These findings are consistent with the results of our study.Enterobacter and Enterococcus, common in various ecological niches and in the gut, were found in most cockroaches.Enterobacter was dominant in wild B. germanica.Although environmental conditions and nutrient supply in the host may generate biased results, bacterial taxa abundant in water and soil (e.g., Desulfovibrio  and Stenotrophomonas), and in the gut (e.g., Bacteroides, Clostridium, Lactobacillus and Lactococcus), were identified in most cockroaches.
The recent development of multi-omics technology and microbiome analysis have raised awareness about cockroaches as a pathogen reservoir (Domínguez-Santos et al. 2021;Marquis et al. 2023).At the genus level, we detected pathogens that may cause opportunistic infections (Fig. 3; Table S3).In particular, Acinetobacter spp., Enterococcus spp.and Pseudomonas spp., which can cause diseases such as bacteremia, pneumonia and catheter-related infection, were frequently found in high abundance.This supports the view that cockroaches may be vectors of pathogen spread in humans.Furthermore, several studies isolated antibiotic-resistant bacteria from B. germanica and P. americana (e.g.Pseudomonas and Staphylococcus), and antibiotic-resistance genes (e.g., aacA-D, tetK and msrA) have been identified (with high resistance to chloramphenicol, clindamycin, gentamicin, methicillin and ofloxacin etc.) (Abdolmaleki et al. 2019;Menasria et al. 2015).We found Acinetobacter baumannii and Enterococcus faecium, which are both on the World Health Organization (WHO) list of multidrug-resistant bacteria in urgent need of new antibiotics (Table S1) (Babu Rajendran et al. 2020).The β-diversity of the microbiome was comparable between species for both wild-collected and laboratory-reared cockroaches.However, wild-collected cockroaches exhibited a higher prevalence of putative pathogens than their laboratory-reared counterparts, as illustrated in Figures 2 and 3. Considering that cockroaches in the laboratory have undergone many generations over a long rearing period (5-28 years), it is plausible that laboratory-reared cockroaches lose harmful bacteria from their microbiome over time (Moran et al. 2019).These results suggest that cockroaches in all human environments may serve as vectors of opportunistic and difficult-to-treat bacterial infections.In addition, the release of pathogens and multidrug-resistant bacteria into the environment through cockroach feces is expected to promote the spread of antibiotic-resistant superbug bacteria (Guzman & Vilcinskas 2020;Solomon et al. 2018).However, our study investigated only the bacterial community of cockroaches using 16S rDNA metabarcoding.The precise identification of bacteria and other microorganisms, as well as pathogenicity experiments, are needed to gain more insights into the risks associated with the spread of putative pathogens from cockroaches.

Conclusion
The comprehensive analysis of microbial communities in cockroaches highlights species-specific patterns and the influence of environmental factors on the composition of bacterial taxa.The detection of putative pathogens underscores the importance of further research to understand and mitigate the associated potential public health risks.

Figure 3
Figure3Heat-map analysis of putative pathogenic bacterial read counts (at the genus level) in collected (blue) and laboratory-reared (gray) cockroaches.

Table 1
Cockroach samples used in the study Abbreviations: N/A, not applicable.SohyeonYUN et al.