Phenotypic Diversity of Lactobacillus casei Group Isolates as a Selection Criterion for Use as Secondary Adjunct Starters

Autochthonous lactic acid bacteria (LAB) play a key role in the development of cheese flavor. As the pasteurization treatment on raw milk causes the elimination of LAB, secondary starter cultures are used in cheese manufacture to obtain cheeses with improved and standardized flavors. In this work, strains of the L. casei group isolated from traditional Italian cheeses were screened for their phenotypic features of technological interest for use as secondary starters. Their milk acidifying performance and the production of volatile compounds when grown in milk were evaluated. Simultaneously, the acetoin metabolic pathway presence was screened in the strains and assessed for its transcriptional activation. The results showed that the analyzed strains, despite belonging to taxonomically-related species, vary greatly according to the measured phenotypes. Four strains among the fourteen screened could be potentially used as adjunct cultures for cheese-making processes. The strain that showed the highest production of acetoin upregulated the aspartate pathway. An increased knowledge of volatile compounds’ production and acidifying properties of LAB strains isolated from traditional dairy products might guide the selection of strains for industrial applications

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

Studio dei determinanti genici coinvolti nell'adattamento all'ospite in Salmonella Derby

Salmonella is the second most commonly cause of food-borne disease in Europe. The majority of Salmonellae causing disease in human and domestic animals belong to S. enterica subspecies enterica, which is divided in approximately 1,500 serovars, based on their unique somatic and flagellar antigenic formulas. Serovars can exhibit different host-tropism: generalist serovars are able to infect a variety of unrelated hosts, host adapted serovars are mainly isolated from a specific host and specialists serovars are restricted to a single host. Epidemiological evidence supports various levels of host adaptation even among isolates belonging to the same serovar. A number of genetic features have been consistently associated to serovars with a specific host range, but the exact mechanism of host adaptation remains still elusive. Discovering the genetic determinants involved in the ability to infect specific hosts is critically important to assess the risk of pathogen transmission along the food-chain. Data from the Emilia-Romagna Reference Centre for Enteropathogens Surveillance show that Salmonella serovar Derby is mainly prevalent in swine (28.2%) while it is only rarely found in human (2.6%). S. Derby isolates were genotyped by Pulsed-Field Gel Electrophoresis (PFGE). Different proportion of human isolates were observed for the two most prevalent PFGE profiles in swine: SXB_BS.0204 is significantly less isolated in human (0.9%) than in swine (9.4%) while SXB_BS.0056 is proportionally isolated in human (13%) as well as in swine (9.7%). Starting from these data, the aim of this thesis was to identify the genetic determinants responsible for different distribution in human of the two PFGE profiles. It was evaluated if the different prevalences in human of SXB_BS.0204 and SXB_BS.0056 isolates were due to disparity in virulence phenotype. C. elegans infection assays did not detect any difference between isolates belonging to distinct PFGE profiles in the ability to kill the nematode. Invasion and replication assays in human INT-407 and swine IPEC-J2 epithelial cell lines were thus performed. The invasion and replication efficiencies in human cells of SXB_BS.0056 isolates are up to 4 logs higher than those of SXB_BS.0204 isolates. In swine cells SXB_BS.0056 isolates are more virulent than SXB_BS.0204 isolates, but differences are significantly reduced to just one log. Isolates were thus defined virulent (V) and non-virulent (NV) based on their infection efficiency. Whole genomes of 45 isolates were sequenced and core single nucleotide polymorphisms (SNPs) were used as input for generating a phylogenetic tree. V and NV isolates cluster separately, suggesting different evolutionary paths. It was assessed if exclusive genes of V isolates were responsible for their virulence phenotype. Mutants deleted for plasmids and genomic regions found only in V isolates showed no changes in infection efficiency compared to the respective wild types. To evaluate the role of different gene allelic variants in virulence, non-synonymous SNPs were selected which discriminate V from NV isolates. Two allelic variants of hilD were detected: HilD is the activator of Salmonella Pathogenic Island (SPI) 1, which encodes for the Type 3 Secretion System (T3SS), responsible for invasion in epithelial cells. In human cells a V isolate (ER1175 WT) carrying the hilD NV allele (hilD_nv) has the same low infection efficiency of NV isolates. Accordingly, a NV isolate (ER278 WT) carrying the V hilD allele (ER278::hilD_v) shows the same high infection efficiency of V isolates. Both strains deleted individually for hilD and invA (encoding for an essential component of T3SS) infect human cells at the same level of NV isolates. In swine cells the introduction of hilD_nv as well as the hilD deletion in ER1175 WT does not cause any changes in the virulence phenotype, whereas the invA deletion triggers a decrease in virulence. No differences were found among ER278 WT, ER278ΔhilD and ER278ΔinvA in swine cells, but hilD_v insertion causes an increase in both invasion and replication compared to ER278 WT. The two hilD allelic variants thus explain virulence differences between V and NV isolates in human cells, but not in swine cells. To assess differences in SPI-1 genes expression caused by hilD allelic variants, a RNA-seq-based transcriptomic analysis was performed extracting RNA in in vitro conditions causing SPI-1 upregulation. There are no differences in genes expression between strains carrying hilD_nv and the respective hilD-deleted strains, whereas SPI-1 genes are upregulated in both ER1175 and ER278 carrying hilD_v. These results prove that hilD_nv produces a non-functional protein. The same results were obtained analyzing the expression of SPI-1 genes in ER1175 WT, ER1175::hilD_nv and ER1175ΔhilD during human cells infection. These 3 strains and ER278 WT were tested in a mouse model of Salmonella infection to assess if hilD variants cause differences in in vivo host-bacterial interaction. No differences among strains were found in cecum bacterial loads as well as in the expression of host genes correlated with inflammation (NOS2 and KC), not significantly different from uninfected mice. Strains seem thus to not induce gut inflammation in mice. No differences in dissemination extent were observed among ER278 WT, ER1175 WT and ER1175::hilD_nv. ER1175ΔhilD accumulates in liver and spleen at higher level than ER1175 WT, suggesting a negative effect of HilD on S. Derby dissemination. Together, this study has provided new information about mechanisms of host adaptation adopted by S. Derby.Salmonella è la seconda causa più comune di zoonosi in Europa. La maggior parte delle Salmonelle che causano malattia nell’uomo e negli animali domestici appartengono a S. enterica sottospecie enterica, divisa in circa 1500 sierotipi in base ai loro specifici antigeni somatici e flagellari. Sierotipi diversi possono esibire un diverso spettro d’ospite: sono stati identificati sierotipi generalisti, in grado di infettare più specie, sierotipi ospite adattati, maggiormente associati a un ospite, e sierotipi specialisti, ristretti a un unico ospite. Evidenze epidemiologiche hanno inoltre evidenziato diversi livelli di adattamento all’ospite anche tra isolati appartenenti allo stesso sierotipo. Anche se alcune caratteristiche genetiche sono state associate a sierotipi con uno spettro di specie infettate ben determinato, i meccanismi coinvolti nell’adattamento all’ospite di questi patogeni rimangono ancora largamente incompresi. Individuare i determinanti genici coinvolti nella capacità di infettare specifici ospiti è determinante nel valutare il rischio di trasmissione del patogeno lungo la catena alimentare. Dati provenienti dal Centro di Referenza dell’Emilia Romagna per la sorveglianza degli enteropatogeni hanno evidenziato che il sierotipo Derby è principalmente isolato in suino (28%) mentre è solo raramente trovato in uomo (2.6%). Gli isolati di S. Derby sono stati genotipizzati mediante Pulsed-Field Gel Electrophoresis (PFGE) ed è emerso che i due profili PFGE più prevalenti in suino hanno diversa distribuzione in uomo: SXB_BS.0204 è significativamente meno isolato in uomo (0.9%) che in suino (9.4%), mentre SXB_BS.0056 è proporzionalmente isolato allo stesso modo in uomo (13%) e in suino (9.7%). Lo scopo della tesi è stato identificare i determinanti genici responsabili della diversa distribuzione in uomo dei due profili PFGE. È stato valutato se le differenze osservate fossero dovute a diversità tra gli isolati SXB_BS.0204 e SXB_BS.0056 nel fenotipo di virulenza. Saggi di infezione su C. elegans non hanno evidenziato alcuna differenza tra isolati con pulsotipi diversi nella capacità di uccidere il nematode. Sono stati quindi eseguiti saggi di invasione e replicazione in linee cellulari epiteliali umane (INT-407) e suine (IPEC-J2). Nelle cellule umane l’efficienza di invasione e replicazione degli isolati SXB_BS.0056 è fino a 4 logaritmi più alta di quella degli isolati SXB_BS.0204. Nelle cellule suine gli isolati SXB_BS.0056 sono più virulenti degli isolati SXB_BS.0204, ma la differenza è ridotta a un logaritmo. Gli isolati sono stati definiti virulenti (V) e non virulenti (NV) in base alla loro efficienza di infezione. 45 isolati sono stati sequenziati per generare un albero filogenetico basato sui polimorfismi a singolo nucleotide (SNPs). Gli isolati NV clusterizzano separatamente dai V, suggerendo diverse strade evolutive. È stato verificato se i geni esclusivi degli isolati V fossero responsabili del loro maggiore capacità di infettare. I mutanti deleti per i plasmidi e le regioni genomiche individuate solo negli isolati V non hanno mostrato fenotipo di virulenza diverso dai rispettivi isolati wild type. È stato quindi valutato se specifiche varianti alleliche discriminanti gli isolati NV dai V fossero implicate nella virulenza. Sono stati individuati due alleli di HilD, l’attivatore dell’Isola di Patogenicità di Salmonella (SPI) 1 che codifica per il sistema di secrezione di tipo 3 (T3SS) responsabile dell’invasione delle cellule epiteliali. In cellule umane un isolato V (ER1175 WT) recante l’allele di hilD degli isolati NV (hilD_nv) ha un fenotipo non virulento. Allo stesso modo un isolato NV (ER278 WT) con l’allele di hilD degli isolati V (hilD_v) ha fenotipo virulento. Entrambi i ceppi deleti per hilD infettano le cellule umane allo stesso livello degli isolati NV. La delezione di hilD così come la sua sostituzione con hilD_nv in ER1175 WT non causano cambiamenti nell’efficienza di infezione delle cellule suine, mentre la delezione di invA (codificante un componente essenziale del T3SS) diminuisce il livello di virulenza. Non sono state riscontrate differenze tra ER278 WT, ER278ΔhilD e ER278ΔinvA in cellule suine, ma l’inserzione di hilD_v causa un aumento di invasione e replicazione del mutante rispetto a ER278 WT. Le due varianti alleliche di hilD sono quindi responsabili delle differenze osservate nelle cellule umane, ma non spiegano quelle osservate nelle cellule suine. Per valutare se la presenza di varianti alleliche diverse di hilD causi una diversa espressione della SPI-1, è stata eseguita un’analisi trascrittomica sull’RNA estratto in condizioni in vitro determinanti l’upregolazione di SPI-1. Non sono state riscontrate differenze nell’espressione genica tra i ceppi recanti hilD_nv e i rispettivi mutanti deleti in hilD, mentre i geni della SPI-1 sono upregolati sia in ER278 che ER1175 recanti hilD_v. I risultati di transcrittomica in vitro provano che hilD_nv esprime una proteina non funzionale. Gli stessi risultati sono stati ottenuti analizzando l’espressione dei geni della SPI-1 in ER1175 WT, ER1175::hilD_nv e ER1175ΔhilD durante infezione delle cellule umane. La virulenza di questi 3 ceppi e di ER278 WT è stata testata sul modello murino di infezione di Salmonella. I ceppi testati si accumulano nell’intestino ceco a livelli simili. Non sono state riscontrate differenze tra topi infettati nel livello di espressione di geni correlati con l’infiammazione (NOS2 and KC), espressi allo stesso livello dei topi di controllo non infettati. S. Derby sembra quindi non causare infiammazione intestinale. ER278 WT, ER1175 WT e ER1175::hilD_nv mostrano livelli di disseminazione non significativamente diversi. ER1175ΔhilD si accumula in fegato e milza a un livello significativamente maggiore rispettivo al ceppo wild type, suggerendo un effetto negativo di hilD sulla disseminazione di S. Derby. Questo studio ha fornito nel suo insieme nuove informazioni riguardo il meccanismo di adattamento all’ospite di S. Derby