Antimicrobial susceptibility of Bacillus strains isolated from primary starters for African traditional bread production and characterization of the bacitracin operon and bacitracin biosynthesis

Abstract

Bacillus spp. are widely used as feed additives and probiotics. However, there is limited information on their resistance to various antibiotics, and there is a growing concern over the transfer of antibiotic resistance genes. The MIC for 8 antibiotics was determined for 85 Bacillus species strains, Bacillus subtilis subsp. subtilis (n = 29), Bacillus licheniformis (n = 38), and Bacillus sonorensis (n = 18), all of which were isolated from starters for Sudanese bread production. All the strains were sensitive to tetracycline (8.0 mg/liter), vancomycin (4.0 mg/liter), and gentamicin (4.0 mg/liter) but resistant to streptomycin. Sensitivity to clindamycin, chloramphenicol, and kanamycin was species specific. The erythromycin resistance genes ermD and ermK were detected by PCR in all of the erythromycin-resistant (MIC, ≥16.0 mg/liter) B. licheniformis strains and one erythromycin-sensitive (MIC, 4.0 mg/liter) B. licheniformis strain. Several amino acid changes were present in the translated ermD and ermK nucleotide sequences of the erythromycin-sensitive strain, which could indicate ErmD and ErmK protein functionalities different from those of the resistance strains. The ermD and ermK genes were localized on an 11.4-kbp plasmid. All of the B. sonorensis strains harbored the bacitracin synthetase gene, bacA, and the transporter gene bcrA, which correlated with their observed resistance to bacitracin. Bacitracin was produced by all the investigated species strains (28%), as determined by ultra-high-definition quadrupole time-of-flight liquid chromatography-mass spectrometry (UHD-QTOF LC/MS). The present study has revealed species-specific variations in the antimicrobial susceptibilities of Bacillus spp. and provides new information on MIC values, as well as the occurrence of resistance genes in Bacillus spp., including the newly described species B. sonorensis.

Fig 1

Erythromycin resistance determinant genes (ermD, ermK) in Bacillus licheniformis and reference strains. Lanes: S, Fermentas 1-kb DNA ladder (SM0313); 0, negative control; DK, B. subtilis pEC 1001, ermD-positive strain. The B. licheniformis strains with an erythromycin MIC of ≥16.0 mg/liter are C34, C38, F33, W43, and W44. B. licheniformis strains with an erythromycin MIC of 4.0 mg/liter are C20, C23, L2, L7, L27, L28, L43, L44, F10, and F15.

Fig 2

Protein sequence alignments of Bacillus licheniformis C8 ErmD and ErmK with ErmD, ErmJ, and ErmK amino acid sequences retrieved from the GenBank database. ErmD and ErmK are the homologous region of the ErmD and ErmK erythromycin resistance proteins. The star with an arrow pointed toward it indicates the position of the termination of protein synthesis in the ErmD and ErmK amino acid sequence.

Fig 3

Agarose gel electrophoresis of plasmid DNA isolated from erythromycin-resistant Bacillus licheniformis strains. Lanes: ST, Fermentas 1-kb DNA ladder (SM0313); 0, negative control; JH, Enterococcus faecalis JH2-2 Tn1545. Gel electrophoresis is also shown for B. licheniformis strains C34, C32, W18, C39, W22, F33, F34, and W17.

Fig 4

(A) PCR detection of the bacitracin synthetase gene bacA in Bacillus species strains. (B) Multiplex PCR detection of bacitracin ABC transporter genes. Band 1, bcrA; band 2, bcrB; band 3, bcrC in Bacillus species strains. The bcrABC-positive reference strains are B. licheniformis DSM 603 (ATCC 10716) and B. licheniformis 5A24 (ATCC 11946); the bcrABC-negative reference strain is B. licheniformis 5A36 (ATCC 14580). Some of the strains studied are B. licheniformis strains C32, C34, C39, W37, and F50, B. subtilis strains C42, C50, F14, F19, and W25, and B. sonorensis strains C1, C2, C3, F1, and F2.

Fig 5

(A) Chromatograms of bacitracin A produced by B. sonorensis strain C2 (19-h incubation in BHI broth) in comparison with a 20-ppm bacitracin standard. (B) Full-scan mass spectrum of bacitracin A standard chromatogram showing the molecular mass of bacitracin A (1,421.8 U). (C) Full-scan mass spectrum of bacitracin A produced by B. sonorensis strain C2 showing the molecular mass of bacitracin A. Triple-charge ions were obtained for bacitracin A, as indicated in the mass spectrum. The bacitracin was detected by UHD-QTOF LC/MS.