Directed Recovery and Molecular Characterization of Antibiotic Resistance Plasmids from Cheese Bacteria

Abstract

Resistance to antimicrobials is a growing problem of worldwide concern. Plasmids are thought to be major drivers of antibiotic resistance spread. The present work reports a simple way to recover replicative plasmids conferring antibiotic resistance from the bacteria in cheese. Purified plasmid DNA from colonies grown in the presence of tetracycline and erythromycin was introduced into plasmid-free strains of Lactococcus lactis, Lactiplantibacillus plantarum and Lacticaseibacillus casei. Following antibiotic selection, the plasmids from resistant transformants were isolated, analyzed by restriction enzyme digestion, and sequenced. Seven patterns were obtained for the tetracycline-resistant colonies, five from L. lactis, and one each from the lactobacilli strains, as well as a single digestion profile for the erythromycin-resistant transformants obtained in L. lactis. Sequence analysis respectively identified tet(S) and ermB in the tetracycline- and erythromycin-resistance plasmids from L. lactis. No dedicated resistance genes were detected in plasmids conferring tetracycline resistance to L. casei and L. plantarum. The present results highlight the usefulness of the proposed methodology for isolating functional plasmids that confer antibiotic resistance to LAB species, widen our knowledge of antibiotic resistance in the bacteria that inhabit cheese, and emphasize the leading role of plasmids in the spread of resistance genes via the food chain.

Phenotypic and Safety Assessment of the Cheese Strain Lactiplantibacillus plantarum LL441, and Sequence Analysis of its Complete Genome and Plasmidome

“…Despite the absence of a recognizable replication protein, pLL441-6 harbored an ORF coding for a DUF536 domain-containing protein, which might account for this function. An identical gene has recently been reported in a plasmid isolated from cheese [40]. Figure 2 shows the phylogenetic relationships of LL441 plasmid-associated proteins to those of other L. plantarum plasmids and to those of well-known plasmids representative of the main plasmid replication families.…”

Antibiotic Resistance/Susceptibility Profiles of <em>Staphylococcus equorum </em>Strains from Cheese, and Genome Analysis for Antibiotic Resistance Genes

“…These results lead us to foresee that the phenotypic resistance to penicillin G is not due to acquired genes. Even though large plasmids have been recently introduced in Gram-positive bacteria by electroporation [75], no colonies were obtained by transforming plasmid DNA from the five strains into S. aureus, suggesting the S. equorum plasmidic BlaZ system is not functional. As reported for S. aureus, sequence variation in the blaR1-blaZI region [47,76], or mutations in the promoter region [77], may also account for the discrepancies observed between genotype and phenotype.…”

The detailed analysis of the microbiome and resistome of artisanal blue-veined cheeses provides evidence on sources and patterns of succession linked with quality and safety traits

“…were the taxa identified as those more frequently associated with tetracycline and erythromycin resistance [23]. Moreover, plasmids harboring determinants of resistance to these antimicrobials have been also characterized [24]. Likewise, a high prevalence of class 1 integrons and determinants of resistance to aminoglycosides, tetracyclines, and β-lactam antibiotics have been reported in Brazilian white soft cheeses [25].…”