ABSTRACT
Ciprofloxacin resistance in Bacillus cereus involves diverse and understudied mechanisms. Here, we present draft genome assemblies of 95 experimentally evolved Bacillus cereus strains that exhibit increased growth in the presence of ciprofloxacin, many containing novel mutations not previously described for this phenotype.
KEYWORDS: Bacillus cereus, antimicrobial resistance, ciprofloxacin, genome assembly, hybrid assembly
ANNOUNCEMENT
Bacillus cereus is a spore-forming, rod-shaped, gram-positive bacterium that can exist as a commensal or pathogenic organism in humans. Although B. cereus infections are most commonly associated with foodborne illness, it is also capable of causing severe infections in the eyes, lungs, and central nervous system. B. cereus is intrinsically resistant to β-lactam antibiotics, often leading to infections being treated with ciprofloxacin or vancomycin (1). However, the inevitable emergence of antibiotic resistance underscores the critical need to study the mechanisms underlying resistance evolution to inform the monitoring, prediction, and mitigation of potential future outbreaks (2).
To elucidate the genotypic changes associated with ciprofloxacin resistance, we have performed the experimental evolution of ciprofloxacin-resistant strains of B. cereus. The susceptible parent strain ATCC 14579 was subjected to 20 rounds of ciprofloxacin exposure in cation-adjusted Mueller Hinton Broth (CAMHB). Exposure occurred in a 96-well plate with ciprofloxacin concentrations ranging from 0.0625–32 µg/mL. The plate was covered with a lid and incubated for 22–24 h at 37°C in a humidified incubator. After 24 h, the wells were inspected for growth, and the modal minimum inhibitory concentration (MIC) was recorded. We then selected the culture at 0.5× MIC at each round for re-exposure. Following each selection cycle, single colonies were isolated from the cultures by plating, and the MICs were determined by the microdilution assay in accordance with CLSI M0-A10 protocols for antibiotic testing of aerobic bacteria. Genomic sequencing was performed on the resulting mutants.
For DNA preparation, bacterial strains were recovered from glycerol stocks by plating on Mueller Hinton agar and incubating overnight at 37°C. Single colonies were transferred to 50 mL of Mueller Hinton broth and grown overnight at 37°C with shaking at 180 rpm. The culture was centrifuged, and the pellet was used for DNA extraction using Qiacube “QIAamp Mini bacterial or yeast DNA with enzymatic lysis V1 protocol.” Enzymatic lysis was performed in 180 μL Tris-EDTA (50 mM Tris-Cl pH 8, 1 mM EDTA) (TE) buffer, 1.2% Triton, and 20 mg/mL lysozyme.
Sequencing libraries were prepared without shearing or size selection and using the Oxford Nanopore native barcoding sequencing kit (SQK-NDB114) and the Illumina Nextera XT kit and sequenced on the GridION vR10.4.1 and Illumina MiSeq (2 × 151-bp v2 kit) instruments, respectively. Base calling, demultiplexing, and adapter trimming for Nanopore data were performed using Dorado v0.5.2 (3) with the super high accuracy model. Additional QC filtering was performed using FiltLong (4) 0.2.1. MiSeq quality filters were used for QC, and adapter sequences were removed from the Illumina data using fastp v0.23.2 (5). All data were assembled using Flye v2.9 (6), with one round of long-read polishing using Medaka (7), polished using Polypolish v0.5.0 (8) and Pypolca v2.1.0 (9). Plasmids were assembled using Plassembler v1.62 (10). The hybrid genome was annotated using Prokka v1.14.5 (11), and reference mapping was performed using Minimap2 (12) for the Nanopore data and BWA (13) for the Illumina data. Default parameters were used for all software except Minimap2 (-ax for long-read data). Assessment using CheckM v1.1.6 and CheckM v2 v1.0.2 estimated that all genomes were >99% complete and <3% contaminated (14).
ACKNOWLEDGMENTS
Funding for this work was provided by the Department of Defense (DoD) Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense (JPEO-CBRND), JPL (CBRND) Enabling Biotechnologies, and Defense Biological Product Assurance Office under NAVSEA contract number N00024-22-D-6404. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the JPEO-CBRND, the Departments of the Army, Navy, or Defense, nor the U.S. Government. References to non-federal entities do not constitute or imply Department of Defense or Army endorsement of any company or organization.
Contributor Information
Rachael Sparklin, Email: Rachael.sparklin@jhuapl.edu.
André O. Hudson, Rochester Institute of Technology, Rochester, New York, USA
DATA AVAILABILITY
The accession numbers and sequence statistics of the 95 genomes across the ciprofloxacin re-exposure cultures are listed in Table 1. Data are deposited in NCBI SRA under BioProject accession number PRJNA1188493 and BioSample accessions SAMN44839382-SAMN44839476, and assembled genomes in Genbank under WGS accessions JBJJPB000000000-JBJJSR000000000.
TABLE 1.
Genome sequence details of B. cereus ciprofloxacin-exposed derivativesa
| Illumina data | ONT data | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Abx exp. no. | Modal MIC (ug/mL) |
Sample no. | SRA accession no. |
Total reads | SRA accession no. |
Total reads | ONT N50 | GenBank WGS accession no. | Assembly size (bp) | No. of contigs |
No. of CDSs | Depth of coverage (X) | Breadth of coverage |
| P1 | 0.125 | 1 | SRR31480868 | 1,702,794 | SRR31480826 | 147,552 | 7,306 | JBJJSR000000000 | 5,450,604 | 3 | 5,438 | 244 | 100 |
| C4 | 0.5 | 13 | SRR31480809-SRR31480814, SRR31480815-SRR31480822 | 937,901.5 ± 388,956.8 | SRR31480923-SRR31480924, SRR31480957-SRR31480963, SRR31480965-SRR31480968 | 87,786.0 ± 48,783.4 | 13,685.9 ± 6,084.6 | JBJJP[W-Z]000000000, JBJJQ[A-I]000000000 | 5,187,781.4 ± 48,861.0 | 4.1 ± 1.8 | 5,175.7 ± 45.2 | 217.7 ± 34.9 | 100.0 ± 0.0 |
| C6 | 2 | 3 | SRR31480806-SRR31480808 | 1,564,483.5 ± 739,312.0 | SRR31480919-SRR31480920, SRR31480922 | 109,370.5 ± 75,253.8 | 11,965.0 ± 7,861.6 | JBJJQ[J-L]000000000 | 5,1095,82.5 ± 74,682.5 | 7.0 ± 2.8 | 5,104.5 ± 74.2 | 228.5 ± 3.5 | 100.0 ± 0.0 |
| C7 | 2 | 5 | SRR31480800-SRR31480801, SRR31480803-SRR31480805 | 1,125,224.6 ± 332,287.8 | SRR31480914-SRR31480918 | 82,699.2 ± 53,570.3 | 10,787.6 ± 6,636.8 | JBJJQ[M-Q]000000000 | 5,059,125.4 ± 96,557.6 | 11.2 ± 7.8 | 5,067.6 ± 82.9 | 183.0 ± 79.4 | 100.0 ± 0.0 |
| C8 | 4 | 21 | SRR31480935-SRR31480937, SRR31480939-SRR31480948, SRR31480950-SRR31480956, SRR31480799 | 1,271,234.1 ± 424,687.7 | SRR31480861-SRR31480862, SRR31480893-SRR31480898, SRR31480900-SRR31480909, SRR31480911, SRR31480913 | 91,375.6 ± 59,276.7 | 12,533.0 ± 4,224.9 | JBJJQ[R-Z]000000000, JBJJR[A-L]000000000 | 5,061,076.9 ± 52,715.8 | 6.9 ± 2.9 | 5,062.6 ± 55.6 | 241.2 ± 50.5 | 100.0 ± 0.0 |
| C9 | 8 | 31 | SRR31480869-SRR31480872, SRR31480874-SRR31480883, SRR31480885-SRR31480892, SRR31480925-SRR31480926, SRR31480928-SRR31480934 | 1,064,004.1 ± 316,069.5 | SRR31480827-SRR31480835, SRR31480837-SRR31480846, SRR31480848-SRR31480857, SRR31480859-SRR31480860 | 99,457.8 ± 76,133.9 | 15,576.9 ± 8,038.4 | JBJJR[M-Z]000000000, JBJJS[A-Q]000000000 | 5,066,966.4 ± 68,586.6 | 8.4 ± 5.8 | 5,069.3 ± 71.8 | 241.4 ± 28.8 | 100.0 ± 0.0 |
| C13 | 16 | 7 | SRR31480938, SRR31480949, SRR31480802, SRR31480813, SRR31480824, SRR31480987-SRR31480988 | 1,217,210.4 ± 325,061.0 | SRR31480984-SRR31480985, SRR31480863-SRR31480867 | 117,176.0 ± 37,654.9 | 9,717.0 ± 3,846.2 | JBJJP[B-H]000000000 | 5,048,248.4 ± 13,234.9 | 5.4 ± 2.4 | 5,035.3 ± 4.3 | 268.3 ± 28.7 | 100.0 ± 0.0 |
| C15 | 16 | 2 | SRR31480927, SRR31480884 | 1,226,321.0 ± 101,181.3 | SRR31480982-SRR31480983 | 142,016.0 ± 15,289.1 | 6,991.0 ± 554.4 | JBJJP[I-J]000000000 | 5,017,976.0 ± 30,578.1 | 7.5 ± 2.1 | 4,979.0 ± 32.5 | 296.0 ± 2.8 | 100.0 ± 0.0 |
| C16 | 32 | 5 | SRR31480921, SRR31480964, SRR31480975, SRR31480986, SRR31480873 | 879,075.8 ± 107,032.1 | SRR31480977-SRR31480981 | 106,502.2 ± 31,171.0 | 10,664.0 ± 3,701.3 | JBJJP[K-O]000000000 | 5,075,772.4 ± 60,766.6 | 10.0 ± 3.2 | 5,069.4 ± 65.5 | 243.0 ± 52.2 | 100.0 ± 0.0 |
| C17 | 32 | 4 | SRR31480847, SRR31480858, SRR31480899, SRR31480910 | 917,156.5 ± 399,580.1 | SRR31480972-SRR31480976 | 160,114.2 ± 29,960.6 | 6,057.8 ± 1,697.5 | JBJJP[P-S]000000000 | 5,035,608.8 ± 47,085.5 | 11.2 ± 7.9 | 5,019.8 ± 59.8 | 229.0 ± 56.2 | 100.0 ± 0.0 |
| C20 | 32 | 3 | SRR31480823, SRR31480825, SRR31480836 | 1,083,012.0 ± 250,219.4 | SRR31480969-SRR31480971 | 228,769.3 ± 65,182.2 | 4,034.0 ± 1,162.9 | JBJJP[T-V]000000000 | 4,858,822.0 ± 156,534.1 | 20.7 ± 17.2 | 4,846.3 ± 158.2 | 182.7 ± 50.5 | 100.0 ± 0.0 |
Antibiotic exposure number (P1 = parental strain, C# = exposure passage number), modal MIC for all colonies sequenced, the number of colonies from each exposure (sample no), and sequencing statistics are provided.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The accession numbers and sequence statistics of the 95 genomes across the ciprofloxacin re-exposure cultures are listed in Table 1. Data are deposited in NCBI SRA under BioProject accession number PRJNA1188493 and BioSample accessions SAMN44839382-SAMN44839476, and assembled genomes in Genbank under WGS accessions JBJJPB000000000-JBJJSR000000000.
TABLE 1.
Genome sequence details of B. cereus ciprofloxacin-exposed derivativesa
| Illumina data | ONT data | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Abx exp. no. | Modal MIC (ug/mL) |
Sample no. | SRA accession no. |
Total reads | SRA accession no. |
Total reads | ONT N50 | GenBank WGS accession no. | Assembly size (bp) | No. of contigs |
No. of CDSs | Depth of coverage (X) | Breadth of coverage |
| P1 | 0.125 | 1 | SRR31480868 | 1,702,794 | SRR31480826 | 147,552 | 7,306 | JBJJSR000000000 | 5,450,604 | 3 | 5,438 | 244 | 100 |
| C4 | 0.5 | 13 | SRR31480809-SRR31480814, SRR31480815-SRR31480822 | 937,901.5 ± 388,956.8 | SRR31480923-SRR31480924, SRR31480957-SRR31480963, SRR31480965-SRR31480968 | 87,786.0 ± 48,783.4 | 13,685.9 ± 6,084.6 | JBJJP[W-Z]000000000, JBJJQ[A-I]000000000 | 5,187,781.4 ± 48,861.0 | 4.1 ± 1.8 | 5,175.7 ± 45.2 | 217.7 ± 34.9 | 100.0 ± 0.0 |
| C6 | 2 | 3 | SRR31480806-SRR31480808 | 1,564,483.5 ± 739,312.0 | SRR31480919-SRR31480920, SRR31480922 | 109,370.5 ± 75,253.8 | 11,965.0 ± 7,861.6 | JBJJQ[J-L]000000000 | 5,1095,82.5 ± 74,682.5 | 7.0 ± 2.8 | 5,104.5 ± 74.2 | 228.5 ± 3.5 | 100.0 ± 0.0 |
| C7 | 2 | 5 | SRR31480800-SRR31480801, SRR31480803-SRR31480805 | 1,125,224.6 ± 332,287.8 | SRR31480914-SRR31480918 | 82,699.2 ± 53,570.3 | 10,787.6 ± 6,636.8 | JBJJQ[M-Q]000000000 | 5,059,125.4 ± 96,557.6 | 11.2 ± 7.8 | 5,067.6 ± 82.9 | 183.0 ± 79.4 | 100.0 ± 0.0 |
| C8 | 4 | 21 | SRR31480935-SRR31480937, SRR31480939-SRR31480948, SRR31480950-SRR31480956, SRR31480799 | 1,271,234.1 ± 424,687.7 | SRR31480861-SRR31480862, SRR31480893-SRR31480898, SRR31480900-SRR31480909, SRR31480911, SRR31480913 | 91,375.6 ± 59,276.7 | 12,533.0 ± 4,224.9 | JBJJQ[R-Z]000000000, JBJJR[A-L]000000000 | 5,061,076.9 ± 52,715.8 | 6.9 ± 2.9 | 5,062.6 ± 55.6 | 241.2 ± 50.5 | 100.0 ± 0.0 |
| C9 | 8 | 31 | SRR31480869-SRR31480872, SRR31480874-SRR31480883, SRR31480885-SRR31480892, SRR31480925-SRR31480926, SRR31480928-SRR31480934 | 1,064,004.1 ± 316,069.5 | SRR31480827-SRR31480835, SRR31480837-SRR31480846, SRR31480848-SRR31480857, SRR31480859-SRR31480860 | 99,457.8 ± 76,133.9 | 15,576.9 ± 8,038.4 | JBJJR[M-Z]000000000, JBJJS[A-Q]000000000 | 5,066,966.4 ± 68,586.6 | 8.4 ± 5.8 | 5,069.3 ± 71.8 | 241.4 ± 28.8 | 100.0 ± 0.0 |
| C13 | 16 | 7 | SRR31480938, SRR31480949, SRR31480802, SRR31480813, SRR31480824, SRR31480987-SRR31480988 | 1,217,210.4 ± 325,061.0 | SRR31480984-SRR31480985, SRR31480863-SRR31480867 | 117,176.0 ± 37,654.9 | 9,717.0 ± 3,846.2 | JBJJP[B-H]000000000 | 5,048,248.4 ± 13,234.9 | 5.4 ± 2.4 | 5,035.3 ± 4.3 | 268.3 ± 28.7 | 100.0 ± 0.0 |
| C15 | 16 | 2 | SRR31480927, SRR31480884 | 1,226,321.0 ± 101,181.3 | SRR31480982-SRR31480983 | 142,016.0 ± 15,289.1 | 6,991.0 ± 554.4 | JBJJP[I-J]000000000 | 5,017,976.0 ± 30,578.1 | 7.5 ± 2.1 | 4,979.0 ± 32.5 | 296.0 ± 2.8 | 100.0 ± 0.0 |
| C16 | 32 | 5 | SRR31480921, SRR31480964, SRR31480975, SRR31480986, SRR31480873 | 879,075.8 ± 107,032.1 | SRR31480977-SRR31480981 | 106,502.2 ± 31,171.0 | 10,664.0 ± 3,701.3 | JBJJP[K-O]000000000 | 5,075,772.4 ± 60,766.6 | 10.0 ± 3.2 | 5,069.4 ± 65.5 | 243.0 ± 52.2 | 100.0 ± 0.0 |
| C17 | 32 | 4 | SRR31480847, SRR31480858, SRR31480899, SRR31480910 | 917,156.5 ± 399,580.1 | SRR31480972-SRR31480976 | 160,114.2 ± 29,960.6 | 6,057.8 ± 1,697.5 | JBJJP[P-S]000000000 | 5,035,608.8 ± 47,085.5 | 11.2 ± 7.9 | 5,019.8 ± 59.8 | 229.0 ± 56.2 | 100.0 ± 0.0 |
| C20 | 32 | 3 | SRR31480823, SRR31480825, SRR31480836 | 1,083,012.0 ± 250,219.4 | SRR31480969-SRR31480971 | 228,769.3 ± 65,182.2 | 4,034.0 ± 1,162.9 | JBJJP[T-V]000000000 | 4,858,822.0 ± 156,534.1 | 20.7 ± 17.2 | 4,846.3 ± 158.2 | 182.7 ± 50.5 | 100.0 ± 0.0 |
Antibiotic exposure number (P1 = parental strain, C# = exposure passage number), modal MIC for all colonies sequenced, the number of colonies from each exposure (sample no), and sequencing statistics are provided.