Invasive and epidemic potential of emergent multiresistant clones of the food-borne pathogen salmonella enterica

Resumo

Salmonella enterica is a food-borne pathogen that causes disease both in humans and animals, being S. Typhimurium and S. Enteritidis the most common non-typhoidal serotypes. In industrialized countries, these serotypes generally cause self-limiting enteritis known as salmonellosis, for which antimicrobial treatment is usually not required. However, in developing countries, mainly in sub-Saharan Africa, non typhoidal serotypes are responsible for severe invasive infections, and even death, in young children with underlying diseases and adults with HIV, being S. Typhimurium and S. Enteritidis the most frequently involved. S. Typhimurium and S. Enteritidis possess serotype-specific virulence plasmids termed pSLT and pSEV, respectively. Besides other virulence genes, both plasmids contain the spv region implicated in the systemic stage of the disease. In recent years, continuous use of antimicrobial agents has contributed to the selection of serotype-specific virulence plasmids that have acquired resistance genes. These virulence-resistance plasmids are of public health concern, because they allow the co-selection of both functions in response to selective pressure. Two of these plasmids, termed pUO-StVR2 and pUO-SeVR1, have been first detected and characterized by our group. pUO-StVR2 is carried by a multidrug resistant clone of S. Typhimurium, showing a long persistence in time and wide geographical dispersion. pUO-SeVR1 is mainly found in multidrug resistant and highly invasive isolates of S. Enteritidis, which have probably originated in Africa. The presence of resistance derivatives of pSLT in S. Typhimurium isolates from Africa, belonging to the invasive ST313 clone, is also of note. This PhD Thesis focused on the study of S. Typhimurium and S. Enteritidis isolates carrying virulence-resistance plasmids, including pUO-StVR2 and pUO-SeVR1, recovered in Asturias (Spain) and Mozambique (sub-Saharan Africa). pUO-StVR2 is a 134 Kb hybrid plasmid derived from pSLT through acquisition of a central resistance island [containing blaOXA-1,catA1, aadA1,sul1 and tet(B) genes which confer resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline, respectively] flanked by conventional right and left regions. The right region contains four ORFs (open reading frames) encoding a putative high-affinity Fe2+ transport system, which function and role in virulence were evaluated in this Thesis. It was proven that these ORFs act as a Fe2+ uptake system under non infective conditions (propagation in culture media) allowing bacterial growth even when iron was restricted. Moreover, their involvement in virulence was also demonstrated, since bacteria carrying this system had higher rates of invasion of epithelial cells and of intracellular replication within macrophages. According to previous observations, all isolates of the pUO-StVR2 corresponding to the 2008-2014 period were fully resistant to nitrofurantoin. In pUO-StVR2 clone, this resistance was caused by inactivation of the chromosomal nfsA and nfsB genes, due to the insertion of one copy of IS1 in the former gene and of six nucleotides in the latter. The nfsA and nfsB genes encode two oxygen-independent nitroreductases. These enzymes are responsible of nitrofurans reduction, yielding highly reactive intermediates that are toxic for the bacteria. Isolates of S. Typhimurium containing pUO-StVR2 were first reported in Asturias during the 90¿s. In later years, they were widely spread in the Iberian Peninsula, and were also detected in other European countries. Its temporal persistence and geographical spread can be attributed, at least in part, to both the chromosomal resistance to nitrofurantoin and the virulence-resistance plasmid, which confers additional resistances and encodes the iron uptake system. Although multi-drug resistance is less frequent in S. Enteritidis than in S. Typhimurium, it has nevertheless been reported due to the presence of resistance or virulence-resistance plasmids. This is the case of pUO-SeVR1, a 110 Kb hybrid plasmid which confers resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline and trimethoprim encoded by the bla TEM-1, catA2, strAB, sul1 and sul2, tet(A) and dfrA7 genes, respectively. Its entire nucleotide sequence, determined in this Thesis, demonstrated that it evolved from pSEV through acquisition of two foreign regions, where resistance genes are located in association with a class one integron and several transposons. pUO-SeVR1 also contains fourteen copies of IS26, which have mediated the acquisition of the resistance regions, as well as the reorganization of the pSEV scaffold, which suffered small deletions as well as large translocations and inversions. The characterization of 491 isolates of S. Enteritidis collected in Asturias between 2008 and 2014 failed to reveal the presence of pUO-SeVR1. However, several blaTEM-1 plasmids, belonging to different incompatibility groups, were detected in ampicillin resistant isolates: IncI (carrying the strAB and sul2 for resistance to streptomycin and sulfonamides, apart from blaTEM-1), IncX (two apparently unrelated plasmids), IncFrepB and ColE1. The IncX plasmids were the most frequent, and they could have been involved in the change of phagetype PT4 to PT6 of the carrying isolates. On the other hand, the plasmids IncI (which contains pSEV genes, including the spv region, together with the resistance genes), and IncFIIA+IncFIB (originated from pSEV through acquisition of the blaTEM gene), at least, are new virulence-resistance plasmids, which were not previously reported in S. Enteritidis. Together with ampicillin resistance, resistance to nalidixic acid and nitrofurantoin were most common in this serotype. In the present Thesis, invasive non-typhoidal isolates obtained from blood samples in Mozambique, and assigned to S. Typhimurium and S. Enteritidis were also characterized. Regarding to S. Typhimurium, resistance were associated with the presence of two virulence-resistant plasmids, both derived from pSLT, which have been previously detected in invasive isolates from others countries of sub-Saharan Africa: pSLT-BT (which confers resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and trimethoprim encoded by the blaTEM-1, catA1, aadA1, strAB, sul1 and sul2, and dfrA1 genes) and pSLT-A130, with two variants differing in size but conferring the same resistance profile (ampicillin, streptomycin and sulfonamides, due to the blaTEM-1/blaOXA-1, aadA1 and sul2 genes). Multilocus sequence typing (MLST) of invasive S. Typhimurium isolates from Mozambique demonstrated the connection of ST313 with multidrug resistant isolates and of ST11 with susceptible isolates. In the other hand, in all multidrug resistant S. Enteritidis isolates from Mozambique, pUO-SeVR1-like plasmids were detected, and a new variant lacking the tet(A) gene was identified. Genomic macrorestriction with XbaI followed by pulsed-field gel electrophoresis reveal the close relationship among invasive S. Enteritidis isolates, with most of them assigned to the ST1479 clone by MLST. In short, the present PhD Thesis deepened the knowledge on clones belonging to the most common non-typhoidal serotypes, S. Typhimurium and S. Enteritidis, notable for their success, and for the risk they pose to human health, both in industrialized and in developing countries. The acquired knowledge may help establish measures which limit the spread of these clones and limit the future selection of new ones.