Genus-Wide Assessment of Antibiotic Resistance in Lactobacillus spp

Abstract

Lactobacillus species are widely used as probiotics and starter cultures for a variety of foods, supported by a long history of safe usage. Although more than 35 species meet the European Food Safety Authority (EFSA) criteria for qualified presumption of safety status, the safety of Lactobacillus species and their carriage of antibiotic resistance (AR) genes is under continuing ad hoc review. To comprehensively update the identification of AR in the genus Lactobacillus, we determined the antibiotic susceptibility patterns of 182 Lactobacillus type strains and compared these phenotypes to their genotypes based on genome-wide annotations of AR genes. Resistances to trimethoprim, vancomycin, and kanamycin were the most common phenotypes. A combination of homology-based screening and manual annotation identified genes encoding resistance to aminoglycosides (20 sequences), tetracycline (18), erythromycin (6), clindamycin (60), and chloramphenicol (42). In particular, the genes aac(3) and lsa, involved in resistance to aminoglycosides and clindamycin, respectively, were found in Lactobacillus spp. Acquired determinants predicted to code for tetracycline and erythromycin resistance were detected in Lactobacillus ingluviei, Lactobacillus amylophilus, and Lactobacillus amylotrophicus, flanked in the genome by mobile genetic elements with potential for horizontal transfer.IMPORTANCELactobacillus species are generally considered to be nonpathogenic and are used in a wide variety of foods and products for humans and animals. However, many of the species examined in this study have antibiotic resistance levels which exceed those recommended by the EFSA, suggesting that these cutoff values should be reexamined in light of the genetic basis for resistance discussed here. Our data provide evidence for rationally revising the regulatory guidelines for safety assessment of lactobacilli entering the food chain as starter cultures, food preservatives, or probiotics and will facilitate comprehensive genotype-based assessment of strains for safety screening.

Keywords: Lactobacillus; antimicrobial resistance; genomics.

FIG 1

Resistance profiles of 182 type strains of the genus Lactobacillus compared with epidemiological cutoff values provided in references , , and . Resistant strains with MIC values higher than the ECOFF are indicated in green, whereas sensitive strains are depicted in gray. Strains are clustered by the phylogroups reported by references and and are demarcated by the colored bar on the left of the heat plot. GM, gentamicin; KM, kanamycin; SM, streptomycin; NM, neomycin; TC, tetracycline; EM, erythromycin; CL, clindamycin; CM, chloramphenicol; AM, ampicillin; PC, penicillin; VA, vancomycin; QD, quinupristin-dalfopristin; LZ, linezolid; TM, trimethoprim; CI, ciprofloxacin; RI, rifampin.

FIG 2

Prevalence of antibiotic-resistant (blue) and antibiotic-susceptible (green) strains within the Lactobacillus phylogroups tested for antimicrobial agents specified in reference , including inhibitors of cell wall synthesis (ampicillin and vancomycin), inhibitors of protein synthesis (erythromycin, clindamycin, chloramphenicol, and tetracycline), and aminoglycosides (gentamicin, kanamycin, and streptomycin).

FIG 3

Diagram showing the genetic organization of the Tn916-like transposon (transp) identified in L. ingluviei DSM 15946^T. Red, AR genes; yellow, genes involved in genetic transfer; gray, ORFs involved in the conjugation process; green, regulatory sequences. Numbers above the diagram refer to loci in Tn916; numbers below are a base pair scale.

FIG 4

Genetic organization of sequences surrounding the tet(W) and erm(B) genes identified in L. ingluviei DSM 15946^T. Red, AR genes; yellow, genes involved in genetic transfer; pink, genes encoding plasmid-associated replication proteins; gray, gene coding for hypothetical proteins. Numbers above the diagram refer to loci; numbers below are a base pair scale. Rep, plasmid replication; int; plasmid integration.

FIG 5

Genetic organization of sequences surrounding the tet(M) (A) and erm(B) (B) genes identified in L. amylophilus DSM 20533^T and L. amylotrophicus DSM 20534^T. Red, AR genes; yellow, genes involved in genetic transfer; green, genes encoding regulatory proteins; gray, gene coding for hypothetical proteins. int, integrase. Numbers below the diagram are a base pair scale.

FIG 6

Phenotype-genotype correlation analysis for the 161 type strains of the genus Lactobacillus. Positive correlations between genomic data and phenotypes observed are presented in green and gray, whereas negative correlations are indicated in yellow and blue. GM, gentamicin; KM, kanamycin; SM, streptomycin; TC, tetracycline; EM, erythromycin, CL, clindamycin; CM, chloramphenicol; AM, ampicillin; VA, vancomycin.