Evaluation of factors influencing the growth of non-toxigenic Clostridium botulinum type E and Clostridium sp. in high-pressure processed and conditioned tender coconut water from Thailand.

Bacterial spores survive high pressure processing (HPP). Group II Clostridium botulinum is an obligate anaerobe spore-forming pathogen that can produce the botulinum neurotoxin under refrigeration. This study assessed nontoxigenic type E C. botulinum and Group II Clostridium sp. growth in raw and HPP (550 MPa, 3 min, 10 °C) Thai coconut water (CCW; pH 5.2). No spore germination or growth occurred in HPP CCW inoculated with 105 CFU/ml after 61 days regardless of oxygen concentration (<0.5 - 11 mg/l) or storage temperature (4 and 20 °C). Spore concentration decreased by 3.0 ± 0.1 log CFU/ml in a worst-case scenario consisting of non-HPP filter-sterilized CCW (pH 7.0) under anoxic incubation at 30 °C during 61 days, suggesting spore germination followed by cellular death. Supplementing filter-sterilized CCW (pH 7.0) with selected germinants and free amino acids did not support spore development, but the addition of nutrient-rich laboratory media (TPGY broth) at low concentrations (6.25%) promoted growth, suggesting that a lack of nutrients prevents C. botulinum development in CCW. Further risk assessment will require evaluating other CCW varieties and toxin production.

Author Correction: Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219.

Clostridium botulinum is an anaerobic sporeforming bacterium that is notorious for producing a potent neurotoxin. Spores of C. botulinum can survive mild food processing treatments and subsequently germinate, multiply, produce toxin and cause botulism. Control of spore germination and outgrowth is therefore essential for the safety of mildly processed foods. However, little is known about the process of spore germination in group II C. botulinum (gIICb), which are a major concern in chilled foods because they are psychrotrophic. The classical model of spore germination states that germination is triggered by the binding of a germinant molecule to a cognate germinant receptor. Remarkably, unlike many other sporeformers, gIICb has only one predicted canonical germinant receptor although it responds to multiple germinants. Therefore, we deleted the gerBAC locus that encodes this germinant receptor to determine its role in germination. Surprisingly, the deletion did not affect germination by any of the nutrient germinants, nor by the non-nutrient dodecylamine. We conclude that one or more other, so far unidentified, germinant receptors must be responsible for nutrient induced germination in gIICb. Furthermore, the gerBAC locus was strongly conserved with intact open reading frames in 159 gIICb genomes, suggesting that it has nevertheless an important function.

Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement.

UNLABELLED: Group II nonproteolytic Clostridium botulinum (gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies with C. botulinum are cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain, bont/E was exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use of pyrE as a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia. IMPORTANCE: The nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophic Clostridium botulinum In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia.

Two Complete and One Draft Genome Sequence of Nonproteolytic Clostridium botulinum Type E Strains NCTC 8266, NCTC 8550, and NCTC 11219.

Group II (gII) nonproteolytic Clostridium botulinum strains are a major cause of foodborne botulism outbreaks. Here, we report two complete genome sequences of gII type E strains NCTC 8266 and NCTC 8550 and one draft genome sequence of type E NCTC 11219.

A combination of polyunsaturated fatty acid, nonribosomal peptide and polyketide biosynthetic machinery is used to assemble the zeamine antibiotics.

The zeamines are a unique group of antibiotics produced by Serratia plymuthica RVH1 that contain variable hybrid peptide-polyketide moieties connected to a common pentaamino-hydroxyalkyl chain. They exhibit potent activity against a broad spectrum of Gram-positive and Gram-negative bacteria. Here we report a combination of targeted gene deletions, high resolution LC-MS(/MS) analyses, in vitro biochemical assays and feeding studies that define the functions of several key zeamine biosynthetic enzymes. The pentaamino-hydroxyalkyl chain is assembled by an iterative multienzyme complex (Zmn10-13) that bears a close resemblance to polyunsaturated fatty acid synthases. Zmn14 was shown to function as an NADH-dependent thioester reductase and is proposed to release a tetraamino-hydroxyalkyl thioester from the acyl carrier protein domain of Zmn10 as an aldehyde. Despite the intrinsic ability of Zmn14 to catalyze further reduction of aldehydes to alcohols, the initially-formed aldehyde intermediate is proposed to undergo preferential transamination to produce zeamine II. In a parallel pathway, hexapeptide-monoketide and hexapeptide-diketide thioesters are generated by a hybrid nonribosomal peptide synthetase-polyketide synthase multienzyme complex (Zmn16-18) and subsequently conjugated to zeamine II by a stand-alone condensing enzyme (Zmn19). Structures for the resulting prezeamines were elucidated using a combination of high resolution LC-MS/MS and 1- and 2-D NMR spectroscopic analyses. The prezeamines are hypothesized to be precursors of the previously-identified zeamines, which are generated by the action of Zmn22, an acylpeptide hydrolase that specifically cleaves the N-terminal pentapeptide of the prezeamines in a post-assembly processing step. Thus, the zeamine antibiotics are assembled by a unique combination of nonribosomal peptide synthetase, type I modular polyketide synthase and polyunsaturated fatty acid synthase-like biosynthetic machinery.

The zeamine antibiotics affect the integrity of bacterial membranes.

The zeamines (zeamine, zeamine I, and zeamine II) constitute an unusual class of cationic polyamine-polyketide-nonribosomal peptide antibiotics produced by Serratia plymuthica RVH1. They exhibit potent bactericidal activity, killing a broad range of Gram-negative and Gram-positive bacteria, including multidrug-resistant pathogens. Examination of their specific mode of action and molecular target revealed that the zeamines affect the integrity of cell membranes. The zeamines provoke rapid release of carboxyfluorescein from unilamellar vesicles with different phospholipid compositions, demonstrating that they can interact directly with the lipid bilayer in the absence of a specific target. DNA, RNA, fatty acid, and protein biosynthetic processes ceased simultaneously at subinhibitory levels of the antibiotics, presumably as a direct consequence of membrane disruption. The zeamine antibiotics also facilitated the uptake of small molecules, such as 1-N-phenylnaphtylamine, indicating their ability to permeabilize the Gram-negative outer membrane (OM). The valine-linked polyketide moiety present in zeamine and zeamine I was found to increase the efficiency of this process. In contrast, translocation of the large hydrophilic fluorescent peptidoglycan binding protein PBDKZ-GFP was not facilitated, suggesting that the zeamines cause subtle perturbation of the OM rather than drastic alterations or defined pore formation. At zeamine concentrations above those required for growth inhibition, membrane lysis occurred as indicated by time-lapse microscopy. Together, these findings show that the bactericidal activity of the zeamines derives from generalized membrane permeabilization, which likely is initiated by electrostatic interactions with negatively charged membrane components.