Search

The transferability of a large plasmid that harbors a tetracycline resistance gene tet(S), to fish and human pathogens was assessed using electrotransformation and conjugation. The plasmid, originally isolated from fish intestinal Lactococcus lactis ssp. lactis KYA-7, has potent antagonistic activity against the selected recipients (Lactococcus garvieae and Listeria monocytogenes), preventing conjugation. Therefore the tetracycline resistance determinant was transferred via electroporation to L. garvieae. A transformant clone was used as the donor in conjugation experiments with three different L. monocytogenes strains. To our knowledge, this is the first study showing the transfer of an antibiotic resistance plasmid from fish-associated lactic bacteria to L. monocytogenes, even if the donor L. garvieae was not the original host of the tetracycline resistance but experimentally created by electroporation. These results demonstrate that the antibiotic resistance genes in the fish intestinal bacteria have the potential to spread both to fish and human pathogens, posing a risk to aquaculture and consumer safety.

Three different molecular techniques were used to characterize bacterial strains isolated from dairy products and phenotypically classified as Lactobacillus casei. Some strains were taxonomically identified as Lb. casei and Lb. paracasei using 23S rRNA-targeted probes specific for Lb. casei and Lb. paracasei. Chromosomal DNA was analyzed by pulsed-field gel electrophoresis. Lb. paracasei strains had a variable number of SfiI fragments harboring rRNA genes, while Lb. casei strains showed more homogeneous patterns. The chromosome size of Lb. paracasei ATCC 334 was evaluated to be 2.17 Mb.

The paper describes two methods that allow rapid and accurate identification of Lactobacillus helveticus strains based on the nucleotide sequence of the gene coding for the surface layer (S-layer) protein. The first method is based on a polymerase chain reaction amplification using primers targeting a specific fragment of the S-layer gene. The second method involved a portion of the S-layer gene of L. helveticus as a probe in a hybridisation test. The specificity of these protocols was checked for DNA samples isolated from various Lactobacillus strains.

Three molecular techniques (RAPD-polymerase chain reaction analysis, plasmid profile and pulsed-field gel electrophoresis) were used for a preliminary approach to type, at strain level, enterococci isolated from a 24-h-old home-made Pecorino Sardo (protected designation of origin) cheese. A high genetic polymorphism was found. Clusters obtained by the RAPD technique and plasmid profile analysis contained different strains. Pulsed-field gel electrophoresis proved to be an efficient and highly reproducible typing method. In addition, by combining the results from plasmid profile analysis and pulsed-field gel electrophoresis, it was possible to identify closely related strains probably belonging to the same clonal lineage.

Lactobacillus gasseri 4B2 is a human isolate characterised by a strong autoaggregating phenotype mediated by APF (aggregation-promoting factor), a secreted protein. Two primer pairs were developed for simultaneous amplification of a specific fragment of the APF gene and a highly conserved region of the 16S rRNA gene. The specificity of this protocol was checked in DNA samples isolated from single and mixed cultures of Lactobacillus. The same amplification protocol was successfully used directly adding whole bacterial cells to PCR reaction tubes. The suitability of this method for in vivo studies was investigated through feeding L. gasseri 4B2 to mice and analysing colony forming units obtained by plating faecal samples on selective medium. The methodology allows a fast and reliable identification of the target strain without any DNA extraction procedure.

A method to obtain gene transfer in lactic acid bacteria is described. When Streptococcus lactis SH4174 and Lactobacillus reuteri DSM20016 protoplasts undergo fusion by exposure to polyethylene glycol, plasmid pAMβ 1 and trehalose-fermenting ability are transferred into L. reuteri .

A method for protoplast formation and regeneration suitable for Lactobacillus reuteri strains was developed. Lysozyme-treated cells formed protoplasts at a high percentage and regenerative ability varied according to the strains considered. Moreover, production and regeneration of protoplasts promoted the loss of plasmids harboured by the strains.