Genetic composition, virulence factors, and antimicrobial resistance profiles of Bacillus cereus and Bacillus subtilis isolates from food vendors in Ondo State, Nigeria: implications for food safety

Abstract

Background

This study investigated Bacillus cereus and Bacillus subtilis from food vendors in Ondo State, Nigeria.

Methods

A comprehensive whole-genome sequencing (WGS) analysis of Bacillus genomes, including genome assembly, plasmid prediction, species identification, antimicrobial resistance (AMR) gene identification, virulence gene identification, and multilocus sequencing typing, was conducted.

Results

The genome assembly revealed a B. cereus genome with 87 contigs, a length of 5,798,917 base pairs, and a GC content of 34.79%, whereas B. subtilis had a genome length of 4,238,143 bp and was composed of 253 contigs with a contig L50 of 24, a contig N50 of 55,053, and a GC content of 43.14904%. Plasmid prediction revealed the absence of prominent plasmids in the assembled B. cereus genome, whereas the repUS12 plasmid was recognized with an identity of less than 95.63% for the B. subtilis genome. Species identification via the average nucleotide identity (ANI) calculation confirmed that Bacillus cereus had a 98.97% ANI value, whereas a 98.39% ANI value was confirmed for B. subtilis WAUSV36. AMR genes were identified, with virulence genes such as the alo, cytK, and hbl genes also detected in B. cereus, whereas clpX, codY, purA, and purB genes were detected in B. subtilis. Multiple-locus sequence typing (MLST) revealed that B. cereus belongs to sequence type 73 with 100% identity, identifying housekeeping gene alleles, including glp_13, gmk_8, and ilv_9, whereas B. subtilis belongs to sequence type 130, with the ilvD gene showing a perfect match and the highest allele length of 471 for the housekeeping genes identified.

Conclusions

This detailed WGS analysis provides valuable insights into the genetic composition, potential virulence factors, and resistance profiles of B. cereus and B. subtilis, enhancing the understanding of their pathogenicity and epidemiology. The genomic analysis of B. cereus and B. subtilis revealed potential genomic applications in the context of food safety.

Background

Bacillus cereus, a common spore-forming facultatively anaerobic gram-positive bacterium, has been isolated from patients suffering from various diseases and foodborne illnesses (Dietrich et al. 2021). It is widely distributed in nature, behaves as an opportunistic pathogen and is often linked to two distinct forms of human foodborne illness: symptoms such as diarrhea and abdominal discomfort and nausea and vomiting. Bacillus subtilis is a gram-positive bacterium with a rod-shaped structure. While it can produce spores that are resistant to heat, it is not known to cause infections in humans. Among the Bacillus species, B. subtilis and B. velezensis have garnered significant interest in the food industry because of their recognized safety and ability to compete with other microorganisms in natural settings, potentially influencing microbiota selection.

While B. cereus may not cause severe issues in healthy individuals, it can pose a significant risk to people with certain underlying conditions, including those who are immunocompromised or in the process of postsurgery recovery (Nguyen and Tallent 2019). Notably, some proteins previously believed to be unique to B. cereus have been discovered in B. thuringiensis isolates. In addition, the cross-talk among these species and the genetics of their association with Bacillus thuringiensis have also interested researchers working on genes that govern their differing roles in nature and pathogenicity (Ehling-Schulz et al. 2019).

Bacillus cereus and Bacillus subtilis are common contaminants in food, posing risks to human health because of their potential to produce toxins and exhibit antimicrobial resistance. Understanding the genetic makeup, virulence factors, and antimicrobial resistance profiles of these isolates is crucial for ensuring food safety and preventing foodborne illnesses, as demonstrated by Adamski et al. (2023). A recent study by Hurtado-Bautista et al. (2021) emphasized the importance of genetic analysis in elucidating bacterial evolution and adaptation to diverse environments. The formation of distinct clades by the B. cereus and B. subtilis lineages indicates the genetic diversity within these bacterial species. The genetic diversity, virulence factors, and antimicrobial resistance profiles vary among different strains of Bacillus species.

Furthermore, the presence of virulence factors in Bacillus species highlights the importance of genetic characterization. A study by Bianco et al. (2021) pinpointed specific virulence genes in B. cereus and B. subtilis that contribute to their pathogenicity. Moreover, the cytK and hbl genes were detected in B. cereus, whereas the clpX, codY, purA, and purB genes were identified in B. subtilis, highlighting the diverse virulence mechanisms employed by these bacteria. In addition to virulence factors, the antimicrobial resistance profiles of Bacillus strains play a critical role in determining their pathogenicity and potential impact on food safety. Research by Tagne et al. (2023) evaluated the antibiotic susceptibility of Bacillus strains isolated from environmental sources, shedding light on the potential influence of seasonal variations on antimicrobial resistance patterns. This underscores the dynamic nature of antimicrobial resistance in Bacillus species and the necessity for ongoing surveillance.

Moreover, the genetic composition of Bacillus strains, including plasmid content and species identification, is vital for understanding their epidemiology and transmission dynamics. The utilization of whole-genome sequencing (WGS) for characterizing B. cereus and B. subtilis isolates offers a comprehensive view of their genetic diversity and evolutionary relationships. Species identification through the average nucleotide identity (ANI) calculation and multilocus sequence typing (MLST) techniques further enhances the classification of these bacterial isolates, as conducted by Bianco et al. (2021).

By elucidating the genetic underpinnings of these bacterial strains, researchers can better evaluate the risks associated with their presence in food environments and develop targeted interventions to mitigate these risks. This study aims to offer insights into the genetic makeup, virulence factors, and antimicrobial resistance profiles of Bacillus cereus and Bacillus subtilis from food vendors to enhance the understanding of their pathogenicity and implications for food safety.

Methods

Isolation and identification of Bacillus species collected from food vendors

The process typically begins with aseptic transfer of 1 g of watermelon, pineapple, cooked cheese, pies, beef, turkey, chicken, and Naira swabs to tenfold serial dilutions of the samples in sterile saline solution as described by the Health Protection Agency (2009). The initial step in the standardized laboratory technique involves the preparation of selective media suitable for the isolation of Bacillus species in HiCrome Bacillus agar, as demonstrated by (Alippi and Abrahamovich 2019), and has been validated for the presumptive identification of Bacillus species. Following the preparation of the selective media, selected diluents of the serially diluted food samples were inoculated onto agar plates following the even distribution of the sample material on the agar surface. The use of Tryptic Soy agar (TSA) and nutrient agar (NA) media, as indicated by (Kim et al. 2022), has been effective in detecting Bacillus species through culture-dependent methods. Hence, we will incorporate these media into our technique to increase the recovery of Bacillus colonies from diverse sample sources.

Genome assembly and annotation of the Bacillus cereus sequence

Unassembled raw reads (R1 and R2) of the whole genome were uploaded in FASTQ format to the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) server to perform assembly of the genome (Olsen et al. 2023). The assembled genome was annotated via the Rapid Annotations using Subsystems Technology (RAST) toolkit on the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) server, which uses a FASTA-format contig file.

Prediction of plasmids in Bacillus cereus and Bacillus subtilis

The plasmid prediction was performed by uploading the assembled genome contig file derived from BV-BRC to the Plasmid Finder 2.1 server. The server enables the detection of plasmids in complete and partially sequenced bacterial isolates by recognizing and characterizing plasmid replicons in whole-genome sequencing (WGS) data (Caratolli and Hasman 2020).

Identification of Bacillus species (ANI calculator)

The average nucleotide identity (ANI) calculator was employed to ensure precise species identification. The ANI calculates ANI values, and the ANI calculates the mean nucleotide identity by considering both the top matches (one-way ANI) and mutually best matches (two-way ANI) between two genomic datasets of genomes of the same species that exhibit a similarity threshold of over 95%. (Yoon et al. 2017).

Determination of antimicrobial resistance genes in Bacillus cereus and Bacillus subtilis

The antimicrobial resistance genes were identified and extracted from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) server and ResFinder 4.1, which is a database for detecting antimicrobial resistance genes within an isolated whole-genome dataset (Florensa et al. 2022).

Determination of virulence genes in Bacillus cereus and Bacillus subtilis

The virulence genes were identified and extracted from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) server.

Determination of multilocus sequence typing (MLST) in Bacillus cereus and Bacillus subtilis

Multilocus sequence typing of the assembled genome was performed via the MLST 2.0 tool. This tool distinguishes the species and strains of the bacteria and the sequence type.

Results

Presumptive and whole-genome sequencing profile of bacterial strains from food samples and naira notes

Presumptively identified Bacillus species were divulged after their genome sequencing analysis as Bacillus cereus AH676, and B. subtilis WAUSV36, respectively, as illustrated in Table 1.

Table 1 The presumptive and sequencing profile of bacterial isolates from food samples and naira notes

Genome assembly and annotation in Bacillus cereus and Bacillus subtilis

The genome identified on the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) server was Bacillus cereus, with a genome ID of 1396.4273. The complete genome has the following genome and annotation statistics, as shown in Tables 2 and 3.

Table 2 Genome statistics for Bacillus cereus

Table 3 Annotation statistics of Bacillus cereus and Bacillus subtilis

Identification of the average nucleotide identity values of Bacillus cereus and Bacillus subtilis

Compared with the reference genome, the Bacillus cereus whole genome has a 98.97% ANI value; the Bacillus cereus AH676 chromosome has an accession number of CM000738, as shown in Fig. 1. Compared with that of Bacillus subtilis WAUSV36, the genome of Bacillus subtilis has a 98.39% ANI value, indicating a high degree of genetic similarity, as illustrated in Fig. 2.

Fig. 1

Average nucleotide identity of Bacillus cereus AH676

Fig. 2

Average nucleotide identity of B. subtilis WAUSV36

Prediction of plasmids in Bacillus cereus and Bacillus subtilis strains

As the PlasmidFinder 2.0 server predicted, the assembled genome containing Zero (0) prominent plasmids in Bacillus cereus and the repUS12 plasmid was recognized by the plasmid finder in Bacillus subtilis. The identity is < 100%, specifically 95.63%, as displayed in Fig. 3.

Fig. 3

Identification of the Plasmid Type in Bacillus subtilis WAUSV36

Determination of antimicrobial resistance genes in Bacillus cereus and Bacillus subtilis strains

The antimicrobial resistance genes identified in Bacillus cereus on the BV-BRC server and the classes in which the resistant drugs exist are recorded in Table 4. Beta-lactam (blaZ), fosfomycin (fosB1 and MurA), aminoglycoside (gidB, S12p), and fluoroquinolones (gyrB and gyrA) are some of the antibiotic resistance genes detected in the Bacillus cereus strain. Bacillus subtilis was resistant to fosfomycin (MurA, dxr), fluoroquinolones (gyrA), and macrolides (RlmA(II), as illustrated in Table 5. Different sets of virulence genes were also found when the result was removed from the BV-BRC.

Table 4 Antimicrobial drug classes and resistance genes detected in Bacillus cereus AH676

Table 5 Predicted antimicrobial resistance genes detected in Bacillus subtilis WAUSV36

Detection of virulence genes in Bacillus cereus and Bacillus subtilis strains

The number of virulence genes identified in the whole genome of Bacillus cereus is presented in Table 6. They include Alo, asbA and clpX. In contrast, B. subtilis WAUSV36 shows a more limited virulence gene profile with only six genes (bslA/yuaB, clpX, codY, purA, purB, recA) were also found to be encoded as virulence factors in Bacillus subtilis when the result was out on BV-BRC, as displayed in Table 7.

Table 6 Virulence genes and their number of occurrences detected in Bacillus cereus AH676

Table 7 Sets of virulence genes detected in Bacillus subtilis WAUSV36

Detection of multilocus sequence typing (MLST) in B. cereus and B. subtilis

The MLST results revealed that the Bacillus cereus strain belongs to sequence type 73 with an identity of 100%. The housekeeping gene alleles were identified as glp_13, gmk_8, ilv_9, pta_14, pur_9, pyc_12 and tpi_31, as shown in Table 8. After following the default procedure on the website, the results revealed that the Bacillus subtilis strain belongs to sequence type 130. Table 9 shows that the ilvD gene had a perfect match, with a percentage identity of 100%. Additionally, alleles for housekeeping genes were successfully identified.

Table 8 Multilocus sequence typing of Bacillus cereus

Table 9 Multilocus sequence typing of Bacillus subtilis AH676

Discussion

Genetic makeup, virulence factors, and antimicrobial resistance profiles enhance the understanding of Bacillus cereus and Bacillus subtilis pathogenicity and implications for food safety. The genome assembly of Bacillus cereus revealed a genome with 87 contigs spanning 5,798,917 base pairs and a GC content of 34.79%, which is consistent with the findings of Bianco et al. (2021), who explored the characterization of Bacillus cereus group isolates from human bacteremia through whole-genome sequencing (WGS). This research emphasizes the effectiveness of WGS in rapidly characterizing B. cereus group strains, providing comprehensive insights into their genetic epidemiology. By utilizing WGS, this study revealed crucial information about the presence of virulence factors and antimicrobial genes within these isolates. The findings shed light on the potential risks associated with these strains, highlighting the importance of understanding and addressing this often underestimated threat in the context of food safety. In contrast, Bacillus subtilis WAUSV36 has a genome length of 4,238,143 base pairs, consisting of 253 contigs with a contig L50 of 24 and a contig N50 of 55,053. It exhibits a higher GC content of 43.15%. This aligns with findings from Ehling-Schulz et al. (2019), who highlighted the need for refining the taxonomic classification and risk assessment of B. cereus AH676 through advancements in computational and microbiological methods. Species identification via the average nucleotide identity (ANI) calculation confirmed that Bacillus cereus AH676 had a 98.97% ANI value and 98.39% ANI value for Bacillus subtilis WAUSV36, similar to the findings of Bianco et al. (2021) on the effectiveness of WGS in rapidly characterizing B. cereus group strains, providing comprehensive insights into their genetic epidemiology.

Plasmid prediction in the assembled B. cereus genome revealed the absence of prominent plasmids, whereas the repUS12 plasmid was identified with less than 95.63% identity in the B. subtilis genome, as similarly reported by Bianco et al. (2021). The presence of virulence factors in Bacillus species underscores the importance of genetic characterization in understanding their pathogenicity. Hurtado-Bautista et al. (2021) conducted a study focusing on the intriguing realm of phenotypic plasticity and the evolution of thermal tolerance in Bacillus species originating from diverse environments as opposed to food sources in this study, specifically temperate and hot springs. This study focused on two bacterial lineages, Bacillus cereus sensu lato and Bacillus subtilis sensu lato, which have evolved in distinct habitats. By examining the growth and reaction norms to temperature of these bacterial strains, research has shed light on how these bacteria adapt to varying thermal conditions. The significant implications of thermal conditions on the risk posed by Bacillus cereus and Bacillus subtilis for food safety highlight the need for comprehensive strategies to mitigate the risks associated with these bacteria. Understanding the adaptive mechanisms, survival strategies, and biofilm-forming abilities of B. cereus and B. subtilis in response to temperature variations in food processing is crucial for implementing effective control measures and ensuring the safety of food products for consumers (Hurtado-Bautista et al. 2021). A study by Bianco et al. (2021) identified the alo, cytK, and hbl genes in B. cereus, whereas the clpX, codY, purA, and purB genes were detected in B. subtilis, highlighting the diverse virulence mechanisms employed by these pathogens. Antimicrobial resistance (AMR) genes were detected in both Bacillus species subjected to whole-genome sequencing (WGS) analysis in this study, covering various drug classes, such as beta-lactams, aminoglycosides, and fluoroquinolones, as similarly reported by Bianco et al. (2021). Notably, virulence genes such as the alo, cytK, and hbl genes were detected in B. cereus, whereas clpX, codY, purA, and purB were detected in B. subtilis, as corroborated by Bianco et al. (2021). This finding highlights the importance of genetic analysis in revealing the virulence factors of Bacillus species and their implications for pathogenicity. Additionally, a study by Qu et al. (2021) revealed the distribution of virulence genes in Bacillus cereus strains isolated from lettuce farms in China, with genes such as nheA, nheB, nheC, hblA, hblC, hblD, entFM, and cytK being prevalent among the strains. This highlights the pathogenic potential of Bacillus cereus strains in foodborne illnesses.

Moreover, in a study by Tagne et al. (2023), the antimicrobial resistance profiles of Bacillus strains isolated from environmental sources were evaluated, shedding light on the potential impact of seasonal variations on antimicrobial resistance patterns. This research emphasizes the dynamic nature of antimicrobial resistance in Bacillus species and underscores the need for continuous surveillance to monitor and address resistance mechanisms. By linking genetic characterization with antimicrobial resistance profiles, a study by Sornchuer et al. (2022) also provided valuable insights into the pathogenic potential of Bacillus strains and their implications for food safety and public health.

Multilocus sequence typing (MLST) further characterized B. cereus AH676 as belonging to sequence type 73 with 100% identity, identifying housekeeping gene alleles, including glp_13, gmk_8, and ilv_9, whereas Bacillus subtilis WAUSV36 was classified as sequence type 130, with the ilvD gene showing a perfect match and the highest allele length of 471 for housekeeping genes identified as similarly reported by Bianco et al. (2021). A study by Bianco et al. (2021) emphasized the importance of whole-genome sequencing and MLST in characterizing Bacillus cereus isolates, highlighting the genetic diversity within the species.

In a study by Yu et al. (2020), the prevalence and characterization of Bacillus cereus in ready-to-eat foods in China revealed antimicrobial resistance patterns, with isolates showing resistance to β-lactam antibiotics and rifamycin. These findings underscore the potential public health risks associated with the presence of antimicrobial-resistant Bacillus cereus strains in food products. Furthermore, Wang et al. (2022) provided insights into Bacillus cereus associated with infant foods in Beijing, identifying various virulence gene carriage patterns in B. cereus strains, with genes such as nhe and entFM being highly prevalent. This study highlights the potential risks posed by Bacillus cereus in food products targeted at vulnerable populations such as infants. Additionally, Hashhash (2023) highlighted the risk assessment of Bacillus cereus in cooked meat products, emphasizing the importance of accurate detection methods such as VITEK®2 and PCR for food safety.

Moreover, Carroll (2020) delved into the evolutionary history of Group III Bacillus cereus sensu lato, elucidating the transition between emetic and diarrheal foodborne pathogens, particularly focusing on cereulide synthetase acquisition and loss events within the species. This research provides valuable insights into the pathogenic mechanisms of Bacillus cereus strains causing foodborne illnesses (Carroll 2020).

Conclusions

By aligning the findings of the genetic makeup of Bacillus cereus and B. subtilis from food vendors in our study, the comprehensive genomic analysis of Bacillus species inferred a more holistic understanding of the genetic underpinnings of pathogenicity, antimicrobial resistance, and virulence in Bacillus strains. This information can contribute to the broader knowledge of Bacillus species, their genetic diversity, and the factors influencing their pathogenic potential, thereby informing strategies for the control and management of these bacteria in various settings. The genomic characteristics, antimicrobial resistance profiles, virulence gene distributions, and clinical implications of Bacillus cereus highlight the diverse nature of this bacterium and its importance in various settings, including food safety, healthcare-associated infections, and environmental contamination. Understanding the genetic makeup and pathogenic potential of Bacillus cereus is crucial for developing effective control and prevention strategies to mitigate the risks associated with this versatile bacterium in food contamination for food safety.