The plasmidome of a Salmonella enterica serovar Derby isolated from pork meat.

The complete nucleotide sequences of four plasmids hosted by a Salmonella enterica serovar. Derby strain 6MK1 isolated from pork were determined by shotgun Sanger sequencing. A 107,637 base pairs (bp) conjugative plasmid pSD107 containing 150 putative coding sequences (CDS) could be assigned to the narrow host range incompatibility group IncI1. A detailed annotation of all CDS was carried out, revealing the presence of genes needed for plasmid replication, conjugal transfer, plasmid partitioning and stability as well as resistance to antimicrobials. The resistance determinants dhfrA1, aadA1, qacEΔ1, sul1 (supplied by a class 1 integron), blaTEM-1b (carried by a truncated Tn2 flanked by IS26), sul2 and strAB confer multidrug resistance to the host bacterium. In addition to pSD107, three small cryptic plasmids pSD4.0, pSD4.6 and pSD5.6 were identified, showing significant sequence similarities to already known replicons of Escherichia coli and S. enterica. In conjugation experiments performed on solid medium, pSD107 was successfully transferred to a nalidixic acid resistant E. coli DH5α, mobilizing pSD4.0 and, more infrequently, also pSD4.6. All transferred plasmids were stably propagated in the recipient strain without selective pressure for approximately 66 generations. The absolute plasmid copy numbers were determined in real time PCR experiments, revealing an approximate 1:1:1:1 ratio of the four replicons compared to the chromosome. The evolutionary position of pSD107 within the IncI1 family of plasmids was inferred from a maximum likelihood phylogenetic tree and by comparison of genetic key elements in a set of 17 IncI1 reference plasmids.

Molecular basis and evolution of multiple drug resistance in the foodborne pathogen Salmonella enterica serovar Ohio.

The molecular basis and evolution of multidrug resistance were established for 54 isolates of Salmonella enterica serovar Ohio, recorded between 1991 and 2005 in Asturias, a northern region of Spain. All isolates were closely related, as shown by cluster analysis of XbaI-BlnI combined profiles. Of these, 33.3% were resistant to one or more unrelated agent(s). Sulphonamides, streptomycin, tetracycline, and trimethoprim, encoded by sul1, aadA1, tet(A) or tet(B), and dfrA1, respectively, were the most common resistances, but ampicillin (bla(TEM-1)), gentamicin (aacC2 or aacC4), kanamycin (aphA1), and chloramphenicol (catA1) were also detected. Two types of complex genetic elements, carried by large conjugative or mobilizable plasmids, were found in isolates resistant to four or more unrelated agents (multidrug resistant), which accounted for 18.5% of the total: (i) a class 1 integron (1600 bp/dfrA1-aadA1) close to a defective Tn10, both inserted within a Tn21-like element that was carried in some cases by Tn9; (ii) Tn3-bla(TEM-1), which was inserted within a defective Tn1721. These elements have been involved in the development and spread of multidrug resistance in S. enterica Ohio, which was detected between 1994 and 2001. The absence of multidrug resistance in later years could have been connected with the European Union strategies for combating antimicrobial resistance and controlling nontyphoid S. enterica in food-producing animals.

[Identification of bacteria through 16S rRNA sequencing: principles, methods and applications in clinical microbiology]. / Identificación bacteriana mediante secuenciación del ARNr 16S: fundamento, metodología y aplicaciones en microbiología clínica.

Phylogenetic relationships among prokaryotes can be inferred from comparisons of their 16S rRNA (or 16S rDNA) sequences. This has had an enormous repercussion on bacterial taxonomy, leading to the currently applied system of classification, and allowing a rapid and precise identification of bacteria. In clinical microbiology, molecular identification based on 16S rDNA sequencing is applied fundamentally to bacteria whose identification by means of other types of techniques turns out impossible, difficult, or requires a lot of time. Amplification of the gene to be sequenced uses preferably DNA extracted from a bacterial pure culture, but can be achieved also directly from a clinical sample. The latter has led to the discovery of new pathogens. Bearing in mind its potential, as the technical resources improve and the prize becomes more competitive, the identification based on 16S rDNA sequencing will certainly find a wider application in the clinical microbiology laboratory.