Insight from the genome sequence of Bacillus cereus BUKA harboring bla2 gene

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Abstract

Introduction. In developing countries, yearly outbreaks of acute gastroenteritis records high rates of morbidity and mortality in children below five years.

Aim: present the whole genome sequence of Bacillus cereus BUKA implicated in acute childhood gastroenteritis.

Materials and methods. The sample was isolated from diarrheal stool of a child diagnosed with acute childhood gastroenteritis from a tertiary hospital in Nigeria.

Results. Antibiotics resistance profiling of the isolate revealed resistance to 79% (11/14) of the tested antibiotics including imipenem, penicillin, cefepime, ceftriaxone which suggests a strong ability to produce extended spectrum beta-lactamases, carbapenemases and cephalosporinases. Whole genome sequence insight confirmed the presence of chromosomally-encoded bla2 gene encoding enzyme that hydrolyze carbapenems, penicillin and cephalosporins. B. cereus encoding bla2 gene may be an emerging pathogen in the yearly incidence of acute childhood gastroenteritis in Nigeria.

Discussion. We herein report and implicate B. cereus harboring bla2 gene capable of hydrolyzing cephalosporins, and carbapenems as an emerging threat in acute childhood gastroenteritis in Nigeria

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Introduction

Worldwide, it is estimated that 1.4–12.0% of food poisoning outbreaks characterised as gastroenteritis are caused by Bacillus cereus [1–3]. There are few reports of childhood diarrheal cases linked to B. cereus in Nigeria and other developing nations [4, 5] due to lack of an effective surveillance system. The fact that the symptoms of food poisoning caused by Clostridium perfringens and Staphylococcus aureus are comparable to that of B. cereus further compounds this problem [6]. For the first time in Nigeria, we recently implicated multi-drug resistant B. cereus predominantly encoding nheA, hblC and cytK enterotoxigenic genes as the possible etiology of the yearly incidence of acute childhood gastroenteritis that occur during “harmattan season” in Nigeria [7]. The widespread use of broad-spectrum antibiotics in the empirical treatment of acute childhood gastroenteritis in Nigeria [8] and other developing countries [9] have exacerbated antibiotic resistance amongst implicated bacteria [10]. B. cereus exhibiting resistance to beta-lactam antibiotics via the bla or bla2 gene with reports of resistance to several antibiotics including ciprofloxacin, tetracycline, amikacin, and chloramphenicol have also been reported [11, 12].

The aim of this study is to present the genome sequence of a Bacillus cereus BUKA harboring metallo-β-lactamase capable of hydrolysing all β-lactams antibiotics.

Materials and methods

The B. cereus was one of 24 isolates from the stool sample of children with acute gastroenteritis as earlier reported [7]. The isolate exhibited strong multi-drug resistance and was therefore isolated for further genomic analysis. Antibiotic susceptibility testing was performed by the disk diffusion method according to the Clinical and Laboratory Standards Institute guidelines 2021 on Muller–Hinton agar [13]. After 24 h of growth at 37°C, the isolate was evaluated for its susceptibility to levofloxacin (5 μg), ceftriaxone (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), amoxicillin-clavulanic acid (20/10 μg), penicillin (10 μg), cefepime (30 μg), azetronam, imipenem (10 μg), meropenem, oxacillin, vancomycin, erythromycin, cefoxitin. The Wizard Genomic Purification kit (Promega) was used to extract the genomic DNA in accordance with the manufacturer's instructions. The bacterial 16S rRNA gene was amplified using pure DNA as a template and the previously known primers 27F (5ʹ AGAGTTTGATCMTGGCTCAG-3ʹ) and 1525R (5′-AAGGAGGTGWTCCARCCGCA-3′) [14]. The purified genomic DNA was used for whole-genome sequencing on Illumina HiSeq2500 (Paired-end 150 nt ~ 20 million reads/sample) (Illumina). The generated paired-end reads were quality controlled and filtered using Fastp v. 0.12.4 [15]. SPAdes v. 3.15.4 was used for de novo assembly of the filtered reads [16]. Completeness and contamination of the assembly were verified with CheckM v. 1.2.0 [17], while the quality of the assembly was evaluated with QUAST v. 5.0.2 [18]. PROKKA v. 1.14.6 [19] was used to annotate the assembled genome. Based on sequence homology, resistomes were predicted using The Comprehensive Antibiotics Resistance Database's Resistant Gene Identifier (CARD- RGI) 6.0.3 [20]. The genomic map was created and displayed using Proksee, as previously mentioned [21]. Pairwise average nucleotide identity (ANI) values, clustering, and dendogram were calculated using other Bacillus spp. whole genomes available in the NCBI database using dRep with FastANI and MASH algorithms [22].

Results

The genome of B. cereus BUKA had a total size of 4.26 Mbp (GC content of 37.15%) and had a chromosome with 3856 coding sequences (81 contigs), 52 tRNA genes, 3 rRNA genes (1 5S, 2 16S), 4 ncRNAs, and 25 pseudogenes; completeness — 99.23%, contamination — 0.04%. The circular genomic map of the chromosomes showing the location of the bla2 gene is presented in Fig. 1. Sequence analysis of this bla2 sequence and other closely related bla2 genes from other B. cereus group could not detect any point mutation (result not shown). The isolate was resistant to 11/14 antibiotics tested which included zidovudine, ceftazidime, ceftriaxone, ceftazidime, cefepime, imipenem, meropenem, oxacillin, erythromycin, amoxicillin-clavulanic acid, cefoxitin; intermediate to 2/14 antibiotics, ciprofloxacin and levofloxacin; susceptible to one 1/14 antibiotic, vancomycin. In the Table, several antibiotics resistant genes clusters were identified, however the major antibiotics resistant genes with more than 80% sequence identity and coverage to the reference sequence were identified as mphL (macrolide phosphotransferase), bla2 (a sub-class B1 beta-lactamase), fosB (fosfomycin thiol transferase), vanR (a vanA glycopepetide resistant cluster) and bc1 (class A Bc beta-lactamase). The ANI-based dendogram placed B. cereus BUKA within the same cluster as B. anthracis (Fig. 2).

 

Fig. 1. Circular genome map showing the location of the bla2 gene.

 

B. cereus antibiotics resistant gene clusters

AMR gene

Identity, %

Coverage, %

AMR gene family

Drug Class

Resistance mechanism

qacJ

40.19

112.15

SMR efflux pump

Disinfecting and antiseptics

Efflux

tetB(P)

45.52

99.23

Tet resistance ribosomal protection

Tetracycline

Target protection

mphL

87.58

98.35

Macrolide phosphotransferase

Macrolide

Activation

vanY (in vanB cluster)

34.87

96.64

Glycopeptide resistance cluster

Glycopeptide

Target alteration

vanW (in vanI cluster)

37.78

81.23

Glycopeptide resistance cluster

Glycopeptide

Target alteration

Bla2

98.05

100

BCll family subclass B1 beta-lactamase

Penem, cephalosporins, penam

Inactivation

fosB

90.58

100

Fosfomycin thiol transferase

Phosphonic acid

Inactivation

vanY (in vanA cluster)

40.69

81.19

Glycopeptide resistance cluster

Glycopeptide

Target alteration

vanT (in vanG cluster)

34.38

57.3

Glycopeptide resistance cluster

Glycopeptide

Target alteration

vanW (in vanI cluster)

28.53

112.6

Glycopeptide resistance cluster

Glycopeptide

Target alteration

vanY (in vanF cluster)

56.36

97.61

Glycopeptide resistance cluster

Glycopeptide

Target alteration

vanY (in vanA cluster)

65.81

42.9

Glycopeptide resistance cluster

Glycopeptide

Target alteration

vanR (in vanA cluster)

90.04

100

Glycopeptide resistance cluster

Glycopeptide

Target alteration

Bc1

88.24

108.5

Class A Bc beta-lactamase

Cephalosporin, penam

Inactivation

qacJ

39.42

100

SMR efflux pump

Disinfecting and antiseptics

efflux

vanT (in vanG cluster)

30.87

54.63

Glycopeptide resistance cluster

Glycopeptide

target alteration

Note. SMR — small multidrug resistance; AMR — antimicrobial resistance.

 

Fig. 2. ANI-based phylogenetic clusters.

 

Discussion

Bla2 is an alternative name for Ambler BcII family belonging to the sub-class B1 metallo-beta- lactamase specifically named BcII-4. Metallo-β-lactamases play a pivotal role in the issue of antibiotic resistance by hydrolyzing carbapenems, penicillins, and cephalosporins [23]. Bla2 is expressed in B. anthracis and strongly in B. cereus and B. thuringiensis [24]. In Bacillus spp., the presence of the two β-lactamase genes, bla1 and bla2 were first reported in a penicillin-susceptible Sterne strain of B. anthracis, while bla2 has cephalosporinase, penicillinase, and carbapenem-hydrolyzing properties, bla1 has penicillinase activity [24, 25]. This supports our ANI-based clustering where our isolate was found closely related to B. anthracis (Fig. 2). In aquaculture, newly emerging MDR virulent B. cereus in M. seheli have been reported to carry bla1 and bla2 genes [26]. Most recently, β-lactam resistance genes, notably bla1, bla2, BcI, BcII, and blaTEM-116, were also detected in B. anthracis strains isolated from various food products in China [27].

The bla2 gene conferring resistance to beta-lactam antibiotics from B. cereus originating from commercial vitamin B2 feed, food additives, ready-to-eat and different food sources in Belgium, Poland and Italy have also been reported [11, 28, 29]. Reports of childhood diarrheal cases linked to B. cereus in Nigeria and other developing nations are lacking due to lack of molecular testing to determine the bacterial species and the genetic determinants of drug resistance. This has created a serious health gap resulting to a yearly incidence of acute childhood gastroenteritis leading to death of children across Nigeria and other countries across Africa. The prevalence of pathogenic B. cereus in cereal-based infant foods and retail foods have been reported in Burkina Faso [30] and Nigeria [31], but genome based antibiotic resistance profiling was not employed. A recent study that investigated B. cereus and B. subtilis from food vendors in Ondo State, Nigeria, employed whole genome sequence analysis but bla2 gene was not detected [32]. Furthermore, the presence of mphl gene (a macrolide) with high percentage identity and coverage justifies the antibiotics resistance observed with erythromycin. In contrast, despite observing the main glycopeptide resistant gene, vanR (in the vanA cluster), the isolate was susceptible to vancomycin, suggesting that the resistant gene was not expressed. Hence, to the best of our knowledge, we herein report and implicate B. cereus harboring bla2 gene capable of hydrolyzing cephalosporins, and carbapenems as an emerging threat in acute childhood gastroenteritis in Nigeria. Further studies on the trans-acting regulators of the bla2 genes [33] is required to reveal possible mechanism of expression, as no point mutation was found within the bla2 gene.

Conclusion

The first version of the whole genome assembly has been deposited in DDBJ/ENA/GenBank under the accession number JAPTHW000000000 (BioProject accession number PRJNA906446 and BioSample accession number SAMN31922703). The B. cereus 16S rRNA sequence was deposited with GenBank accession number OP650104 as strain BU14, while the antibiotics resistant genes were deposited with the accession numbers, PP801245, PP801246, PP801247, PP801248, PP801249 for mphL, bla2, fosB, vanR and Bc1 respectively. The data are simultaneously made available to other INSDC databases, the European Nucleotide Archive and the DNA Data Bank of Japan.

×

About the authors

Ebuka David

Alex Ekwueme Federal University Ndufu Alike Ikwo

Author for correspondence.
Email: david.ebuka@funai.edu.ng
ORCID iD: 0000-0001-6903-6392

PhD, senior lecturer, Department of biochemistry

Nigeria, Ikwo

Ikechukwu Igwenyi

Ebonyi State University

Email: igwenyike@yahoo.com
ORCID iD: 0000-0002-2056-751X

PhD, Professor, Department of biochemistry

Nigeria, Abakaliki

Ifeanyichukwu Iroha

Ebonyi State University

Email: iriroha@yahoo.com
ORCID iD: 0000-0002-8198-3955

PhD, Professor, Department of microbiology

Nigeria, Abakaliki

Uchenna Ezeilo

Alex Ekwueme Federal University Ndufu Alike Ikwo

Email: uchenna.ezeilo@funai.edu.ng
ORCID iD: 0000-0001-7022-8922

PhD, senior lecturer, Department of biochemistry

Nigeria, Ikwo

Moses Ogbanshi

Alex Ekwueme Federal University Ndufu Alike Ikwo

Email: ogbanshi.moses@funai.edu.ng
ORCID iD: 0000-0001-9955-2785

PhD, Associate Professor, Department of biochemistry

Nigeria, Ikwo

David Obasi

David Umahi Federal University of Health Sciences

Email: obasidc@dufuhs.edu.ng
ORCID iD: 0000-0003-4612-196X

PhD, senior lecturer, Head, Department of biochemistry

Nigeria, Uburu

Jerius Ejeje

Alex Ekwueme Federal University Ndufu Alike Ikwo

Email: ejeje.jerius@funai.edu.ng
ORCID iD: 0000-0001-8224-9579

PhD, lecturer, Department of biochemistry

Nigeria, Ikwo

Shedrack Ogunwa

Alex Ekwueme Federal University Ndufu Alike Ikwo

Email: ogunwa.shedrack@funai.edu.ng
ORCID iD: 0009-0009-8438-610X

PhD, lecturer, Department of biochemistry

Nigeria, Ikwo

Tochukwu Onyemuche

Alex Ekwueme Federal University Ndufu Alike Ikwo

Email: tochukwu.onyemuche@funai.edu.ng
ORCID iD: 0009-0007-0296-7134

PhD, lecturer, Department of biochemistry

Nigeria, Ikwo

Shedrach Kanu

Alex Ekwueme Federal University Ndufu Alike Ikwo

Email: kanu.shedrack@funai.edu.ng
ORCID iD: 0000-0001-7927-8432

PhD, lecturer, Department of biochemistry

Nigeria, Ikwo

Chidinma Ezennaya

Alex Ekwueme Federal University Ndufu Alike Ikwo

Email: felicia.ezennaya@funai.edu.ng
ORCID iD: 0000-0002-2945-4925

lecturer, Department of biochemistry

Nigeria, Ikwo

Stanley Alugbuo

Alex Ekwueme Federal University Ndufu Alike Ikwo

Email: alugbuo.udochukwu@funai.edu.ng
ORCID iD: 0000-0002-1074-1553

lecturer, Department of biochemistry

Nigeria, Ikwo

Sunday Igboke

Alex Ekwueme Federal University Ndufu Alike Ikwo

Email: igboke.sunday@funai.edu.ng
ORCID iD: 0009-0002-6200-2004

lecturer, Department of biochemistry

Nigeria, Ikwo

Chidinma David

Universidade Federal de São Paulo

Email: nnenne.nnaji@gmail.com
ORCID iD: 0000-0002-2550-4614

PhD Fellow, Escola Paulista de Medicina

Brazil, São Paulo

Aline da Silva

Universidade Federal de São Paulo

Email: almsilva@iq.usp.br
ORCID iD: 0000-0001-9249-4922

PhD, Professor, Department of biochemistry, Institute of Chemistry, Escola Paulista de Medicina

Brazil, São Paulo

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Supplementary files

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2. Fig. 1. Circular genome map showing the location of the bla2 gene.

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3. Fig. 2. ANI-based phylogenetic clusters.

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Copyright (c) 2026 David E., Igwenyi I., Iroha I., Ezeilo U., Ogbanshi M., Obasi D., Ejeje J., Ogunwa S., Onyemuche T., Kanu S., Ezennaya C., Alugbuo S., Igboke S., David C., da Silva A.

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