Background: Pseudomonas aeruginosa is an opportunistic pathogen with a high incidence of hospital infections that represents a threat to immune compromised patients. Genomic studies have shown that, in contrast to other pathogenic bacteria, clinical and environmental isolates do not show particular genomic differences. In addition, genetic variability of all the P. aeruginosa strains whose genomes have been sequenced is extremely low. This low genomic variability might be explained if clinical strains constitute a subpopulation of this bacterial species present in environments that are close to human populations, which preferentially produce virulence associated traits. Results: In this work, we sequenced the genomes and performed phenotypic descriptions for four non-human P. aeruginosa isolates collected from a plant, the ocean, a water-spring, and from dolphin stomach. We show that the four strains are phenotypically diverse and that this is not reflected in genomic variability, since their genomes are almost identical. Furthermore, we performed a detailed comparative genomic analysis of the four strains studied in this work with the thirteen previously reported P. aeruginosa genomes by means of describing their core and pan-genomes. Conclusions: Contrary to what has been described for other bacteria we have found that the P. aeruginosa core genome is constituted by a high proportion of genes and that its pan-genome is thus relatively small. Considering the high degree of genomic conservation between isolates of P. aeruginosa from diverse environments, including human tissues, some implications for the treatment of infections are discussed. This work also represents a methodological contribution for the genomic study of P. aeruginosa, since we provide a database of the comparison of all the proteins encoded by the seventeen strains analyzed.

P. aeruginosa is one of the most metabolically versatile bacteria having the ability to survive in multiple environments through its accessory genome. An important hallmark of P. aeruginosa is the high level of antibiotic resistance, which often makes eradication difficult and sometimes impossible. Evolutionary forces have led to this bacterium to develop high antimicrobial resistance with a variety of elements contributing to both intrinsic and acquired resistance. The objectives were to genetically and phenotypically characterizer P. aeruginosa strains isolated from companion animals of different species. We characterized a collection of 39 P. aeruginosa strains isolated from infected animals. The genetic characterization was in relation to chromosomal profile by PFGE; content of virulence gene; presence of genomic islands (GIs); genes of the cytotoxins exported by T3SS: exoU, exoS, exoT and exoY; and type IV pili allele. The phenotypic characterization was based on patterns of susceptibility to different antimicrobials. Each strain had a PFGE profile, a high virulence genes content, and a large accessory genome. However, most of the strains presented high sensitivity to almost all antimicrobials tested, showing no acquired resistance (no β-lactamases). The exception to this lack of resistance was seen with penicillin. P. aeruginosa could be a naturally sensitive bacterium to standard antimicrobials but could rapidly develop intrinsic and acquired resistance when the bacterium is exposed to pressure exerted by antibiotics, as observed in hospital settings.

The Helicobacter pylori vapD gene is transcribed and expressed when the bacteria are within the gastric cell. In this current study, we investigated how vapD knockout affects the survival of H. pylori inside human gastric adenocarcinoma cells. We constructed an H. pylori 26695 vapD (Hp ΔvapD) mutant strain. H. pylori 26695 wt and Hp ΔvapD strains were grown in synthetic media and were co-cultured with AGS cells. From the start, the growth curve, total protein concentration and colony-forming units (CFUs) of each strain were measured. From each co-culture, CFUs and total RNA were obtained, and transcript levels of GAPDH, vapD, vacA, ureA, and 16s Hp were measured by qRT-PCR. Hp ΔvapD did not affect the growth rate of the strain in synthetic media, showing that the vapD gene is not necessary when the bacteria grow outside eukaryote cells. However, in the intracellular environment, the number of CFUs recovered from the Hp ΔvapD strain from AGS cells decreased after 36 h. Transcription levels of the vacA gene from the Hp ΔvapD strain were 10,000-fold lower than those of H. pylori wt, to the point of being undetectable. The results suggest that the vapD gene contributed to maintaining H. pylori inside gastric cells.

The Helicobacter pylori vapD gene is transcribed and expressed when the bacteria are within the gastric cell. In this current study, we investigated how vapD knockout affects the survival of H. pylori inside human gastric adenocarcinoma cells. We constructed an H. pylori 26695 vapD (Hp ΔvapD) mutant strain. H. pylori 26695 wt and Hp ΔvapD strains were grown in synthetic media and were co-cultured with AGS cells. From the first, the growth curve, total protein concentration and colony-forming units (CFU) of each strain were measured. From each co-culture, CFU and total RNA were obtained, transcript levels of GAPDH, vapD, vacA, ureA, and 16s Hp were measured by qRT-PCR. Hp ΔvapD did not affect the growth rate of the strain in synthetic media showing that the vapD gene is not necessary when the bacteria grow outside eukaryote cells. However, in the intracellular environment, the number of CFU recovered from the Hp ΔvapD strain from AGS cells decreases after 36 hours. Transcription levels of the vacA gene from the Hp ΔvapD strain were 10,000-fold lower than those of H. pylori wt to the point of being undetectable. The results suggest that the vapD gene contributed to maintaining H. pylori inside gastric cells.

Helicobacter pylori can persist intracellularly, offering protection against the immune system and antimicrobial treatments, which promote chronic infection. Previous studies revealed that the virulence-associated protein D ( vapD ) gene is transcriptionally induced during H. pylori intracellular infection and was associated with bacterial persistence, as deletion of vapD impairs bacterial intracellular survival. However, whether VapD protein is expressed and localized during intracellular infection had not been demonstrated. The aim of this study was to detect the VapD protein when H. pylori is inside eukaryotic cells. Polyclonal antibodies against VapD and immunofluorescence microscopy were used to detect the VapD expression in co-cultures of H. pylori strain 26695 and AGS cells. The H. pylori strain Tx30a, which lacks the vapD gene, was used as a negative control. VapD expression was detected exclusively in AGS cells infected with strain 26695. Confocal microscopy confirmed the intracellular localization of the VapD signal, which coincided with the localization of H. pylori within AGS cells. In addition, a high proportion of H. pylori -infected AGS cells were positive for VapD signal. Together, these results provide first direct evidence of VapD expression during H. pylori intracellular infection and extend previous transcriptional and genetic studies by confirming VapD production at the protein level, supporting its association with the intracellular state of H. pylori .