Identification and characterization of immuno-active factors of Enterococcus spp. involved in immunomodulatory effects of Probiotic Enterococcus faecium SF68

E. faecium SF68 is a licensed probiotic, which has been shown to ameliorate symptoms of inflammation in enteritis and to reduce the incidence and severity of diarrhea in humans and animals. In vivo trials with weaning piglets performed in our laboratory or in cooperation with our laboratory, yielded controversial observations on the anti-inflammatory effects of E. faecium SF68. Supplementation with the probiotic in animals was reported to contribute to reduced expression of immune-associated genes in intestinal tissues and associated lymphoid organs. Moreover, these animals had lower amounts of serum IgG and fecal IgA, and reduced CD8+ intraepithelial lymphocyte populations. Piglets supplemented with E. faecium SF68 showed higher colonization and greater pathogen shedding in Salmonella infection studies. Since the alterations observed were mainly associated with local innate immune responses, this doctoral study focused mainly on the direct immunomodulatory effects of E. faecium SF68 on intestinal epithelial cells in an in vitro model. NF-κB, one of the key initiators of innate immune responses in intestinal epithelial cells encountering microbiota and invasive enteric pathogens, was selected as a reporter for determination of the effects of E. faecium SF68 on immune response modulation in intestinal epithelial cells. Cell-free, whole bacterial lysates of E. faecium SF68 inhibited NF-κB activation in intestinal epithelial cells from different host species. This inhibitory effect occurred in a reversible manner; after removal of the lysate and addition of fresh culture medium, cells recovered their NF-κB activity. Cells treated with the bacterial lysates ceased to proliferate, based on the results of host cell cycle protein Ki67 staining. Porcine intestinal epithelial cells treated with the probiotic lysate contained lower levels of phosphorylated p65 subunit of NF-κB at serine 536 compared to untreated cells. Phosphorylation at this amino acid is one of the most studied modifications involved in the transcriptional activity of p65 subunit. Moreover, fluorescence microscopy images of porcine intestinal cells suggested a dysregulation in the trafficking of NF-κB between the cytoplasm and nucleus. Ammonium sulfate (AS) fractionation of proteins of the E. faecium SF68 lysates and analysis of the proteins present in different AS-fractions revealed that several enzymes involved in arginine metabolism are present in the active AS-fraction. Other commensal and clinical E. faecium isolates, as well as isolates of other enterococcal species possessing enzymes of arginine deiminase pathway including E. faecalis, E. hirae and E. durans, exhibited the same inhibitory effect on NF-κB activity of IPEC-J2 cells. E. gallinarum and E. casseliflavus species were the two exceptions. These two species are the only motile enterococcal species expressing flagellin which is a strong stimulant of inflammatory pathways such as NF-κB, particularly in our in vitro cell model. Experiments examining pre- and co-incubation of host cells with E. faecium SF68 lysates and different Toll-like receptor (TLR)- and NOD-like receptor (NLR)-ligands showed that cells pre-treated with the E. faecium SF68 bacterial lysates were severely impaired in their NF-κB activation responses to these pathogen-associated molecular pattern (PAMP) signals. Lysates from strains of other enterococcal species with no AD pathway activity (E. avium, E. cecorum and E. raffinosus) did not inhibit the NF-κB activity of treated IPEC-J2 cells. The arcA gene of E. faecium SF68 encoding arginine deiminase was cloned into an E. coli-Enterococcus shuttle vector and introduced into an E. avium strain which showed no inhibitory effects on NF-κB activity. Transformants of E. avium constitutively expressing arginine deiminase of E. faecium SF68 showed the same NF-κB-inhibitory effects on porcine and human intestinal epithelial cell lines as E. faecium SF68. Comparison of results with the Streptococcus suis 10 strain, another bacterial genus possessing AD-pathway enzymes, exhibited the same results. Furthermore, an arcA knockout mutant of S. suis 10 no longer showed inhibition of NF-κB activity of host cells and purified recombinant arginine deiminase of S. suis 10 was shown to have the same inhibitory effects on NF-κB activity as the cell-free, whole bacterial lysates of S. suis 10 and E. faecium SF68. Finally, arginine supplementation of the cell culture medium eliminated the inhibitory effects on NF-κB activity mediated by arginine deiminase or bacterial lysates containing arginine deiminase. In summary, we postulate that the reported anti-inflammatory effects of E. faecium SF68 in vitro and likely in vivo, are associated with arginine deiminase-mediated arginine depletion. Beneficial bacteria in general can have a large number of molecules and metabolites, allowing them to interact with intestinal epithelial and immune cells. Therefore, the immunomodulatory effects elicited by beneficial microbes in the gut may not be limited to a single factor. Additionally, the health condition of the recipients, their age and immune status can determine the outcome of beneficial microbe administration. For instance, neonates with underdeveloped immune systems, not fully shaped microbiota and higher metabolic activity are more sensitive to arginine depletion (Flynn et al. 2002). Moreover, the concurrent presence of an intestinal pathogen such as Salmonella or Giardia, which use arginine depletion as a defensive strategy to hinder the host iNOS production and proliferation of immune cells, can aggravate the clinical condition. These pathogens promote arginine depletion by their own AD-pathway enzymes or induction of arginine consuming enzymes in the host cells. The mechanisms of action of beneficial microbes should be studied well and in detail. More importantly, the assessments and decisions on the administration of a beneficial microbe as therapeutic or preventive measure should be made cautiously and individually for each case to optimize the use of these advantageous biological tools.E. faecium SF68 ist ein lizenziertes Probiotikum, dass nachweislich Entzündungssymptome bei Enteritis lindert und die Häufigkeit und Schwere von Durchfall bei Menschen und Tier reduziert. In einigen In vivo-Studien, die in unserem Labor oder in Zusammenarbeit mit unserem Labor durchgeführt wurden, wurden kontroverse Beobachtungen über entzündungshemmende Effekte von E. faecium SF68 bei Ferkeln gemacht. Es wurde berichtet, dass die Supplementierung von Tieren mit dem Probiotikum zu einer geringeren Expression von immun-assoziierten Genen des Darmgewebes und der assoziierten lymphoiden Organe beiträgt. Außerdem hatten diese Tiere ein niedrigeres Serum-IgG und fäkales IgA. Darüber hinaus wurden bei diesen Tieren geringere CD8+ intraepitheliale Lymphozyten-populationen nachgewiesen. Ferkel, die gleichzeitig mit Salmonellen infiziert wurden, hatten eine höhere Kolonisierung und eine größere Ausscheidung der Infektionserreger. Da die beobachteten Veränderungen hauptsächlich mit lokalen angeborenen Immunantworten in Verbindung gebracht wurden, lag der Fokus dieser Doktorarbeit auf der Analyse direkter immunmodulatorischer Effekte von E. faecium SF68 auf intestinale Epithelzellen in einem in vitro Modell. NF-κB, einer der wichtigsten Regulatoren angeborener Immunantworten in Darmepithelzellen, die auf Mikrobiota und invasive enterische Pathogene treffen, wurde als Kriterium für die Bestimmung der Modulationen der Immunantwort in Darmepithelzellen ausgewählt. Das zellfreie Gesamtlysat von E. faecium SF68 hemmte die NF-κB-Aktivierung in intestinalen Epithelzellen, die von verschiedenen Wirtsspezies stammen. Dieser inhibitorische Effekt trat reversibel auf. Durch Entfernen des Lysats und Zugabe von frischem Kulturmedium erlangten die Zellen ihre NF-κB-Aktivität wieder. In Schweine-Darmepithelzellen, die mit dem probiotischen Lysat behandelt wurden, kam die phosphorylierte p65-Untereinheit von NF-κB an Serin 536 im Vergleich zu unbehandelten Zellen in geringerer Menge vor. Darüber hinaus deuteten fluoreszenzmikroskopische Aufnahmen von Schweine-Darmzellen auf eine Behinderung des Trafficking von NF-κB zwischen Zytoplasma und Zellkern hin. Zudem waren die mit dem bakteriellen Lysat behandelten Zellen nicht proliferativ, was mittels Zellproliferationsassay und Ki67-Färbung nachgewiesen wurde. Die Ammoniumsulfat-Fraktionierung von E. faecium SF68-Lysat und die Vergleichsanalyse der in den verschiedenen AS-Fraktionen vorhandenen Proteine ergab, dass mehrere am Arginin-Stoffwechsel beteiligte Enzyme in der aktiven AS-Fraktion vorhanden waren. Andere kommensale und klinische E. faecium-Isolate sowie Isolate anderer Enterokokken-Spezies, die Enzyme des Arginin-Deiminase-Pfads besitzen, einschließlich E. faecalis, E. hirae und E. durans, zeigten die gleiche inhibitorische Wirkung auf die NF-κB-Aktivität von IPEC-J2-Zellen. Die beiden Ausnahmen waren die Spezies E. gallinarum und E. casseliflavus. Diese beiden Spezies sind die einzigen motilen Enterokokken-Spezies, also Flagellin exprimieren. Flagellen sind ein starker Stimulator von Entzündungswegen, wie z.B. NF-κB, insbesondere in unserem In vitro Zellmodell. Darüber hinaus zeigten unsere Co-Inkubationsexperimente von Zellen mit E. faecium SF68-Lysat und verschiedenen TLR- und NLR-Liganden, dass die Verabreichung von Flagellin auf Zellen, die mit dem bakteriellen Lysat von E. faecium SF68 vorbehandelt wurden, die NF-κB-Aktivierung zusätzlich zu der inhibitorischen Wirkung des Lysats stimuliert. Lysat von Stämmen anderer Enterokokken-Spezies, die keine Enzyme des AD-Wegs enthalten, wie E. avium, E. cecorum und E. raffinosus, hingegen hemmten die NF-κB-Aktivität der behandelten IPEC-J2-Zellen nicht. Im nächsten Schritt wurde das arcA-Gen von E. faecium SF68, das Arginin-Deiminase exprimiert, in einen E. avium-Stamm kloniert, der keine hemmende Wirkung auf die NF-κB-Aktivität der Zellen besaß. E. avium, das die Arginin-Deiminase von E. faecium SF68 konstitutiv exprimiert, hatte die gleichen NF-κB-hemmenden Effekte auf porcine und humane intestinale Epithelzelllinien wie E. faecium SF68. Darüber hinaus zeigten Tests mit S. suis 10-Stamm aus anderen Bakteriengattungen, die AD-Weg-Enzyme besitzt, die gleichen Ergebnisse. Außerdem konnte die arcA-Knockout-Mutante von S. suis 10 die NF-κB-Aktivität der Zellen nicht beeinträchtigen. Schließlich wurde gezeigt, dass überexprimierte und gereinigte rekombinante Arginin-Deiminase von S. suis 10 die gleiche hemmende Wirkung hatte wie das zellfreie Gesamtlysat von S. suis 10 und E. faecium SF68. Die Arginin-Supplementierung des Kulturmediums wiederrum eliminierte die suppressiven Effekte auf die NF-κB-Aktivität, die durch Arginin-Deiminase oder bakterielle Lysate, die Arginin-Deiminase enthalten, vermittelt werden. Insgesamt postulieren wir, dass die entzündungshemmenden Effekte von E. faecium SF68 In vitro und möglicherweise auch In vivo mit dem Arginin-Deiminase-vermittelten Arginin-Abbau verbunden sind. Allerdings haben probiotische Bakterien im Allgemeinen eine große, komplizierte Anzahl von Molekülen und Metaboliten, wodurch sie mit intestinalen Epithel- und Immunzellen interagieren können. Daher können die immunmodulatorischen Effekte, die probiotische Mikroben im Darm hervorrufen, nicht auf einen einzigen Faktor zurückgeführt werden. Darüber hinaus können der Gesundheitszustand der Empfänger, ihr Alter und ihr Immunstatus für das Ergebnis der Verabreichung von nützlichen Mikroben ausschlaggebend sein. So sind zum Beispiel Neugeborene mit einem unterentwickelten Immunsystem, einer nicht vollständig ausgebeildeten Mikrobiota und einer höheren Stoffwechselaktivität empfindlicher gegenüber einer Arginin-Verarmung. Darüber hinaus kann das gleichzeitige Vorhandensein eines Darmpathogens, wie Salmonellen oder Giardien, die die Arginin-Depletion als Verteidigungsstrategie zur Behinderung der iNOS-Produktion und der Proliferation von Immunzellen im Wirt nutzen, den klinischen Zustand verschlimmern. Diese Erreger fördern den Arginin-Abbau durch ihre eigenen AD-Weg-Enzyme oder die Induktion von Arginin-verbrauchenden Enzymen in den Wirtszellen. Die Wirkungsmechanismen probiotischer Mikroben sollten gut und detailliert untersucht werden. Noch wichtiger ist, dass die Bewertungen und Entscheidungen über die Verabreichung einer probiotischen Mikrobe als therapeutische oder präventive Maßnahme in jedem Fall vorsichtig und individuell getroffen werden sollten, um den Einsatz dieser vorteilhaften biologischen Werkzeuge zu optimieren

A molecular study of hemotropic mycoplasmas (hemoplasmas) in cats in Iran

BackgroundThree feline hemoplasma species are recognized: Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum', and Candidatus Mycoplasma turicensis'. These species can cause anemia in cats and have a worldwide distribution. ObjectivesThere was no previous information on hemotropic mycoplasma spp in cats in Iran and the Middle East. Accordingly, we investigated the molecular presence, and clinical signs and hematological profile in cats infected with these microorganisms in Iranian cats. MethodsPolymerase chain reaction (PCR) assays and cytology were performed on 100 blood samples collected from Iranian Shorthair cats. ACBC and case history were also collected for each sample. ResultsBy PCR, 22 (22%; 14-30%, 95% CI) samples were positive. The prevalence of M haemofelis, Ca M haemominutum', and Ca M turicensis' was 63.63% (14/22), 54.54% (12/22), and 18.18% (4/22), respectively. Some double and triple co-infections were also found. Using PCR as the reference method, cytology had poor sensitivity (27%) and reasonable specificity (89.74%). Male cats were at a higher risk of infection (P=.001). Cats older than 8years were more frequently infected than the younger cats (P=.0018). Lower HCT (P=.018), RBC count (P=.028) and HGB concentration (P=.003) were also associated with hemoplasma PCR-positive status. ConclusionsBased on this study, the most prevalent feline hemoplasma species in Iranian cats was M haemofelis, but double and triple co-infections are also documented. Age and sex, as well as reduced RBC parameters, were predisposing factors for hemoplasma infection

Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs

Strategies to reduce economic losses associated with post-weaning diarrhea in pig farming include high-level dietary zinc oxide supplementation. However, excessive usage of zinc oxide in the pig production sector was found to be associated with accumulation of multidrug resistant bacteria in these animals, presenting an environmental burden through contaminated manure. Here we report on zinc tolerance among a random selection of intestinal Escherichia coli comprising of different antibiotic resistance phenotypes and sampling sites isolated during a controlled feeding trial from 16 weaned piglets: In total, 179 isolates from “pigs fed with high zinc concentrations” (high zinc group, [HZG]: n = 99) and a corresponding “control group” ([CG]: n = 80) were investigated with regard to zinc tolerance, antimicrobial- and biocide susceptibilities by determining minimum inhibitory concentrations (MICs). In addition, in silico whole genome screening (WGSc) for antibiotic resistance genes (ARGs) as well as biocide- and heavy metal tolerance genes was performed using an in-house BLAST-based pipeline. Overall, porcine E. coli isolates showed three different ZnCl2 MICs: 128 μg/ml (HZG, 2%; CG, 6%), 256 μg/ml (HZG, 64%; CG, 91%) and 512 μg/ml ZnCl2 (HZG, 34%, CG, 3%), a unimodal distribution most likely reflecting natural differences in zinc tolerance associated with different genetic lineages. However, a selective impact of the zinc-rich supplemented diet seems to be reasonable, since the linear mixed regression model revealed a statistically significant association between “higher” ZnCl2 MICs and isolates representing the HZG as well as “lower ZnCl2 MICs” with isolates of the CG (p = 0.005). None of the zinc chloride MICs was associated with a particular antibiotic-, heavy metal- or biocide- tolerance/resistance phenotype. Isolates expressing the 512 μg/ml MIC were either positive for ARGs conferring resistance to aminoglycosides, tetracycline and sulfamethoxazole-trimethoprim, or harbored no ARGs at all. Moreover, WGSc revealed a ubiquitous presence of zinc homeostasis and – detoxification genes, including zitB, zntA, and pit. In conclusion, we provide evidence that zinc-rich supplementation of pig feed selects for more zinc tolerant E. coli, including isolates harboring ARGs and biocide- and heavy metal tolerance genes – a putative selective advantage considering substances and antibiotics currently used in industrial pork production systems

A virulence factor as a therapeutic: the probiotic Enterococcus faecium SF68 arginine deiminase inhibits innate immune signaling pathways

The probiotic bacterial strain Enterococcus faecium SF68 has been shown to alleviate symptoms of intestinal inflammation in human clinical trials and animal feed supplementation studies. To identify factors involved in immunomodulatory effects on host cells, E. faecium SF68 and other commensal and clinical Enterococcus isolates were screened using intestinal epithelial cell lines harboring reporter fusions for NF-κB and JNK(AP-1) activation to determine the responses of host cell innate immune signaling pathways when challenged with bacterial protein and cell components. Cell-free, whole-cell lysates of E. faecium SF68 showed a reversible, inhibitory effect on both NF-κB and JNK(AP-1) signaling pathway activation in intestinal epithelial cells and abrogated the response to bacterial and other Toll-like receptor (TLR) ligands. The inhibitory effect was species-specific, and was not observed for E. avium, E. gallinarum, or E. casseliflavus. Screening of protein fractions of E. faecium SF68 lysates yielded an active fraction containing a prominent protein identified as arginine deiminase (ADI). The E. faecium SF68 arcA gene encoding arginine deiminase was cloned and introduced into E. avium where it conferred the same NF-κB inhibitory effects on intestinal epithelial cells as seen for E. faecium SF68. Our results indicate that the arginine deiminase of E. faecium SF68 is responsible for inhibition of host cell NF-κB and JNK(AP-1) pathway activation, and is likely to be responsible for the anti-inflammatory and immunomodulatory effects observed in prior clinical human and animal trials. The implications for the use of this probiotic strain for preventive and therapeutic purposes are discussed

Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification–based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa

Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification–based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa

High-Zinc Supplementation of Weaned Piglets Affects Frequencies of Virulence and Bacteriocin Associated Genes Among Intestinal Escherichia coli Populations

To prevent economic losses due to post-weaning diarrhea (PWD) in industrial pig production, zinc (Zn) feed additives have been widely used, especially since awareness has risen that the regular application of antibiotics promotes buildup of antimicrobial resistance in both commensal and pathogenic bacteria. In a previous study on 179 Escherichia coli collected from piglets sacrificed at the end of a Zn feeding trial, including isolates obtained from animals of a high-zinc fed group (HZG) and a corresponding control group (CG), we found that the isolate collection exhibited three different levels of tolerance toward zinc, i.e., the minimal inhibitory concentration (MIC) detected was 128, followed by 256 and 512 mu g/ml ZnCl2. We further provided evidence that enhanced zinc tolerance in porcine intestinal E. coli populations is clearly linked to excessive zinc feeding. Here we provide insights about the genomic make-up and phylogenetic background of these 179 E. coli genomes. Bayesian analysis of the population structure (BAPS) revealed a lack of association between the actual zinc tolerance level and a particular phylogenetic E. coli cluster or even branch for both, isolates belonging to the HZG and CG. In addition, detection rates for genes and operons associated with virulence (VAG) and bacteriocins (BAG) were lower in isolates originating from the HZG (41 vs. 65% and 22 vs. 35%, p < 0.001 and p = 0.002, resp.). Strikingly, E. coli harboring genes defining distinct pathotypes associated with intestinal disease, i.e., enterotoxigenic, enteropathogenic, and Shiga toxin-producing E. coli (ETEC, EPEC, and STEC) constituted 1% of the isolates belonging to the HZG but 14% of those from the CG. Notably, these pathotypes were positively associated with enhanced zinc tolerance (512 mu g/ml ZnCl2 MIC, p < 0.001). Taken together, zinc excess seems to influence carriage rates of VAGs and BAGs in porcine intestinal E. coli populations, and high-zinc feeding is negatively correlated with enteral pathotype occurrences, which might explain earlier observations concerning the relative increase of Enterobacterales considering the overall intestinal microbiota of piglets during zinc feeding trials while PWD rates have decreased

Phenotypic zinc resistance does not correlate with antimicrobial multi-resistance in fecal E. coli isolates of piglets

Background: Following the ban on antimicrobial usage for growth promotion in animal husbandry in the EU, nonantimicrobial agents including heavy metal ions (e.g. zinc and copper), prebiotics or probiotics have been suggested as alternatives. Zinc has extensively been used in pig farming, particularly during weaning of piglets to improve animal health and growth rates. Recent studies, however, have suggested that high dietary zinc feeding during weaning of piglets increases the proportion of multi-drug resistant E. coli in the gut, contraindicating the appropriateness of zinc as an alternative. The underlying mechanisms of zinc effects on resistant bacteria remains unclear, but coselection processes could be involved. In this study, we determined whether E. coli isolates from intestinal contents of piglets that had been supplemented with high concentrations of zinc acquired a higher tolerance towards zinc, and whether multi-drug resistant isolates tolerated higher zinc concentrations. In addition, we compared phenotypic zinc and copper resistance of E. coli isolates for possible correlation between phenotypic resistance/tolerance to different bivalent ionic metals. Results: We screened phenotypic zinc/copper tolerance of 210 isolates (including antimicrobial resistant, multi-drug resistant, and non-resistant E. coli) selected from two, independent zinc-feeding animal trials by determining a zinc/ copper minimal inhibitory concentration (Merlin, Bornheim-Hersel, Germany). In both trials, groups of piglets were supplemented either with high dietary zinc (> 2000 ppm) or control (50–70 ppm, background) concentrations. Our observations showed that high concentration zinc exposure did not have an effect on either zinc or copper phenotypic tolerance of E. coli isolates from the animals. No significant association was found between antimicrobial resistance and phenotypic zinc/copper tolerance of the same isolates. Conclusion: Our findings argue against a co-selection mechanism of antimicrobial drug-resistance and zinc tolerance after dietary zinc supplementation in weaning piglets. An explanation for an increase in multi-drug resistant isolates from piglets with high zinc dietary feeding could be that resistant bacteria to antimicrobial agents are more persistent to stresses such as zinc or copper exposure.Peer Reviewe