Molecular and Biochemical Characterization of the Chromosome-Encoded Class A β-Lactamase BCL-1 from Bacillus clausii

Abstract

A chromosomal ␤-lactamase gene from Bacillus clausii NR, which is used as a probiotic, was cloned and expressed in Escherichia coli. It encodes a clavulanic acid-susceptible Ambler class A ␤-lactamase, BCL-1, with a pI of 5.5 and a molecular mass of ca. 32 kDa. It shares 91% and 62% amino acid identity with the chromosomally encoded PenP penicillinases from B. clausii KSM-K16 and Bacillus licheniformis, respectively. The hydrolytic profile of this ␤-lactamase includes penicillins, narrow-spectrum cephalosporins, and cefpirome. This chromosome-encoded enzyme was inducible in B. clausii, and its gene is likely related to upstreamlocated regulatory genes that share significant identity with those reported to be upstream of the penicillinase gene of B. licheniformis. The bla BCL-1 gene was located next to the known chromosomal aadD2 gene and the erm34 gene, which encode resistance to aminoglycosides and macrolides, respectively. Similar genes were found in a collection of B. clausii reference strains.Bacillus species are used as probiotics in both humans and animals for the prevention of infectious bacterial diarrhea (8,10,18,28) or preneoplastic lesions (29). Enterogermina (SanofiAventis, Milan, Italy) is a pharmaceutical preparation containing an aqueous suspension of a mix of viable antibiotic-resistant alkaliphilic Bacillus clausii strains (NR, OC, SIN, and T) (10), initially identified as Bacillus subtilis (37). B. clausii spores are able to germinate after exposure to acidic pH in the stomach and to grow in the presence of bile and with a limited amount of oxygen, consistent with the beneficial health effect of probiotics (8). In their vegetative form, B. clausii strains are able to induce an immune response (39), as recently described for "Bacillus polyfermenticus" (22). Bacillus spores are used as probiotics, although they may carry antibiotic resistance determinants and express toxins (14,19). Resistance to erythromycin is one of the reported features of B. clausii (3, 10), due to the presence of an erm-related gene (5). B. clausii strains are also resistant to penicillin G, cephalothin, and cefotaxime (4).Therefore, it was interesting to determine the resistance determinants responsible for resistance phenotype in B. clausii. The study was initiated with one of the strains, B. clausii NR, present in the Enterogermina mixture and with the B. clausii reference strain ATCC 21537. A ␤-lactamase with an uncommon hydrolytic profile that included cefpirome was characterized. MATERIALS AND METHODSBacterial strains. B. clausii NR, SIN, T, OC, and DSM 8716 were from Sanofi-Aventis. B. clausii ATCC 21537 was used as a reference strain. Most of the work reported below was performed in parallel with B. clausii NR and ATCC 21537 strains. Escherichia coli DH10B and E. coli BL21(DE3) (Invitrogen, Life Technologies, Cergy-Pontoise, France) were used for cloning experiments and ␤-lactamase overexpression, respectively (17).Antimicrobial agents and MIC determinations. The antimicrobial agents and their sources are described ...

Bacillusprobiotics: an alternative to antibiotics for livestock production

“…Another concern that should be taken into consideration is the potential of some Bacillus strains to transfer antibiotic resistance genes within the GITs that might take place between probiotics and opportunistic or pathogenic bacteria. Genes conferring resistance to aminoglycosides (aadD2), macrolides (erm34), b-lactams (bla BCL-1 ) and chloramphenicol (cat Bcl ) were found in probiotic strains of B. clausii (Bozdogan et al 2003(Bozdogan et al , 2004Girlich et al 2007;Galopin et al 2009). b-lactams, chloramphenicol and tetracycline resistance determinants have also been reported in probiotic strains of B. cereus (Hoa et al 2000).…”

Antibiotic resistance in probiotic bacteria

“…For instance, the erm (34) gene has been identified in the probiotic Bacillus clausii DSM8716 strain (Bozdogan et al, 2004). Probiotic Bacillus clausii strains also harbor specific antibiotic-defense mechanisms, such as an aminoglycoside resistance gene ( aadD2 ), a chloramphenicol acetyltransferase gene, cat (Bcl) or a β-lactamase (BCL-1; Bozdogan et al, 2003; Girlich et al, 2007; Galopin et al, 2009). …”

Presence of blaCTX-M antibiotic resistance gene in Lactobacillus spp. isolated from Hirschsprung diseased infants with stoma

“…In connection to it, a few βlactam resistance genes naming blaZ, mecA and blaBCL-1 were reported in Gram-positive organisms, among which blaZ was found in Lactobacillus spp. [18,19]. Regardless the availability of the clinical data about the resistance pattern of different antibiotics in Lactobacillus spp.…”