DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens.

We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors.

A translocation motif in relaxase TrwC specifically affects recruitment by its conjugative type IV secretion system.

Type IV secretion system (T4SS) substrates are recruited through a translocation signal that is poorly defined for conjugative relaxases. The relaxase TrwC of plasmid R388 is translocated by its cognate conjugative T4SS, and it can also be translocated by the VirB/D4 T4SS of Bartonella henselae, causing DNA transfer to human cells. In this work, we constructed a series of TrwC variants and assayed them for DNA transfer to bacteria and human cells to compare recruitment requirements by both T4SSs. Comparison with other reported relaxase translocation signals allowed us to determine two putative translocation sequence (TS) motifs, TS1 and TS2. Mutations affecting TS1 drastically affected conjugation frequencies, while mutations affecting either motif had only a mild effect on DNA transfer rates through the VirB/D4 T4SS of B. henselae. These results indicate that a single substrate can be recruited by two different T4SSs through different signals. The C terminus affected DNA transfer rates through both T4SSs tested, but no specific sequence requirement was detected. The addition of a Bartonella intracellular delivery (BID) domain, the translocation signal for the Bartonella VirB/D4 T4SS, increased DNA transfer up to 4% of infected human cells, providing an excellent tool for DNA delivery to specific cell types. We show that the R388 coupling protein TrwB is also required for this high-efficiency TrwC-BID translocation. Other elements apart from the coupling protein may also be involved in substrate recognition by T4SSs.

Transfer of R388 derivatives by a pathogenesis-associated type IV secretion system into both bacteria and human cells.

Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A ΔvirB mutant strain was transfer deficient, while a ΔtrwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs.

Comparison of phenotypical and genetic identification of Aeromonas strains isolated from diseased fish.

Phenotypicaly identified Aeromonas strains (n=119) recovered mainly from diseased fish were genetically re-identified and the concordance between the results was analysed. Molecular characterization based on the GCAT genus specific gene showed that only 90 (75.6%) strains belonged to the genus Aeromonas. The 16S rDNA-RFLP method identified correctly most of the strains with the exception of a few that belonged to A. bestiarum, A. salmonicida or A. piscicola. Separation of these 3 species was correctly assessed with the rpoD gene sequences, which revealed that 5 strains with the RFLP pattern of A. salmonicida belonged to A. piscicola, as did 1 strain with the pattern of A. bestiarum. Correct phenotypic identification occurred in only 32 (35.5%) of the 90 strains. Only 14 (21.8%) of the 64 phenotypically identified A. hydrophila strains belonged to this species. However, coincident results were obtained in 88% (15/17) of the genetically identified A. salmonicida strains. Phenotypic tests were re-evaluated on the 90 genetically characterized Aeromonas strains and there were contradictions in the species A. sobria for a number of previously published species-specific traits. After genetic identification, the prevailing species were A. sobria, A. salmonicida, A. bestiarum, A. hydrophila, A. piscicola and A. media but we could also identify a new isolate of the recently described species A. tecta. This work emphasizes the need to rely on the 16S rDNA-RFLP method and sequencing of housekeeping genes such as rpoD for the correct identification of Aeromonas strains.

Aeromonas taiwanensis sp. nov. and Aeromonas sanarellii sp. nov., clinical species from Taiwan.

Two clinical Aeromonas strains (A2-50(T) and A2-67(T)) recovered from the wounds of two patients in Taiwan could not be assigned to any known species of this genus based on their 16S rRNA gene sequences, which showed similarities of 99.6-99.8 % to those of the type strains of Aeromonas caviae, A. trota and A. aquariorum. The rpoD phylogenetic tree allocated these strains to two novel and independent phylogenetic lines, the neighbouring species being A. caviae, the type strain of which showed 93.2 % similarity (56 bp differences) to strain A2-50(T) and 92.2 % (63 bp differences) to strain A2-67(T). A multilocus phylogenetic analysis of five housekeeping genes (gyrB, rpoD, recA, dnaJ and gyrA; 3684 bp) confirmed that the two strains formed independent phylogenetic lineages within the genus. These data, together with phenotypic characterization and DNA-DNA reassociation results, revealed that these strains represent novel Aeromonas species, for which the names Aeromonas taiwanensis sp. nov. (type strain A2-50(T) =CECT 7403(T) =LMG 24683(T)) and Aeromonas sanarellii sp. nov. (type strain A2-67(T) =CECT 7402(T) =LMG 24682(T)) are proposed.

Aeromonas fluvialis sp. nov., isolated from a Spanish river.

A Gram-stain-negative, facultatively anaerobic bacterial strain, designated 717(T), was isolated from a water sample collected from the Muga river, Girona, north-east Spain. Preliminary analysis of the 16S rRNA gene sequence showed that this strain belonged to the genus Aeromonas, the nearest species being Aeromonas veronii (99.5 % similarity, with seven different nucleotides). A polyphasic study based on a multilocus phylogenetic analysis of five housekeeping genes (gyrB, rpoD, recA, dnaJ and gyrA; 3684 bp) showed isolate 717(T) to be an independent phylogenetic line, with Aeromonas sobria, Aeromonas veronii and Aeromonas allosaccharophila as the closest neighbour species. DNA-DNA reassociation experiments and phenotypic analysis identified that strain 717(T) represents a novel species, for which the name Aeromonas fluvialis sp. nov. is proposed, with type strain 717(T) (=CECT 7401(T) =LMG 24681(T)).

Clinical relevance of the recently described species Aeromonas aquariorum.

Twenty-two human extraintestinal isolates (11 from blood) and three isolates recovered from patients with diarrhea were genetically characterized as Aeromonas aquariorum, a novel species known only from ornamental fish. The isolates proved to bear a considerable number of virulence genes, and all were resistant to amoxicillin (amoxicilline), cephalothin (cefalotin), and cefoxitin. Biochemical differentiation from the most relevant clinical species is provided.

Development of imipenem resistance in an Aeromonas veronii biovar sobria clinical isolate recovered from a patient with cholangitis.

Several imipenem-susceptible and -resistant Aeromonas veronii biovar sobria isolates with different morphologies and antimicrobial susceptibilities recovered from bile samples of a patient with cholangitis were analysed. These isolates belonged to the same clone and the imipenem-resistant strains showed overexpression of the imiS gene, encoding a chromosomal carbapenemase. These results should make clinicians aware of the possible emergence of multidrug-resistant A. veronii biovar sobria, perhaps as a consequence of previous treatment of a urinary tract infection with amoxicillin plus clavulanic acid.

Surgical site infection due to Aeromonas species: report of nine cases and literature review.

Gastrointestinal and wound infections are the most common clinical presentation of Aeromonas. Surgical site infections (SSIs) due to this microorganism are rare. We studied the clinical and microbiological characteristics of 9 cases that appeared at 2 Spanish hospitals and reviewed 15 cases available in the literature. All patients (including our cases) had gastrointestinal or biliary diseases. 21 patients (91.3%) developed SSIs after abdominal or pelvic surgery. The mean duration from surgery to the onset of wound infection was 2.2 d in our 9 patients. The infection was polymicrobial in 17 patients (77.2%) and 19 cases were nosocomial (95%). Clinical outcome of all cases was uniformly good after treatment except for 2 patients. Two patients were cured only with surgical drainage. In conclusion, SSIs due to Aeromonas species have a probable endogenous source after abdominal or pelvic surgery and the onset is rapid in most cases. Clinical outcome is good after antibiotic treatment but surgical drainage without antibiotic therapy can be sufficient to clear the infection in some cases.

Phylogenetic evidence suggests that strains of Aeromonas hydrophila subsp. dhakensis belong to the species Aeromonas aquariorum sp. nov.

Three strains of Aeromonas hydrophila subspecies dhakensis, including the type strain, were subjected to phylogenetic analysis by sequencing gyrB, rpoD, and 16S rRNA genes and compared with all known Aeromonas species. The obtained gyrB and rpoD phylogenetic trees clearly suggested that these A. hydrophila subsp. dhakensis strains indeed belong to the species A. aquariorum. This finding may indicate that, at the time of "dhakensis" subspecies description, the strains were incorrectly identified as A. hydrophila by a polyphasic approach that included DNA-DNA hybridization.

Analysis of 16S rRNA gene mutations in a subset of Aeromonas strains and their impact in species delineation.

Characterization of 999 Aeromonas strains using a published 16S rDNA RFLP identification method showed that 8.1% of the strains produced unexpected (hereafter called "atypical") restriction patterns, making their identification uncertain. Atypical patterns were due to the presence of nucleotide polymorphisms among the rrn operons of the 16S rRNA gene (so-called microheterogeneities). Double sequencing signals at certain positions revealed the nucleotide composition was responsible for the microheterogeneities. Although the number of microheterogeneities was relatively low (0.06-0.66%), trees inferred from the 16S rRNA gene led either to a misidentification or to an inconclusive result for the majority of these strains. Strains with atypical patterns were, however, correctly identified using the rpoD gene sequences, as belonging to Aeromonas caviae, A. veronii, and A. media. All of them, but particularly the two former species, are associated with human disease. Microheterogeneities in 16S rRNA gene sequence were significantly (P 0.01) more prevalent in clinical than in environmental strains. This work also analyzed the effects of these microheterogeneities on the taxonomic position of the investigated strains. The results suggest the need for recording microheterogeneities in the 16S rRNA gene.

Analysis of 16S rRNA gene mutations in a subset of Aeromonas strains and their impact in species delineation

Characterization of 999 Aeromonas strains using a published 16S rDNA RFLP identification method showed that 8.1% of the strains produced unexpected (hereafter called «atypical») restriction patterns, making their identification uncertain. Atypical patterns were due to the presence of nucleotide polymorphisms among the rrn operons of the 16S rRNA gene (so-called microheterogeneities). Double sequencing signals at certain positions revealed the nucleotide composition was responsible for the microheterogeneities. Although the number of microheterogeneities was relatively low (0.06-0.66%), trees inferred from the 16S rRNA gene led either to a misidentification or to an inconclusive result for the majority of these strains. Strains with atypical patterns were, however, correctly identified using the rpoD gene sequences, as belonging to Aeromonas caviae, A. veronii, and A. media. All of them, but particularly the two former species, are associated with human disease. Microheterogeneities in 16S rRNA gene sequence were significantly (P 0.01) more prevalent in clinical than in environmental strains. This work also analyzed the effects of these microheterogeneities on the taxonomic position of the investigated strains. The results suggest the need for recording microheterogeneities in the 16S rRNA gene (AU)

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Aeromonas hemolytic uremic syndrome. A case and a review of the literature.

Although rarely, hemolytic uremic syndrome can be induced by Aeromonas. We report a case in a 40-year-old Spanish female produced by Aeromonas veronii bv. sobria and review the previous cases described in the literature. This is the 2nd case described in adults.