Biochemical, biophysical and structural characterization of Lactococcus lactis bacteriophage proteins involved in Homologous Recombination and Abortive Infection mechanisms

I batteriofagi virulenti del gruppo 936 e P335 sono predominanti nelle infezioni verso Lactococcus lactis, un batterio Gram-positivo molto diffuso nell’industria casearia. Tra i meccanismi adottati dal batterio contro l’infezione virale, ritroviamo i meccanismi abortivi antivirali (Abi), di cui poco si conosce riguardo alla loro modalità d’azione. Sotto la pressione selettiva dei sistemi Abi, la ricombinazione omologa con sequenze presenti nel cromosoma batterico (Bouchard et al 2000 et 2002, Moineau et al 1995) produce fagi mutati che sono stati ritrovati in fermentazioni ad esito negativo e perciò recanti enormi perdite economiche. Riguardo il sistema AbiK, Bouchard et al (Bouchard et al 2004) hanno identificato quattro proteine denominate Sak, a bassa similarità di sequenza, ma tutte implicate nella sensibilità verso AbiK. Tra queste proteine, Ploquin et al (Ploquin et al 2008) hanno dimostrato che la ORF252 del fago ul36 (specie P335), denominata Sak, é una “DNA Single Strand Annealing Protein (SSAP)“, omologa della proteina eucariotica RAD52. Sak3 del fago p2 (specie 936) é invece la proteina di riferimento del terzo gruppo di geni sak che non mostrano alcuna similarità di sequenza con le SSAPs. Indipendentemente da questo, le proteine Sak sono sicuramente coinvolte nel meccanismo di ricombinazione omologa poiché i loro geni sono localizzati in « recombination cassette » insieme a geni di altre proteine chiaramente implicate in questi meccanismi (Bouchard and Moineau, 2004). La prima parte dei nostri studi é stata focalizzata sulla caratterizzazione biochimica e strutturale delle due proteine Sak : Sak del fago UL36 e Sak3 del fago p2. Di queste ultime abbiamo studiato lo stato di oligomerizzazione attraverso un approccio multi-tecnica, abbiamo visualizzato le tipiche strutture ad anello (Cryo Microscopie Electronique) ed allestito test funzionali per chiarire la loro appartenenza alla famiglia delle SSAP. La loro capacità di legare il DNA é stata inoltre testata e studiata attraverso « Electrophoretic Mobility Shift Assays, EMSA », Microscopia a Forza Atomica (AFM) e « Surface Plasmon Resonance, SPR » ; la relazione con altri partners nel processo di ricombinazione omologa é stata inoltre testata tramite SPR. Nella seconda parte del nostro studio, abbiamo caratterizzato la “Single-Strand Binding protein (SSB)” del fago p2, partner di Sak3 nella ricombinazione omologa. A questo proposito sono stati studiati il livello di espressione di SSB durante l’infezione fagica, la sua capacità di legare il DNA a singolo filamento, le sue implicazioni nel meccanismo di ricombinazione omologa ed abbiamo determinato la sua struttura tridimensionale. I nostri risultati costituiscono gli elementi che permetteranno la comprensione di alcune funzioni sconosciute delle proteine Sak, in particolare del legame tra gli avvenimenti del processamento del DNA ed il sistema AbiK, dell’implicazione di altre proteine correlate come le SSBs in questi meccanismi.Virulent phages of the 936 and P335 group are predominant in infections to Lactococcus lactis, a Gram-positive bacterium widely used in the dairy industry. Among the mechanisms adopted by the bacterium against virus infection, abortive infection mechanisms (Abi) were found, but little is known about their molecular way of action. Under the selective pressure of Abi systems, homologous recombination with phage-related sequences present in the host chromosome (Bouchard et al 2000 and 2002, Moineau et al 1995) produces phage mutants which are found in unsuccessful fermentations that lead to huge economical looses. In particular for the AbiK system, Bouchard et al (Bouchard et al 2004) have identified four non similar proteins involved in the sensibility to AbiK named Sak. Among these proteins, Ploquin et al (Ploquin et al 2008) demonstrated that ORF252 of phage ul36 (species P335), renamed Sak, is a DNA Single-Strand Annealing Protein (SSAP), homologue of the eukaryotic protein RAD52. Sak3 from phage p2 (species 936) is reported to be the reference for the third group of identified sak genes that do not show significant sequence similatity to SSAPs. However, Sak proteins were suspected to be involved in homologous recombination due to their genetic localization in “recombination cassette” with other genes of proteins clearly involved in these processes (Bouchard and Moineau, 2004).The first part of our studies present the biochemical and structural characterization of two Sak proteins: the Sak of phage UL36 and the Sak3 of phage p2. In particular, multi-technique approaches were performed to investigate the oligomerization state and to visualise ring structures (Cryo Electron Microscopy), while functional tests were used to elucidate the SSAP family membership. DNA binding properties were also tested by gel mobility shift assays (EMSA), atomic force microscopy (AFM) and surface plasmon resonance (SPR), and the relationship with their partners in homologous recombination was determined. In the second part of our studies the phage p2 single-strand binding protein (SSB), a Sak3 partner in homologous recombination/replication, was studied. Expression level during phage infection, binding properties to ssDNA, involvement in homologous recombination processes and tridimensional structure were elucidated. Our studies might complete and help to understand some aspects of the Sak function leading to the definition of a linkage between DNA processing events and the antiviral system AbiK, and might elucidate the implication of correlated recombinant proteins like SSBs in these processes

A new nomenclature for the livestock-associated Mycobacterium tuberculosis complex based on phylogenomics

Background: The bacteria that compose the Mycobacterium tuberculosis complex (MTBC) cause tuberculosis (TB) in humans and in different animals, including livestock. Much progress has been made in understanding the population structure of the human-adapted members of the MTBC by combining phylogenetics with genomics. Accompanying the discovery of new genetic diversity, a body of operational nomenclature has evolved to assist comparative and molecular epidemiological studies of human TB. By contrast, for the livestock-associated MTBC members, Mycobacterium bovis, M. caprae and M. orygis, there has been a lack of comprehensive nomenclature to accommodate new genetic diversity uncovered by emerging phylogenomic studies. We propose to fill this gap by putting forward a new nomenclature covering the main phylogenetic groups within M. bovis, M. caprae and M. orygis. Methods: We gathered a total of 8,747 whole-genome sequences (WGS) from public sources and 39 newly sequenced strains, and selected a subset of 839 WGS, representative of the worldwide diversity of M. bovis, M. caprae and M. orygis. We used phylogenetics and genetic diversity patterns inferred from WGS to define groups. Results: We propose to divide M. bovis, M. caprae and M. orygis, in three main phylogenetic lineages, which we named La1, La2 and La3, respectively. Within La1, we identified several monophyletic groups, which we propose to classify into eight sublineages (La1.1-La1.8). These differed in geographic distribution, with some being geographically restricted and others globally widespread, suggesting different expansion abilities. To ease molecular characterization of these MTBC groups by the community, we provide phylogenetically informed, single nucleotide polymorphisms that can be used as barcodes for genotyping. These makers were implemented in a new test suit in KvarQ, a platform-independent, open-source tool. Conclusions: Our results contribute to an improved classification of the genetic diversity within the livestock-associated MTBC, which will benefit future molecular epidemiological and evolutionary studies

Detection of Extended- Spectrum Beta-Lactamase producing Escherichia coli from mesenteric lymph nodes of wild boars (Sus scrofa)

Wild boars (Sus scrofa) are increasing in several European countries, including Italy. In areas with intensive animal farming, like the Italian Emilia-Romagna region, they are likely to be exposed to antimicrobialresistant (AMR) bacteria of livestock origin. In 2017-2018, 108 mesenteric lymph nodes samples were collected from 108 wild boars hunted in Parma province, Emilia-Romagna region, to be tested for ESBL- and carbapenemase-producing Escherichia coli. One isolate (WB-21L) out of 108 (0.9%) was phenotypically confirmed as ESBLproducing E. coli. The strain WB-21L was tested by PCR for the genes blaSHV, blaCTX-M, blaTEM, blaAmpC, blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48, blaSPM, blaBIC, blaSIM, blaDIM, blaGIM, blaAIM, resulting positive for TEM β-lactamase. Resistance to ampicillin, amoxicillin/clavulanic acid, streptomycin, sulfasomidine, tetracycline and trimethoprim confirmed the multi-resistance nature of the strain WB-21L. Nine E. coli isolates showed resistance to meropenem by the Kirby Bauer test but none of them showed Meropenem MIC values indicative of resistance. In conclusion, the present study shows the presence of ESBL E. coli in wild boars and the possible risk of transfer to game meat handlers and consumers. Future studies are needed to better evaluate the sources of AMR bacteria in wildlife

Klebsiella pneumoniae carrying multiple alleles of antigen 43-encoding gene of Escherichia coli associated with biofilm formation

A clinical strain of Klebsiella pneumoniae typed as sequence type 307 carrying three different alleles of the flu gene encoding the Escherichia coli virulence factor antigen 43 associated with biofilm formation was detected and characterized. The flu alleles are located in the chromosome inside putative integrative conjugative elements. The strain displays the phenotypes associated with Ag43, i.e. bi-phasic colony morphology and enhanced biofilm production. Furthermore, the strain produces low amount of capsule known to affect Ag43 function. Analysis of 1431 worldwide deposited genomes revealed that 3.7% Klebsiella pneumoniae carry one or two flu alleles

Loggerhead Sea Turtle as Possible Source of Transmission for Zoonotic Listeriosis in the Marine Environment

Listeria monocytogenes is an ubiquitous pathogen isolated from different host species including fish, crustaceans, and molluscs, but it is rarely a pathogenic microorganism to marine reptiles. In particular, only two cases of fatal disseminated listeriosis have been described in the loggerhead sea turtle (Caretta caretta). In this study, we describe a lethal case of L. monocytogenes infection in a loggerhead sea turtle. The turtle was found alive, stranded on a beach in North-eastern Italy, but perished soon after being rescued. The autoptic examination revealed that heart, lung, liver, spleen, and urinary bladder were disseminated with multiple, firm, 0.1–0.5 mm sized, nodular, white-green lesions. Microscopically, these lesions corresponded with heterophilic granulomas with Gram+ bacteria within the necrotic center. Furthermore, the Ziehl–Neelsen stain was negative for acid-fast organisms. Colonies isolated from heart and liver were tested through MALDI-TOF for species identification, revealing the presence of L. monocytogenes. Whole Genome Sequencing on L. monocytogenes isolates was performed and the subsequent in silico genotyping revealed the belonging to Sequence Type 6 (ST 6); the virulence profile was evaluated, showing the presence of pathogenicity islands commonly observed in ST 6. Our results further confirm that L. monocytogenes should be posed in differential diagnosis in case of nodular lesions of loggerhead sea turtles; thus, given the zoonotic potential of the microorganism, animals should be treated with particular caution. In addition, wildlife animals can play an active role as carriers of possibly pathogenetic and virulent strains and contribute to the distribution of L. monocytogenes in the environment

A Fatal Case of Pseudomonas aeruginosa Community-Acquired Pneumonia in an Immunocompetent Patient: Clinical and Molecular Characterization and Literature Review

Rare cases of Pseudomonas aeruginosa community-acquired pneumonia (PA-CAP) were reported in non-immunocompromised patients. We describe a case of Pseudomonas aeruginosa (PA) necrotizing cavitary CAP with a fatal outcome in a 53-year-old man previously infected with SARS-CoV-2, who was admitted for dyspnea, fever, cough, hemoptysis, acute respiratory failure and a right upper lobe opacification. Six hours after admission, despite effective antibiotic therapy, he experienced multi-organ failure and died. Autopsy confirmed necrotizing pneumonia with alveolar hemorrhage. Blood and bronchoalveolar lavage cultures were positive for PA serotype O:9 belonging to ST1184. The strain shares the same virulence factor profile with reference genome PA01. With the aim to better investigate the clinical and molecular characteristics of PA-CAP, we considered the literature of the last 13 years concerning this topic. The prevalence of hospitalized PA-CAP is about 4% and has a mortality rate of 33–66%. Smoking, alcohol abuse and contaminated fluid exposure were the recognized risk factors; most cases presented the same symptoms described above and needed intensive care. Co-infection of PA-influenza A is described, which is possibly caused by influenza-inducing respiratory epithelial cell dysfunction: the same pathophysiological mechanism could be assumed with SARS-CoV-2 infection. Considering the high rate of fatal outcomes, additional studies are needed to identify sources of infections and new risk factors, along with genetic and immunological features. Current CAP guidelines should be revised in light of these results

Mutation of hilD in a Salmonella Derby lineage linked to swine adaptation and reduced risk to human health

Salmonella enterica variants exhibit diverse host adaptation, outcome of infection, and associated risk to food safety. Analysis of the distribution of Salmonella enterica serovar Derby (S. Derby) subtypes in human and swine identified isolates with a distinct PFGE profile that were significantly under-represented in human infections, consistent with further host adaptation to swine. Here we show that isolates with this PFGE profile form a distinct phylogenetic sub-clade within S. Derby and exhibit a profound reduction in invasion of human epithelial cells, and a relatively small reduction in swine epithelial cells. A single missense mutation in hilD, that encodes the master-regulator of the Salmonella Pathogenicity Island 1 (SPI-1), was present in the adapted lineage. The missense mutation resulted in a loss of function of HilD that accounted for reduced invasion in human epithelial cells. The relatively small impact of the mutation on interaction with swine cells was consistent with an alternative mechanism of invasion in this pathogen-host combination

Salmonella detection and counting on pig carcasses and cutting lines in Italian slaughterhouses

During 2014-2015, 300 pig carcasses before chilling and 85 food contact surfaces (FCSs) at cutting lines were tested for Salmonella in three slaughterhouses (namely A, B and C) of Northern Italy. In slaughterhouses A and B, four carcass sites of 100 cm2 each (from both the exterior and interior side) were swabbed with a single sponge. In abattoir C, four 100-cm2 sites of the exterior and the interior side were swabbed with two independent sponges. The population average prevalence of Salmonella-positive carcasses (which takes into account the structure of the study design, with multiple samples collected in a single day) in slaughterhouses A and B was 12.3%, while in slaughterhouse C it was 11.2%. Presence of Salmonella on exterior and interior side of carcasses showed a low level of concordance (only 3/12 of the contaminated carcasses where positive on both sides). No significant difference was found for FCSs contamination in the three slaughterhouses, with a population average prevalence of Salmonella-positive FCSs of 19.9%. In addition, we found that the clustering due to the day of sampling account for more than 36% and 60% of the overall prevalence variation on carcasses and FCSs, respectively. Eight serovars were identified, with S. Derby as the most common type. The counting of Salmonella on carcasses showed large variability. It was low (< 0.0075 MPN/cm2) in 46.6% of the carcasses and as high as 2.7 MPN/cm2 in 4.7%. Specifically, we found that counts on carcasses fit with “heavy tailed” distributions (log-normal and Weibull with a small shape parameter), suggesting not negligible probability of episodes of high Salmonella contamination. The mean values of contamination obtained with the two distributions ranged from 0.235 to 0.435 MPN/cm2

A novel IncA plasmid carrying blaVIM-1 in a Kluyvera cryocrescens strain

Kluyvera is considered a benign saprophyte commonly present in the environment and also in the human gastrointestinal tract. Kluyvera infections in humans are rare and the pathogenic role of Kluyvera remains uncertain.1 In recent years, sporadic cases of Kluyvera species carrying class A carbapenemase genes (blaGES-5 and blaKPC-2) have been reported.2,3 Herein we describe the complete nucleotide sequence of an IncA plasmid carrying blaVIM-1 recovered from a Kluyvera cryocrescens strain isolated in Italy. This is, to our knowledge, the first report about a Kluyvera strain with an acquired class B carbapenemase