2. Universidad Cardenal Herrera-CEU

Different studies have reported the prevalence and antibiotic resistance of Salmonella in dromedaries’ camels and its role in camelid-associated salmonellosis in humans, but little is known about the epidemiology of Campylobacter in dromedaries. Here we investigate the prevalence, genetic diversity and antibiotic resistance of Campylobacter and Salmonella in dromedary camels (Camelus dromedarius). A total of 54 individuals were sampled from two unique dromedary farms located in Tenerife (Canary Islands, Spain). Whilst all the samples were Campylobacter-negative, Salmonella prevalence was 5.5% (3/54) and the only serovar isolated was S. Frintrop. The pulsed field gel electrophoresis analysis revealed a low genetic diversity, with all isolates showing a nearly identical pulsotype (similarity > 95%). Our results indicate that dromedaries’ camels could not be a risk factor for Campylobacter human infection, but seems to be a reservoir for Salmonella transmission. Since camel ride has become one of the main touristic attractions in several countries and its popularity has considerably risen in the last years, a mandatory control, especially for zoonotic pathogens, such as Campylobacter and Salmonella should be implemented.

The emergence of new pathogens is a major threat to public and veterinary health. Changes in bacterial habitat such as a switch in host or disease tropism are typically accompanied by genetic diversification. Staphylococcus aureus is a multi-host bacterial species associated with human and livestock infections. A microaerophilic subspecies, Staphylococcus aureus subsp. anaerobius, is responsible for Morel’s disease, a lymphadenitis restricted to sheep and goats. However, the evolutionary history of S. aureus subsp. anaerobius and its relatedness to S. aureus are unknown. Population genomic analyses of clinical S. aureus subsp. anaerobius isolates revealed a highly conserved clone that descended from a S. aureus progenitor about 1000 years ago before differentiating into distinct lineages that contain African and European isolates. S. aureus subsp. anaerobius has undergone limited clonal expansion, with a restricted population size, and an evolutionary rate 10-fold slower than S. aureus. The transition to its current restricted ecological niche involved acquisition of a pathogenicity island encoding a ruminant host-specific effector of abscess formation, large chromosomal re-arrangements, and the accumulation of at least 205 pseudogenes, resulting in a highly fastidious metabolism. Importantly, expansion of ~87 insertion sequences (IS) located largely in intergenic regions provided distinct mechanisms for the control of expression of flanking genes, including a novel mechanism associated with IS-mediated anti-antisense decoupling of ancestral gene repression. Our findings reveal the remarkable evolutionary trajectory of a host-restricted bacterial pathogen that resulted from extensive remodelling of the S. aureus genome through an array of diverse mechanisms in parallel.

Conjugation has classically been considered the main mechanism driving plasmid transfer in nature. Yet bacteria frequently carry so-called non-transmissible plasmids, raising questions about how these plasmids spread. Interestingly, the size of many mobilisable and nontransmissible plasmids coincides with the average size of phages (~40 kb) or that of a family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs, ~11 kb). Here, we show that phages and PICIs from Staphylococcus aureus can mediate intra- and inter-species plasmid transfer via generalised transduction, potentially contributing to non-transmissible plasmid spread in nature. Further, staphylococcal PICIs enhance plasmid packaging efficiency, and phages and PICIs exert selective pressures on plasmids via the physical capacity of their capsids, explaining the bimodal size distribution observed for non-conjugative plasmids. Our results highlight that transducing agents (phages, PICIs) have important roles in bacterial plasmid evolution and, potentially, in antimicrobial resistance transmission.

The carriage of two important pathogens of pigs, i.e. enterotoxigenic E. coli (ETEC) and Clostridioides difficile, was investigated in 104 cloacal samples from wild griffon vultures (Gyps fulvus) fed on pig carcasses at supplementary feeding stations (SFS), along with their level of antimicrobial resistance (AMR). E. coli was isolated from 90 (86.5%) samples but no ETEC was detected, likely because ETEC fimbriae confer the species specificity of the pathogen. Resistance to at least one antimicrobial agent was detected in 89.9% of E. coli isolates, being AMR levels extremely high (>70%) for tetracycline and streptomycin, and very high (>50%) for ampicillin and sulfamethoxazole-trimethoprim. Resistance to other critically important antimicrobials such as colistin and extended-spectrum cephalosporins was 2.2%, and 1.1%, respectively, and was encoded by the mcr-1 and blaSHV-12 genes. Multidrug resistance was displayed by 80% of the resistant E. coli and blaSHV-12 gene shared plasmid with other AMR genes. In general, resistance patterns in E. coli from vultures mirrored those found in pigs. C. difficile was detected in three samples (2.9%), two of them belonged to PCR-ribotype 078 and one to PCR-ribotype 126, both commonly found in pigs. All C. difficile isolates were characterized by a moderate to high level of resistance to fluoroquinolones and macrolides but susceptible to metronidazole or vancomycin, similar to what is usually found in C. difficile isolates from pigs. Thus, vultures may contribute somewhat to the environmental dissemination of some pig pathogens through their acquisition from pig carcasses and, more importantly, of AMR for antibiotics of critical importance for humans. However, the role of vultures would likely be much lesser than that of disposing pig carcasses at the SFS. The monitoring of AMR, and particularly of colistin resistant and ESLB-producing E. coli, should be considered in pig farms used as sources of carcasses for SFS.

The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.

Most human listeriosis outbreaks are caused by Listeria monocytogenes evolutionary lineage I strains which possess four exotoxins: a phosphatidylinositol-specific phospholipase C (PlcA), a broad-range phospholipase C (PlcB), listeriolysin O (LLO) and listeriolysin S (LLS). The simultaneous contribution of these molecules to virulence has never been explored. Here, the importance of these four exotoxins of an epidemic lineage I L. monocytogenes strain (F2365) in virulence was assessed in chicken embryos infected in the allantoic cavity. We show that LLS does not play a role in virulence while LLO is required to infect and kill chicken embryos both in wild type transcriptional regulator of virulence PrfA ( PrfAWT) and constitutively active PrfA (PrfA*) backgrounds. We demonstrate that PlcA, a toxin previously considered as a minor virulence factor, played a major role in virulence in a PrfA* background. Interestingly, GFP transcriptional fusions show that the plcA promoter is less active than the hly promoter in vitro, explaining why the contribution of PlcA to virulence could be observed more importantly in a PrfA* background. Together, our results suggest that PlcA might play a more important role in the infectious lifecycle of L. monocytogenes than previously thought, explaining why all the strains of L. monocytogenes have conserved an intact copy of plcA in their genomes.

While many bacterial pathogens are restricted to single host species, some have the capacity to undergo host switches, leading to the emergence of new clones that are a threat to human and animal health. However, the bacterial traits that underpin a multihost ecology are not well understood. Following transmission to a new host, bacterial populations are influenced by powerful forces such as genetic drift that reduce the fixation rate of beneficial mutations, limiting the capacity for host adaptation. Here, we implement a novel experimental model of bacterial host switching to investigate the ability of the multihost pathogen Staphylococcus aureusto adapt to new species under continuous population bottlenecks. We demonstrate that beneficial mutations accumulated during infection can overcome genetic drift and sweep through the population, leading to host adaptation. Our findings highlight the remarkable capacity of some bacteria to adapt to distinct host niches in the face of powerful antagonistic population forces.

Phage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gramnegative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.

We have recently proposed that the trimeric staphylococcal phage encoded dUTPases (Duts) are signaling molecules that act analogously to eukaryotic G-proteins, using dUTP as a second messenger. To perform this regulatory role, the Duts require their characteristic extra motif VI, present in all the staphylococcal phage coded trimeric Duts, as well as the strongly conserved Dut motif V. Recently, however, an alternative model involving Duts in the transfer of the staphylococcal islands (SaPIs) has been suggested, questioning the implication of motifs V and VI. Here, using state-of the-art techniques, we have revisited the proposed models. Our results confirm that the mechanism by which the Duts derepress the SaPI cycle depends on dUTP and involves both motifs V and VI, as we have previously proposed. Surprisingly, the conserved Dut motif IV is also implicated in SaPI derepression. However, and in agreement with the proposed alternative model, the dUTP inhibits rather than inducing the process, as we had initially proposed. In summary, our results clarify, validate and establish the mechanism by which the Duts perform regulatory functions.