Correction: Zinc is required to ensure the expression of flagella and the ability to form biofilms in Salmonella enterica sv Typhimurium

: Correction for 'Zinc is required to ensure the expression of flagella and the ability to form biofilms in Salmonella enterica sv Typhimurium' by Serena Ammendola et al., Metallomics, 2016, DOI: 10.1039/c6mt00108d

Salmonella enterica serovar typhimurium exploits inflammation to modify swine intestinal microbiota

Salmonella enterica serovar Typhimurium is an important zoonotic gastrointestinal pathogen responsible for foodborne disease worldwide. It is a successful enteric pathogen because it has developed virulence strategies allowing it to survive in a highly inflamed intestinal environment exploiting inflammation to overcome colonization resistance provided by intestinal microbiota. In this study, we used piglets featuring an intact microbiota, which naturally develop gastroenteritis, as model for salmonellosis. We compared the effects on the intestinal microbiota induced by a wild type and an attenuated S. Typhimurium in order to evaluate whether the modifications are correlated with the virulence of the strain. This study showed that Salmonella alters microbiota in a virulence-dependent manner. We found that the wild type S. Typhimurium induced inflammation and a reduction of specific protecting microbiota species (SCFA-producing bacteria) normally involved in providing a barrier against pathogens. Both these effects could contribute to impair colonization resistance, increasing the host susceptibility to wild type S. Typhimurium colonization. In contrast, the attenuated S. Typhimurium, which is characterized by a reduced ability to colonize the intestine, and by a very mild inflammatory response, was unable to successfully sustain competition with the microbiota

Development of a framework for genotyping bovine-derived Cryptosporidium parvum, using a multilocus fragment typing tool

Background: There is a need for an integrated genotyping approach for C. parvum; no sufficiently discriminatory scheme to date has been fully validated or widely adopted by veterinary or public health researchers. Multilocus fragment typing (MLFT) can provide good differentiation and is relatively quick and cheap to perform. A MLFT tool was assessed in terms of its typeability, specificity, precision (repeatability and reproducibility), accuracy and ability to genotypically discriminate bovine-derived Cryptosporidium parvum. Methods: With the aim of working towards a consensus, six markers were selected for inclusion based on their successful application in previous studies: MM5, MM18, MM19, TP14, MS1 and MS9. Alleles were assigned according to the fragment sizes of repeat regions amplified, as determined by capillary electrophoresis. In addition, a region of the GP60 gene was amplified and sequenced to determine gp60 subtype and this was added to the allelic profiles of the 6 markers to determine the multilocus genotype (MLG). The MLFT tool was applied to 140 C. parvum samples collected in two cross-sectional studies of UK calves, conducted in Cheshire in 2004 (principally dairy animals) and Aberdeenshire/Caithness in 2011 (beef animals). Results: Typeability was 84 %. The primers did not amplify tested non-parvum species frequently detected in cattle. In terms of repeatability, within- and between-run fragment sizes showed little variability. Between laboratories, fragment sizes differed but allele calling was reproducible. The MLFT had good discriminatory ability (Simpson’s Index of Diversity, SID, was 0.92), compared to gp60 sequencing alone (SID 0.44). Some markers were more informative than others, with MS1 and MS9 proving monoallelic in tested samples. Conclusions: Further inter-laboratory trials are now warranted with the inclusion of human-derived C. parvum samples, allowing progress towards an integrated, standardised typing scheme to enable source attribution and to determine the role of livestock in future outbreaks of human C. parvum

Multiple identification of most important waterborne protozoa in surface water used for irrigation purposes by 18S rRNA amplicon-based metagenomics

[EN] Understanding waterborne protozoan parasites (WPPs) diversity has important implications in public health. In this study, we evaluated a NGS-based method as a detection approach to identify simultaneously most important WPPs using 18S rRNA high-throughput sequencing. A set of primers to target the V4 18S rRNA region of WPPs such as Cryptosporidium spp., Giardia sp., Blastocystis sp., Entamoeba spp, Toxoplasma sp. and free-living amoebae (FLA) was designed. In order to optimize PCR conditions before sequencing, both a mock community with a defined composition of representative WPPs and a real water sample inoculated with specific WPPs DNA were prepared. Using the method proposed in this study, we have detected the presence of Giardia intestinalis, Acanthamoeba castellanii, Toxoplasma gondii, Entamoeba histolytica and Blastocystis sp. at species level in real irrigation water samples. Our results showed that untreated surface irrigation water in open fields can provide an important source of WPPs. Therefore, the methodology proposed in this study can establish a basis for an accurate and effective diagnostic of WPPs to provide a better understanding of the risk associated to irrigation water.This work was supported through the project funded by the Spanish Ministry of Economy and Competitiveness (MINECO) in the frame of the collaborative international consortium JPIW2013-095-C03-02 of the Water Challenges for a Changing World Joint Programming Initiative (Water JPI) Pilot Call. R. Perez acknowledges support from MINECO program "Promocion de Empleo Joven e Implantacion de la Garantia Juvenil en I + D + i".Moreno Trigos, MY.; Moreno-Mesonero, L.; Amoros, I.; Pérez-Santonja, R.; Morillo, J.; Alonso Molina, JL. (2018). Multiple identification of most important waterborne protozoa in surface water used for irrigation purposes by 18S rRNA amplicon-based metagenomics. International Journal of Hygiene and Environmental Health. 221(1):102-111. https://doi.org/10.1016/j.ijheh.2017.10.008S102111221

Study of host-pathogen-microbiota interactions on a Salmonella enterica serovar Typhimurium piglet model

Salmonella enterica serovar Typhimurium is a Gram negative gastrointestinal pathogen responsible for food poisoning worldwide. Upon entry into the host, Salmonella must overcome the robust line of defense provided by the innate immune system and by microbiota-mediated colonization resistance. It is a fascinating example of pathogen able not only to evade immune response, but also to exploit multiple aspects of it to colonize intestine. S. Typhimurium has evolved strategies allowing to thrive in the inflamed gut at the expense of the resident microbiota, taking advantage of the dramatic environmental conditions induced by inflammation. To understand the complex dynamics of interaction among S. Typhimurium-host-microbiota, is extremely important for the possible implications in the development strategies resulting in the reduction of colonization and/or frequency of salmonellosis. This thesis aimed at investigating the contribution of virulence, inflammation and microbiota to S. Typhimurium infection using piglet as model for salmonellosis. Firstly, we evaluated the capability of S. Typhimurium to exploit inflammation to promote its own colonization in vivo and in vitro. Our findings have shown that lipopolysaccharide (LPS) treatment-induced inflammation enhances the progression of S. Typhimurium, making the pathogen more capable to colonize at higher numbers both piglets and porcine enterocytes (IPEC-J2) and monocytes/macrophages cells. Secondly, we investigated the impact of Salmonella virulence on the porcine intestinal microbiota. To this end, we compared the effects on the microbiota induced by two different strains of Salmonella, a wild type strain and its isogenic attenuated mutant. We observed the existence of a link among virulence, inflammation and microbiota composition. S. Typhimurium wild type induces a strong inflammation which results in the reduction of some members of microbiota (i.e. SCFA-producing bacteria), normally involved in the maintaining of intestinal homeostasis and in the inhibition of pathogen growth. On the contrary, S. Typhimurium attenuated strain determines a milder inflammation and is correlated to an increase of protective species of microbiota which could shift the competition between the pathogen and microbiota in favour of the latter explaining the reduced ability of the attenuated strain to colonize host. Finally, we focused on the physiological alterations of porcine cecum caused by wild type and attenuated S. Typhimurium strains. Our results suggest that host could adopt a “nutriprive mechanism” in which deprives environment of nutrients and energy sources, creating intestinal conditions that are detrimental for Salmonella growth. Moreover, the minor reduction of metabolic and energetic status of the host upon infection with Salmonella wild type in comparison with the attenuated strain could indicate that Salmonella wild type is more capable to oppose to the nutriprive mechanism. Overall, by investigating the interaction dynamics among S. Typhimurium-host-microbiota, we have provided insights that the three factors are strictly interrelated and multiple aspects of each of them contribute to determine the outcome of infection.Salmonella enterica serovar Typhimurium è un batterio Gram negativo, patogeno enterico responsabile di una tossinfezione alimentare a diffusione mondiale. In seguito alla penetrazione nell’ospite, Salmonella deve superare la robusta linea di difesa rappresentata dal sistema dell’immunità innata e dalla resistenza alla colonizzazione mediata dal microbiota. Salmonella è un affascinante esempio di patogeno capace oltre che di eludere la risposta immunitaria, anche di sfruttarne i molteplici aspetti per colonizzare l’intestino. Ha sviluppato, infatti, strategie di virulenza che gli consentono di prosperare nell’intestino infiammato a discapito del microbiota residente, traendo vantaggio dalle condizioni ambientali fortemente alterate indotte dall’infiammazione. Comprendere le complesse dinamiche di interazione tra S. Typhimurium-ospite-microbiota, è estremamente importante per le possibili implicazioni nello sviluppo di strategie vòlte a ridurre la colonizzazione e/o la frequenza della salmonellosi. Scopo di questa tesi è stato caratterizzare il contributo della virulenza, dell’infiammazione e del microbiota all’infezione da Salmonella, utilizzando il suino come modello sperimentale per la salmonellosi. In primo luogo, abbiamo valutato l’abilità di S. Typhimurium di trarre vantaggio dall’infiammazione per favorire la propria colonizzazione, in vivo e in vitro. I nostri risultati hanno mostrato che l’infiammazione indotta dal trattamento con lipopolisaccaride (LPS) migliora la progressione dell’infezione, rendendo il patogeno capace di colonizzare con una carica più elevata sia i suinetti sia gli enterociti (IPEC-J2) e i monociti/macrofagi di origine suina. Successivamente, abbiamo valutato l’impatto che la virulenza del patogeno ha sul microbiota intestinale suino. A tale scopo, abbiamo comparato gli effetti causati da due differenti ceppi di Salmonella, un ceppo wild type e il suo mutante isogenico attenuato, sulla composizione del microbiota. Abbiamo osservato l’esistenza di un collegamento diretto tra virulenza, infiammazione e composizione del microbiota. S. Typhimurium wild type induce una forte risposta infiammatoria che determina la riduzione di membri del microbiota (ad es. i batteri che producono SCFA) normalmente implicati nel mantenimento dell’omeostasi intestinale e nell’inibizione della crescita dei patogeni. Al contrario, il ceppo attenuato di S. Typhimurium causa una debole infiammazione che è invece associata ad un aumento di specie protettive del microbiota e ciò potrebbe spostare la competizione tra patogeno e microbiota in favore di quest’ ultimo, spiegando così la ridotta abilità di questo ceppo a colonizzare l’ospite. Infine, la nostra attenzione si è focalizzata sulla risposta dell’ospite ed in particolare sulle modificazioni pato-fisiologiche che verificano a livello del cieco in seguito ad infezione con i due ceppi di S. Typhimurium, wild type e attenuato. I nostri risultati suggeriscono l’induzione di un “meccanismo nutriprivo” attraverso il quale l’ospite riduce la disponibilità di nutrienti e di fonti di energia per creare condizioni intestinali che risultano svantaggiose per la crescita di Salmonella. Inoltre, è stata osservata una minore riduzione dello stato metabolico ed energetico dell’ospite dopo infezione con S. Typhimurium wild type rispetto a quella indotta dal ceppo attenuato che potrebbe indicare una maggiore capacità del ceppo wild type a contrastare il meccanismo nutriprivo. In conclusione, studiando le dinamiche di interazione tra S. Typhimurium-ospite-microbiota, abbiamo fornito ulteriori evidenze del fatto che i tre fattori sono strettamente correlati e che l’esito dell’infezione è il risultato del contributo fornito dai molti aspetti che li caratterizzano

Treatment of hydatid bronchobiliary fistulas: 30 years of experience

Background: Bronchobiliary fistula (BBF) is an uncommon but severe complication of hydatid disease of the liver. Operation is considered the treatment of choice but the most appropriate operation is uncertain. The aim of this study was to evaluate the early and long-term outcomes following different surgical procedures. Methods: A retrospective evaluation of 31 patients with BBF was performed. Surgical access consisted of laparotomy, thoracotomy or a thoracoabdominal (TA) incision. Surgical procedures for the treatment of the cyst were classified as conservative or radical. Results: Radical treatment including lung resection and pericystectomy was performed in all patients in whom the surgical exposure was obtained by either thoracotomy or TA. Of the patients treated by laparotomy, two had a pericystectomy, and four had drainage of the cyst. There were two deaths among the seven thoracotomy patients and one among the 18 TA patients. Pleural effusion was observed in six of the TA, two of the thoracotomy, and three of the laparotomy patients. Biliary fistula occurred in two of the five thoracotomy patients surviving operation and in two laparotomy patients (2/6). Progression of the lung disease was observed in four laparotomy patients and in one thoracotomy patient. Conclusions: The better outcome achieved in TA patients is the result of the simultaneous radical treatment of all the pathological aspects of BBF

Zinc is required to ensure the expression of flagella and the ability to form biofilms in: Salmonella enterica sv Typhimurium

Zinc is known to play a central role in bacterial physiology and pathogenesis. Here, we report that the accumulation of FliC, the structural subunit of Salmonella phase 1 flagella, is sharply reduced in a znuABC Salmonella enterica sv. Typhimurium strain grown in zinc-poor media. Consequently, this mutant strain lacks motility, unless it grows in zinc-replete environments. This phenotype is the consequence of a general downregulation of all the genes involved in the biosynthesis of flagella, suggesting that zinc is the cofactor of proteins involved in the initiation of the transcriptional regulatory cascade leading to flagella assembly. Competition experiments in mice demonstrated that aflagellated (fliBfljC) and znuABC strains are outcompeted by the wild type strain in the gastrointestinal tract. The fliBfljC strain overgrows a fliCfljBznuABC mutant strain, but the difference in gut colonization between these two strains is less striking than that between the wild type and the znuABC strains, suggesting that the downregulation of flagella contributes to the loss of virulence of Salmonella znuABC. The absence of either flagella or ZnuABC also impairs the ability of S. Typhimurium to produce biofilms. Zinc suppresses this defect in the znuABC mutant but not in the aflagellated strains, highlighting the role of flagella in biofilm organization. We have also observed an increased production of the quorum sensing signal AI-2 in the znuABC strain sensing zinc deprivation, that may further contribute to the reduced ability to form biofilms. On the whole, our study reveals novel roles of zinc in Salmonella motility and intercellular communication