Plasmids encoding resistance and virulence properties in multidrug resistant (MDR) Salmonella enterica (S.) serovar Typhimurium monophasic variant 4,[5],12:i:- isolates recovered from pigs and humans (2006-2008) in Europe were characterised. The isolates were selected based on the detection by PCR-amplification of S. Typhimurium virulence plasmid pSLT genes and were analysed by multi-locus sequence typing (MLST). The resistance genes present in the isolates and the association of these genes with integrons, transposons and insertion sequences were characterised by PCR-sequencing, and their plasmid location was determined by alkaline lysis and by S1-nuclease pulsed-field gel electrophoresis (PFGE) Southern-blot hybridisation. Plasmids were further analysed by replicon typing, plasmid MLST and conjugation experiments. The 10 S. 4,[5],12,i:- selected isolates belonged to ST19. Each isolate carried a large plasmid in which MDR with pSLT-associated virulence genes were located. After analysis, eight different plasmids of three incompatibility groups (IncA/C, IncR and IncF) were detected. Two IncA/C plasmids represented novel variants within the plasmid family of the S. 4,[5],12:i:- Spanish clone, and carried an empty class 1 integron with a conventional qacEΔ1-sul1 3′ conserved segment or an In-sul3 type III with estX-psp-aadA2-cmlA1-aadA1-qacH variable region linked to tnpA440-sul3, part of Tn2, Tn21 and Tn1721 transposons, and ISCR2. Four newly described IncR plasmids contained the resistance genes within In-sul3 type I (dfrA12-orfF-aadA2-cmlA1-aadA1-qacH/tnpA440-sul3) and part of Tn10 [tet(B)]. Two pSLT-derivatives with FIIs-ST1+FIB-ST17 replicons carried cmlA1-[aadA1-aadA2]-sul3-dfrA12 and bla_TEM-1 genes linked to an In-sul3 type I integron and to Tn2, respectively. In conclusion, three emerging European clones of S. 4,[5],12:i:- harboured MDR plasmids encoding additional virulence functions that could contribute significantly to their evolutionary success.

To broaden knowledge of the molecular bases and genetics of multidrug resistance in clinical isolates of Salmonella enterica serotype 4,5,12:i:− belonging to the Spanish clone. The relatedness of the isolates was determined by phage typing and XbaI-PFGE. Resistance genes, integrons and transposable elements were identified by PCR amplification and sequencing. Plasmids were characterized by alkaline lysis, S1-PFGE, conjugation, replicon typing and Southern blot hybridization. The isolates were closely related and resistant to five to seven antimicrobials (ampicillin, chloramphenicol, gentamicin, streptomycin/spectinomycin, sulphonamides, trimethoprim and tetracycline, arranged in different combinations). Most of the responsible genes were provided by a conventional class 1 integron with the dfrA12-orfF-aadA2 variable region, an atypical class 1 integron containing sul3 next to the estX-psp-aadA2-cmlA1-aadA1 variable region and a truncated Tn1721 transposon carrying tet(A). A defective Tn21 with the mer operon and ISVsa3 associated with sul2 were also detected. All resistance genes and mobile genetic elements were located on large, non-conjugative and highly variable plasmids carrying one (A/C) or two (A/C and N) replicons, as well as virulence genes of pSLT. IncA/C plasmids are responsible for multidrug resistance in an increasing number of relevant human and animal bacterial pathogens, and hence are regarded as an important threat to public health. Those found in the Spanish clone of Salmonella 4,5,12:i:− constitute a relevant example of short-term evolution, and could have been involved in the successful adaptation of this pathogen.

Salmonella enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium incapable of expressing the second-phase flagellar antigen (fljAB operon), and it is recognized to be one of the most prevalent serovars causing human infections. A clonal lineage characterized by phage type DT193, PulseNet PFGE profile STYMXB.0131 and multidrug resistance to ampicillin, streptomycin, sulphonamides and tetracycline (R-type ASSuT) is commonly circulating in Europe. In this study we determined the deletions affecting the fljAB operon and the resistance region responsible for the R-type ASSuT. in a strain of Salmonella enterica serovar 4,5,12:i:- DT193/STYMXB.0131, through an approach based on PCRs and Southern blot hybridization of genomic DNA. Using a set of nine specific PCRs, the prevalence of the resistance region was assessed in a collection of 144 S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 strains isolated from Germany, Switzerland and Italy. A 28 kb-region is embedded between the loci STM2759 and iroB, replacing the DNA located in between, including the fljAB operon. It encompasses the genes blaTEM-1, strA-strB, sul2 and tet(B) responsible for the R-type ASSuT together with genes involved in plasmid replication and orfs of unknown function characteristically located on IncH1 plasmids. Its location and internal structure is fairly conserved in S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 strains regardless of the isolation source or country. Hence, in the S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 clonal lineage widespread in Germany, Switzerland and Italy, a resistance region derived from IncH1 plasmids has replaced the chromosomal region encoding the second flagellar phase and is an example of the stabilization of new plasmid-derived genetic material due to integration into the bacterial chromosome.

Pseudomonas aeruginosa is an opportunistic pathogen with a multidrug-resistant profile that has become a critical threat to global public health. It is one of the main causes of severe nosocomial infections, including ventilator-associated pneumonia, chronic infections in patients with cystic fibrosis, and bloodstream infections in immunosuppressed individuals. Development of vaccines against P. aeruginosa is a major challenge owing to the high capacity of this bacterium to form biofilms, its wide arsenal of virulence factors (including secretion systems, lipopolysaccharides, and outer membrane proteins), and its ability to evade the host immune system. This review provides a comprehensive historical overview of vaccine development efforts targeting this pathogen, ranging from early attempts in the 1970s to recent advancements, including vaccines based on novel proteins and emerging technologies such as nanoparticles and synthetic conjugates. Despite numerous promising preclinical developments, very few candidates have progressed to clinical trials, and none have achieved final approval. This panorama highlights the significant scientific efforts undertaken and the inherent complexity of successfully developing an effective vaccine against P. aeruginosa.

Salmonella enterica subsp. enterica 4,[5],12:i:− is one of the most prevalent serovars associated with human infections worldwide. Two multidrug-resistant clones, designated Spanish and European clones, are recognized as having importance for public health and are subject to control measures in the European Union. In this study, 23 clinical isolates belonging to the Spanish clone were characterized by multilocus sequence typing, multiple-locus variable number tandem repeat analysis (MLVA), PCR amplification and sequencing, and a DNA microarray targeting 263 genes, in order to provide new insights into their origins and further evolution. The derived data were compared with information available from other studies for S. 4,[5],12:i:− isolates of both the Spanish and the European clones, to identify differential molecular markers which could be potentially used as surveillance tools in the control of dissemination of this serovar. The isolates analyzed were assigned to sequence type 19 and to 17 MLVA patterns, with 3-13-16-NA-311 being the most prevalent. Highly similar virulence, metabolic, and prophage-associated gene profiles were identified, but DNA mobility markers distinguished five genotypes. Two types of deletions, caused by insertion of IS26, presumably donated by pUO-STmR/RV1-like plasmids typically found in the Spanish clone, affected the fljAB operon and surrounding DNA. The Spanish and European clones differ in sequence type, MLVA patterns, gene repertoire, and fljAB deletion type. The observed variability supports an independent evolution of the two successful monophasic clones from different Salmonella enterica serovar Typhimurium ancestors and can be taken into consideration for epidemiological surveillance.