High Pressure Processing Applications in Plant Foods

High pressure processing (HPP) is a cold pasteurization technology by which products, prepacked in their final package, are introduced to a vessel and subjected to a high level of isostatic pressure (300–600 MPa). High-pressure treatment of fruit, vegetable and fresh herb homogenate products offers us nearly fresh products in regard to sensorial and nutritional quality of original raw materials, representing relatively stable and safe source of nutrients, vitamins, minerals and health effective components. Such components can play an important role as a preventive tool against the start of illnesses, namely in the elderly. An overview of several food HPP products, namely of fruit and vegetable origin, marketed successfully around the world is presented. Effects of HPP and HPP plus heat on key spoilage and pathogenic microorganisms, including the resistant spore form and fruit/vegetable endogenous enzymes are reviewed, including the effect on the product quality. Part of the paper is devoted to the industrial equipment available for factories manufacturing HPP treated products.

Identification of a novel regulator of Clostridioides difficile cortex formation

Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. C. difficile infections are transmitted by its metabolically dormant, aerotolerant spore form. Functional spore formation depends on the assembly of two protective layers: a thick layer of modified peptidoglycan known as the cortex layer and a multilayered proteinaceous meshwork known as the coat. We previously identified two spore morphogenetic proteins, SpoIVA and SipL, that are essential for recruiting coat proteins to the developing forespore and making functional spores. While SpoIVA and SipL directly interact, the identities of the proteins they recruit to the forespore remained unknown. We used mass spectrometry-based affinity proteomics to identify proteins that interact with the SpoIVA-SipL complex. These analyses identified the Peptostreptococcaceae family-specific, sporulation-induced bitopic membrane protein CD3457 (renamed SpoVQ) as a protein that interacts with SipL and SpoIVA. Loss of SpoVQ decreased heat-resistant spore formation by ∼5-fold and reduced cortex thickness∼2-fold; the thinner cortex layer of ΔspoVQ spores correlated with higher levels of spontaneous germination (i.e., in the absence of germinant). Notably, loss of SpoVQ in either spoIVA or sipL mutants prevented cortex synthesis altogether and greatly impaired the localization of a SipL-mCherry fusion protein around the forespore. Thus, SpoVQ is a novel regulator of C. difficile cortex synthesis that appears to link cortex and coat formation. The identification of SpoVQ as a spore morphogenetic protein further highlights how Peptostreptococcaceae family-specific mechanisms control spore formation in C. difficile.ImportanceThe Centers for Disease Control has designated Clostridioides difficile as an urgent threat because of its intrinsic antibiotic resistance. C. difficile persists in the presence of antibiotics in part because it makes metabolically dormant spores. While recent work has shown that preventing the formation of infectious spores can reduce C. difficile disease recurrence, more selective anti-sporulation therapies are needed. The identification of spore morphogenetic factors specific to C. difficile would facilitate the development of such therapies. In this study, we identified SpoVQ (CD3457) as a spore morphogenetic protein specific to the Peptostreptococcaceae family that regulates the formation of C. difficile’s protective spore cortex layer. SpoVQ acts in concert with the known spore coat morphogenetic factors, SpoIVA and SipL, to link formation of the protective coat and cortex layers. These data reveal a novel pathway that could be targeted to prevent the formation of infectious C. difficile spores.

Omadacycline compared to vancomycin when combined with germinants to disrupt the life cycle of Clostridioides difficile

Clostridioides difficile (C. difficile) infections (CDI) are commonly treated with antibiotics that do not impact the dormant spore form of the pathogen. CDI-directed antibiotics, such as vancomycin and metronidazole, can destroy the vegetative form of C. difficile and protective microbiota. After treatment, spores can germinate into vegetative cells causing clinical disease relapse and further spore shedding. This in vitro study compares the combination of germinants with vancomycin or omadacycline to antibiotics alone in eradicating C. difficile spores and vegetative cells. Among the four strains in this study, omadacycline minimum inhibitory concentrations (0.031-0.125 mg/L) were lower than vancomycin (1-4 mg/L). Omadacycline nor vancomycin in media alone reduced spore counts. In three of the four strains, including the epidemic ribotype 027, spore eradication with germinants was 94.8-97.4% with vancomycin and 99.4-99.8% with omadacycline (p<0.005). In ribotype 012, either antibiotic combined with germinants resulted in 100% spore eradication at 24 hours. The addition of germinants with either antibiotic did not result in significant toxin A or B production, which were below the limit of detection (<1.25 ng/mL) by 48 hours. Limiting the number of spores present in patient GI tracts at the end of therapy may be effective at preventing recurrent CDI and limiting spore shedding in the healthcare environment. These results with germinants warrant safety and efficacy evaluations in animal models.

Diversity of Endophytic fungi in liana, Celastrus paniculatus collected from few sites of Jhargram and Paschim Medinipur districts, West Bengal, India

To determine the identity and diversity of endophytic fungi associated with the liana from five different forest localities of Jhargram and West Medinipur districts of West Bengal. On the basis of differentiation of weather and microclimate, I have to select the regions. Between the two regions, the distance is at least 25-30 km, the microclimate and moisture under the canopy will differ, it affects mainly on the presence of endophytes. Leaf, fruit and stem segments were collected randomly in summer, winter and monsoon in 2018. It is impossible to take all leaves and other organs of a plant because plant parts should be collected sustainably so that minimum damage of stock occurs. Surfaces of all samples were sterilised just before putting on pot5ato dextrose agar (PDA) media for the growth of endophytic fungal mycelia and their isolation. Fungi were isolated and identified based on the morphology of its colony, and mycelial form and morphology, sexual and asexual reproductive structures and their characters, spore-form and nature of attachment, cultural conditions etc. were taken in consideration to identify them. Total 1125 samples were used for endophytic growth. The total of 1558 endophytic fungi were isolated from 797 sample segments of Celastrus paniculatus. The dominant endophytic fungi belong to genera Fusarium sp., Aspergillus sp., Chaetomium sp., Beltrania sp., Pestalotiopsis sp., Verticillium sp., Arthrinium sp, Penicillium sp., Podospora sp., Alternarium sp., Acrocylindrium sp. etc. Maximum endophytic isolates were obtained from leaf segments followed by fruit and then stem. In monsoon, colonization frequency shows highest (80.53%) and in summer, it is lowest (61.87%) from the plant samples of all locations. The examples from Chilkigarh shows the highest colonization frequency (90.22%) and from Nayagram, it is the lowest (61.78%). The leaf’s colonization frequency is maximum (84.53%) and the stem is minimum (62.4%). Most of the isolated endophytic fungi were found under the group Deuteromycetes. Endophytic fungi show a wide range of Shannon-Weiner and Simpson’s indices. These indicators point to an equal and throughout distribution of different species. The findings add to our knowledge of the identity and diversity of endophytic fungi, which are expected to have a variety of interactions with their host plants.

Influence of antibacterial agents on vaccine strains of Anthrax

Neutralization of spore-forming pathogenic microflora is carried out by solutions of disinfectants repeatedly. Antibacterial agents when used uncontrolled can reduce the sensitivity of microorganisms and as a result cause severe complications. Of considerable interest is the combined use of antibiotics with antibacterial drugs of plant origin. The use of antimicrobial agents of plant origin is due to their low toxicity, the possibility of long-term use, greater availability and ability to biodegradation, while synthetic drugs: antibiotics, fluoroquinolones, antiseptics have strong activity. Therefore, of great interest is the combined use of antibiotics with antibacterial drugs of plant origin. The object of our study were vaccine strains of the anthrax pathogen: B. anthracis K-79Z, B. anthracis 34F2 and anthrax-like bacilli B. cereus 8035, in spore form, disinfectants – sterilium (classic pur), ethanol 96%, alcohol solutions chlorophyllipt (1%) and propolis (7%). Studies have shown that bacteria of the strain B. cereus 8035 were insensitive to disinfectants of non-vegetable origin in the native and diluted state at exposures of 30, 60, 120 minutes and 24 hours of incubation, as evidenced by the intensive growth of the culture on meat-peptone agar (MPA). In the study of the disinfecting effect of 96% ethanol and sterile on the B. anthracis K-79Z strain, it was found that after exposure for 30, 60, 120 minutes and 24 hours, bacterial growth is recorded when using the native and disinfectant. Strains B. anthracis K-79Z and B. cereus 8035 were found to be more resistant to the action of disinfectants of plant and synthetic origin. In the future, we plan to continue the study of strains of the anthrax pathogen and anthrax bacilli on the sensitivity to disinfectants and to determine the relationship between the toxigenic characteristics of the strains.

Rapid Ultrasensitive Detection of Clostridiodes difficile Toxins in Stool Samples Using A Single-Molecule Counting Method

Background:Clostridiodes difficile infection is considered an urgent antibiotic resistance threat by the CDC, accounting for ∼225,000 hospitalizations, 12,800 deaths, and ∼$1 billion in healthcare costs in the United States in 2017. The presence of the secreted toxins that cause the devastating symptoms of this gastrointestinal infection are diagnostic of C. difficile infection (CDI). However, the rapid testing methods currently used to detect CDI lack accuracy. Enzyme immunoassays are specific but lack sensitivity because they do not detect CDI patients that have low levels of the toxins. Nucleic acid amplification tests (NAATs) are sensitive, but they lack specificity because they detect patients colonized with C. difficile in the dormant spore form that does not produce the toxins. This insufficiency has resulted in the adoption of complex multitest algorithms for C. difficile diagnosis. We present results for a new toxin test that demonstrates both high clinical sensitivity and clinical specificity for C. difficile toxin B on a fully automated benchtop platform. Methods: The detection technology uses nonmagnified digital imaging to count single toxin molecules that tether together target-specific magnetic and fluorescent particles. The 30-minute method includes the use of a dye cushion to eliminate wash steps and the need for time-consuming specimen preparation steps. We determined analytical performance characteristics of the test using negative clinical stool samples spiked with purified toxin. To assess clinical performance, we tested 785 stool samples from 5 clinical sites and compared the results with the cellular cytotoxicity neutralization assay (CCNA). Results: The test’s limit of detection for toxin B was 60 pg/mL. A comparison of the new test to the CCNA reference method gave 98% positive percentage agreement (83 of 85 samples) and 95% negative percentage agreement (667 of 700 samples). Conclusions: The new method demonstrated 96% accuracy compared to the gold standard for C. difficile toxin tests. The results also demonstrate an analytical sensitivity (limit of detection, 60 pg/mL). Thus, the test has the potential to detect CDI patients missed by enzyme immunoassay (EIA) tests due to their low analytical sensitivity. Because the test detects toxins directly, it is expected to have a lower false-positive rate than NAAT methods, which detect patients colonized with the non–toxin-producing spore form. A single accurate test for toxin-producing C. difficile could eliminate the need for multitest algorithms.Funding: First Light Diagnostics, Inc., provided support for this study.Disclosures: Donald Straus reports that he is the founder and chief scientific officer of First Light Diagnostics (FLDx) with salary and ownership interest in the form of stocks, stock options, and warrants. Adam Williams reports salary from First Light Diagnostics.

Treatment issues in recurrent Clostridioides difficile infections and the possible role of germinants

ABSTRACT Clostridioides difficile is the number one cause of hospital-acquired infections in the United States and one of the CDC's urgent-level pathogen threats. The inflammation caused by pathogenic C. difficile results in diarrhea and pseudomembranous colitis. Patients who undergo clinically successful treatment for this disease commonly experience recurrent infections. Current treatment options can eradicate the vegetative cell form of the bacteria but do not impact the spore form, which is impervious to antibiotics and resists conventional environmental cleaning procedures. Antibiotics used in treating C. difficile infections (CDI) often do not eradicate the pathogen and can prevent regeneration of the microbiome, leaving them vulnerable to recurrent CDI and future infections upon subsequent non-CDI-directed antibiotic therapy. Addressing the management of C. difficile spores in the gastrointestinal (GI) tract is important to make further progress in CDI treatment. Currently, no treatment options focus on reducing GI spores throughout CDI antibiotic therapy. This review focuses on colonization of the GI tract, current treatment options and potential treatment directions emphasizing germinant with antibiotic combinations to prevent recurrent disease.

Role of SpoIVA ATPase Motifs during Clostridioides difficile Sporulation

ABSTRACT The nosocomial pathogen Clostridioides difficile is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved spore protein SpoIVA and the clostridial-organism-specific spore protein SipL, which directly interact. Mutations that disrupt their interaction cause the coat to mislocalize and impair spore formation. In Bacillus subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in the C. difficile SpoIVA ATPase motifs impact functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104-fold less severe than the equivalent mutations in B. subtilis. Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased the SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but maintain ATP binding enhanced the SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele specific, our data imply that SipL recognizes the ATP-bound form of SpoIVA and highlight the importance of this interaction for functional C. difficile spore formation. IMPORTANCE The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins SpoIVA and SipL regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in the C. difficile SpoIVA ATPase motifs result in relatively minor defects in spore formation, in contrast with Bacillus subtilis. Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in the SipL C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

Role of SpoIVA ATPase Motifs During Clostridioides difficile Sporulation

The nosocomial pathogen, Clostridioides difficile, is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved coat protein, SpoIVA, and the clostridial-specific coat protein, SipL, which directly interact. Mutations that disrupt their interaction cause coat to mislocalize and decrease functional spore formation. In B. subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to help drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in C. difficile’s SpoIVA ATPase motifs impair functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104 less severe than the equivalent mutations in B. subtilis. Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but retain binding to ATP enhanced SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele-specific, our data implies that SipL recognizes the ATP-bound form of SpoIVA and highlights the importance of this interaction for functional C. difficile spore formation.ImportanceThe aerotolerant spores formed by the major nosocomial pathogen Clostridioides difficile are its primary infectious particle. However, the mechanism by which this critical cell type is assembled remains poorly characterized, especially with respect to its protective coat layer. We previously showed that binding between the spore morphogenetic proteins, SpoIVA and SipL, regulates coat assembly around the forespore. SpoIVA is widely conserved among spore-forming bacteria, and its ATPase activity is essential for Bacillus subtilis to form functional spores. In this study, we determined that mutations in C. difficile SpoIVA’s ATPase motifs result in relatively minor defects in spore formation in contrast with B. subtilis. Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in SipL’s C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

Differential effects of ‘resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination

Clostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of hospital-acquired gastroenteritis. C. difficile infections begin when its spore form germinates in the gut upon sensing bile acids. These germinants induce a proteolytic signaling cascade controlled by three members of the subtilisin-like serine protease family, CspA, CspB, and CspC. Notably, even though CspC and CspA are both pseudoproteases, they are nevertheless required to sense germinants and activate the protease, CspB. Thus, CspC and CspA are part of a growing list of pseudoenzymes that play important roles in regulating cellular processes. However, despite their importance, the structural properties of pseudoenzymes that allow them to function as regulators remain poorly understood. Our recently solved crystal structure of CspC revealed that its pseudoactive site residues align closely with the catalytic triad of CspB, suggesting that it might be possible to ‘resurrect' the ancestral protease activity of the CspC and CspA pseudoproteases. Here, we demonstrate that restoring the catalytic triad to these pseudoproteases fails to resurrect their protease activity. We further show that the pseudoactive site substitutions differentially affect the stability and function of the CspC and CspA pseudoproteases: the substitutions destabilized CspC and impaired spore germination without affecting CspA stability or function. Thus, our results surprisingly reveal that the presence of a catalytic triad does not necessarily predict protease activity. Since homologs of C. difficile CspA occasionally carry an intact catalytic triad, our results indicate that bioinformatic predictions of enzyme activity may underestimate pseudoenzymes in rare cases.