Control of bacterial pathogens during processing of cold-smoked and dried salmon strips.

Microbiological and chemical changes were determined during the smoking and drying of salmon strips processed at 29 to 31 degrees C for 4 days at a facility in Alaska in 1993. During the process, Staphylococcus aureus populations increased to more than 10(5) CFU/g after 2 to 3 days of processing. Subsequent laboratory studies showed that a pellicle (dried skinlike surface) formed rapidly on the strips when there was rapid air circulation in the smokehouse and that bacteria embedded in or under the pellicle were able to grow even when heavy smoke deposition occurred. Under these conditions, an inoculum of 26 CFU/g of S. aureus increased to 10(5) CFU/g after 3 days of processing. Elimination of preprocess drying and reduction in air flow during smoking resulted in smoke deposition before pellicle formation and enabled the product to reach levels of water-phase salt and water activity that inhibit the growth of S. aureus and Listeria monocytogenes. In 1994, these modifications were then applied during processing at an Alaskan facility, and S. aureus could not be detected in the finished product. L. monocytogenes was detected in the raw product area, on the processing tables, and on the raw salmon strips, but it was not detected in the finished product when the smoke was applied before pellicle formation.

Competitive inhibition between different Clostridium botulinum types and strains.

Mixtures of proteolytic and nonproteolytic strains of toxigenic Clostridium botulinum types A, B, and F; nonproteolytic types B, E, and F; Clostridium sporogenes; and nontoxic E-like organisms resembling nonproteolytic C. botulinum were tested against each other for the purpose of selecting a mixture of compatible C. botulinum strains for inoculated pack studies on the basis of their sensitivity to bacteriophages and bacteriocin-like agents. All of the proteolytic strains produced bacteriocin-like agents that were inhibitory to three or more of the other proteolytic types and C. sporogenes. When selected strains of proteolytic types A and B were grown together, type A cultures produced neurotoxin, but type B toxin production was inhibited. Nonproteolytic strains of C. botulinum also produced bacteriocin-like agents against each other. Of these, type E strain EF4 produced bacteriocin-like agents against both proteolytic and nonproteolytic types of C. botulinum and C. sporogenes. EF4, however, was not inhibitory to the nontoxigenic E-like strains. When EF4 was grown with type A strain 62A, it had an inhibitory effect on type A toxin production. Strain 62A inactivated the type E toxin of EF4 after 7 to 21 days at 30 degrees C. On the basis of the production of these bacteriocin-like agents by different strains of C. botulinum and their potential effect on neurotoxin production, it is very important that compatible strains are used in mixtures for inoculated pack studies to determine the safety of a food process or product.

Control of nonproteolytic Clostridium botulinum types B and E in crab analogs by combinations of heat pasteurization and water phase salt.

Water phase sodium chloride (WPS) levels of 1.8 to 3.0% in combination with heat pasteurization for 15 min at temperatures of 75, 80, 85, and 90 degrees C were evaluated as methods for the inactivation or inhibition of nonproteolytic, psychrotrophic Clostridium botulinum types B and E in crab analogs (imitation crab legs) subsequently stored at 10 and 25 degrees C. Samples inoculated with 10(2) type B or E spores per g prior to pasteurization remained nontoxic for 120 days at 10 degrees C and for 15 days at 25 degrees C. With 10(4) type E spores per g and 80 degrees C pasteurization, > or = 2.4 and 2.7% WPS was required for inhibition at 10 and 25 degrees C storage, respectively. Pasteurization at 85 degrees C decreased the inhibitory level of WPS to 2.1% at 10 degrees C and to 2.4% at 25 degrees C. When the inoculum was 10(4) type B spores per g, samples with 2.7% WPS were toxic after 80 days of storage at 10 degrees C. Samples inoculated with 10(3) type B spores per g and processed at 85 degrees C remained nontoxic for 15 days at 25 degrees C with a WPS of > or = 2.4%. When pasteurization was carried out before inoculation and packaging, 1.8% WPS prevented toxin production by 10(2) and 10(4) type E spores per g for 30 days at 10 degrees C, and this time period increased as the WPS concentrations increased. Three percent WPS prevented toxin production by 10(4) type E spores per g in vacuum-packaged analogs stored 110 days at 10 degrees C. Pasteurization processes used in these experiments, however, do not inactivate the heat-resistant proteolytic types of Clostridium botulinum. Therefore, the most important factor controlling the growth of this bacterium is continuous refrigeration below 3.0 degrees C or frozen storage of the finished product.

Heat-Pasteurization Process for Inactivation of Nonproteolytic Types of Clostridium botulinum in Picked Dungeness Crabmeat.

Development of a heat-pasteurization process is described for picked meat of Dungeness crabs ( Cancer magister ) contained in oxygen-impermeable flexible pouches, For each time-temperature treatment, 30 samples, each inoculated with an equal mixture of three strains of C. botulinum nonproteolytic type B, for a total of 107 spores, provided the basis for calculation of the thermal resistance (a 7D process). Following heat processing, the crabmeat was removed from the pouches and transferred to enrichment medium where it was incubated anaerobically for 150 days. Endpoints at which spores survived were determined by the presence of toxin in the enrichment medium. Process times ranged from 90 min at 88.9°C to 20.3 min at 94.4°C. D values (the time at each temperature required to reduce the inoculum by 1 log) ranged from 12.9 for the 88.9°C process to 2.9 for the 94.4°C process. The relative sterilization value, F0 was .054 and the pasteurization value, , was 240. This pasteurization process safely extends refrigerated shelf life by inactivating spores of Clostridium botulinum nonproteolytic types B, E, and F and also non-spore-forming pathogens such as Listeria monocytogenes . The process does not, however, inactivate the heat-resistant proteolytic strains of C. botulinum or other more heat-resistant spore-formers. The packages and master cartons of the pasteurized product, therefore, should be labeled "Keep refrigerated-Continuous refrigeration below 38°F (3.3°C) required."

Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion.

Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD, was identified by its ability to complement the pilD mutation in P. aeruginosa. Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes (tapABCD) that are homologous to P. aeruginosa type IV pilus biogenesis genes (pilABCD). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N-methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila.

Botulinal neurotoxin C1 complex genes, clostridial neurotoxin homology and genetic transfer in Clostridium botulinum.

The botulinal neurotoxins (BoNT) associate with non-toxic proteins (ANTP) by non-covalent bonds to form large complexes. In C. botulinum C, the BoNT/C1 locus consists of six genes which are organized in three clusters. Cluster 1 encompasses the genes of BoNT/C1 and ANTP/139 which could be involved in the resistance of the BoNT/C1 to the acidic pH and protease degradation. The second cluster consists of three genes which encode hemagglutinin components. The last gene encodes a DNA binding protein (Orf22) which might regulate the BoNT/C1 complex gene expression. BoNT and tetanus toxin (TeTx) display similar structure and mechanism of action at the molecular level. Their identity at the amino acid level range from 34 to 96.8%, indicating that the clostridial neurotoxin genes probably derive from a common ancestor. The fact that Clostridium other than C. botulinum such as C. butyricum and C. baratii can produce a BoNT suggests that the BoNT genes can be transferred between Clostridium strains. The toxigenic C. butyricum strains seem to derive from originally non-toxic strains by neurotoxin gene transfer from C. botulinum E, probably including a mobile DNA element. In C. botulinum C and D the gene encoding the exoenzyme C3 has been localized in a transposon-like element of 21.5 kbp. Transposons could be involved in BoNT gene transfer in C. botulinum.

Organization of the botulinum neurotoxin C1 gene and its associated non-toxic protein genes in Clostridium botulinum C 468.

A 12.3 kb DNA fragment encompassing the botulinum neurotoxin C1 (BoNT/C1) gene and an upstream flanking region was sequenced from Clostridium botulinum C 468 phage 1C. The resulting bont/C1 locus includes six genes which are organized into three transcriptional units. Cluster 1 encompasses the bont/C1 gene and an upstream gene encoding a non-toxic protein associated with the toxin (Antp139/C1). Transcriptional analysis revealed that these two genes form an operon; the bont/C1 gene can be transcribed alone or co-transcribed with antp139/C1. Cluster 2 encompasses three genes (antp33/C1, antp17/C1 and antp70/C1), which also form an operon. The corresponding proteins are similar to components of the hemagglutinin complex associated with BoNT/A and BoNT/B of C. botulinum A and B. In addition, Antp33/C1 is identical to HA-33, an hemagglutinin encoded by C. botulinum C-Stockholm phage C-St; Antp70/C1 displays some relatedness to C. perfringens enterotoxin. The third transcriptional unit consists of orf-22, which encodes a basic protein showing 29% identity with the gene product of uviA, a plasmid-encoded protein of 22 kDa which has been identified as a positive regulator of the bacteriocin production in C. perfringens. Orf-22 could be an effector controlling the expression of the bont/C1 and its antp genes in C. botulinum C 468.

Comparative analysis of C3 and botulinal neurotoxin genes and their environment in Clostridium botulinum types C and D.

The C3 exoenzyme gene is located on a bacteriophage in Clostridium botulinum types C and D (M. R. Popoff, D. Hauser, P. Boquet, M. W. Eklund, and D. M. Gill, Infect. Immun. 59:3673-3679, 1991). A derivative CN phage from phage C of C. botulinum Stockholm (C-St) (K. Oguma, H. Iida, and K. Inoue, Jpn. J. Microbiol. 19:167-172, 1975), isolated as neurotoxin negative, also does not produce exoenzyme C3. The botulinal neurotoxin C1 gene is present on the CN phage but contains a stop mutation in the DNA region encoding the N-terminal part of the heavy chain (codon 553). The putative truncated botulinal neurotoxin C1 protein was not recovered in a C. botulinum strain harboring the CN phage. We found that the C3 gene is localized on a 21.5-kbp DNA fragment flanked by the core motif 5'-AAGGAG-3' in DNAs of phage C of C. botulinum 468 (C-468), C-St phage, and phage D of C. botulinum 1873 (D-1873). The 21.5-kbp DNA fragment is deleted in CN phage DNA, and the motif 5'-AAGGAG-3' is present only in one copy at the deletion junction, but the deletion in the CN phage could be nonspecific, since this phage was obtained by nitrosoguanidine treatment. These findings could indicate that the C3 gene is localized on a 21.5-kbp mobile element. C. botulinum type C strain 003-9 produces a C3 exoenzyme (Y. Nemoto, T. Namba, S. Kozaki, and S. Narumiya, J. Biol. Chem. 266:19312-19319, 1991), and Staphylococcus aureus E1 produces a related C3 enzyme which is named epidernmal cell differentiation inhibitor (S. Inoue, M. Sugai, Y. Murooka, S. Y. Paik, Y. M. Hong, H. Oghai, and H. Suginaka, Biochem. Biophys. Res. Comm. 174:459-464, 1991) and which shares 80.6 and 56.6% similarity, respectively with the C3 enzymes from C-468 or C-St and D-1873 phages athe amino acid level. The features of the putative 21.5-kbp transposon were not found in C. botulinum 003-9 and S. aureus E1, as determined by analysis of the C3 and epidermal cell differentiation inhibitor gene-flanking DNA regions. These data suggest a common ancestral origin and divergent evolution of the C3 genes in these three groups of bacterial strains and dissemination of a 21.5-kbp element carrying the C3 gene C-468, C-St, and D-1873 phages.

Characterization of the C3 gene of Clostridium botulinum types C and D and its expression in Escherichia coli.

Clostridium botulinum type C and D strains produce exoenzyme C3, which ADP-ribosylates the Rho protein, a 21-kDa regulatory GTP-binding protein. In a previous work, we demonstrated that the C3 gene is encoded by bacteriophages C and D of C. botulinum by using DNA-DNA hybridizations with oligonucleotides deduced from the C3 protein N-terminal sequence. The C3 coding gene was cloned and sequenced, but its upstream DNA region could not be studied because of its instability in Escherichia coli. In this work, the upstream DNA region of the C3 gene was directly amplified by the polymerase chain reaction and sequenced. The C3 gene encodes a polypeptide of 251 amino acids (27,823 Da) consisting of a 40-amino-acid signal peptide and a mature protein of 211 amino acids (23,546 Da). The C3 mature protein was expressed in E. coli under the control of the trc promoter. The recombinant polypeptide obtained was recognized by C3 antibodies and ADP-ribosylated the Rho protein. The C3 gene nucleotide sequence is identical on C and D phage DNAs. At the amino acid sequence level, no similarity was found among C3, other ADP-ribosylating toxins, or tetanus or botulinal A, C1, and D neurotoxins.

Clostridium botulinum type-C intoxication associated with consumption of processed alfalfa hay cubes in horses.

An episode of nervous system dysfunction was observed in horses on 17 premises in 4 counties of southern California. Thirty-eight horses were affected, and 31 of those died. The common clinical signs of disease in the affected horses were: increased appetite; anxious attitude; rythmic, intermittent muscle tremors in the area of the tricep muscles; decreased palpebral tone; mydriasis; small hard fecal balls; and tendency to become sternally recumbent with the neck extended. The temporal distribution of cases on all 17 premises suggested a relationship between exposure to a common batch of alfalfa hay cubes and manifestations of similar clinical signs of disease in affected horses. Fifteen horses were submitted for necropsy. Diagnosis of botulism was established on the basis of detection of type-C1 toxin in the feed, in intestinal contents of 1 horse, and in the liver of the aforementioned horse and another horse. Toxigenic strains of Clostridium botulinum type-C were isolated from intestinal contents of 5 affected horses, one of which also contained type-C1 and type-C2 toxins. Seven of 10 horses treated with type-C antitoxin and plasma obtained from horses hyperimmunized with C botulinum type-C toxoids survived.

Evidence for plasmid-mediated toxin and bacteriocin production in Clostridium botulinum type G.

A single 81-megadalton plasmid was previously isolated from each of six toxigenic strains of Clostridium botulinum type G (M. S. Strom, M. W. Eklund, and F. T. Poysky, Appl. Environ. Microbiol. 48:956-963, 1984). In this study, nontoxigenic derivatives isolated from each of the toxigenic strains following consecutive daily transfers in Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract-glucose broth at 44 degrees C simultaneously ceased to produce type G neurotoxin and to harbor the resident 81-megadalton plasmid. The nontoxigenic derivatives also ceased to produce bacteriocin and lost their immunity to the bacteriocin produced by the toxigenic strains. In contrast, all of the toxigenic isolates continued to carry the resident plasmid and to produce both bacteriocin and type G neurotoxin. This is the first evidence suggesting that the production of neurotoxin and bacteriocin by C. botulinum is mediated by a plasmid.

Feasibility of a Heat-Pasteurization Process for the Inactivation of Nonproteolytic Clostridium botulinum types B and E in Vacuum-Packaged, Hot-Process (Smoked) Fish.

This study demonstrates the feasibility of a heat-pasteurization process for certain vacuum-packaged hot-smoked fishery products for inactivation of the spores of the nonproteolytic Group II Clostridium botulinum types B, E, and F. This process permits the use of lower concentrations of salt and other inhibitors without jeopardizing safety and quality of the products during prolonged refrigerated storage. The pasteurization treatment was developed based upon the inactivation of nonproteolytic types B or E in hot-process (smoked) salmon. Smoke was not applied to the samples inoculated with types B and E because of its possible inhibitory effects. After processing in the smokehouse, each sample was cooled to 34°F (1.1°C), injected with 106 spores, vacuum-packaged, and then heat-pasteurized in a water bath held at a constant temperature. A total of 85, 65, and 55 min in the 185°F (85°C), 192°F (88.9°C), and 198°F (92.2°C) baths, respectively, prevented toxin production by type E during 21 d of incubation at 25°C. Longer times, 175, 85, and 65 min, respectively, were required to prevent toxin production by nonproteolytic type B. Toxin production by type E during 120 d of storage at 10°C was prevented by a 45-minute treatment in the 198°F (92.2°C) bath. When heat-pasteurized samples were transferred into TPGY broth and incubated anaerobically for 150 d at 25°C, outgrowth and toxin production by type E was prevented by a 55-minute process at 198°F (92.2°C) and type B was prevented by a 65-minute process. This process does not, however, inactivate the more heat-resistant proteolytic strains of C. botulinum Group I or other spore-formers. The packages and master cartons of these pasteurized products therefore should follow the existing recommendations for smoked fishery products and be labeled "Keep refrigerated - Store below 38°F (3.3°C)."

Inhibition of Clostridium botulinum Type E Toxin Formation by Potassium Chloride and Sodium Chloride in Hot-Process (Smoked) Whitefish ( Coregonus clupeaformis ).

Whitefish steaks were brined in NaCl, KCl, or equimolar NaCl:KCl to contain similar chloride ion concentration and inoculated intramuscularly with 10 or 100 spores of Clostridium botulinum type E per gram of fish. Steaks were then heated in a simulated (i.e., without smoke) hot-smoke process to internal temperatures of 62.8° to 76.7°C (145°-170°F) for the final 30 min of a 2- to 3-h process, packaged under vacuum in oxygen-impermeable film, and stored at abuse temperature of 25°C. During 7 d of storage, toxin production was inhibited in steaks containing more than 0.66 ionic strength NaCl, 0.64 KCl, or 0.71 equimolar NaCl:KCl. The results indicate that it is feasible to substitute KCl for NaCl in hot-process smoked fish for inhibition of outgrowth and toxin production by Clostridium botulinum type E.

Plasmids in Clostridium botulinum and related Clostridium species.

Toxigenic Clostridium botulinum and nontoxigenic C. sporogenes, C. subterminale, and C. botulinum-like organisms from a variety of sources were screened for plasmids. Of the 68 toxigenic C. botulinum isolates, 56% carried one or more plasmids, ranging in mass from 2.1 to 81 megadaltons. Within individual groups (based on the type of neurotoxin produced), many strains showed identical plasmid banding patterns on agarose gels. Of the 15 nontoxigenic strains tested, 40% also carried one or more plasmids ranging from 1.7 to 25.0 megadaltons, with both unique and common banding patterns represented. A total of 67 plasmids from both toxigenic and nontoxigenic strains were detected. At this time, no phenotypic functions have been assessed for these plasmids, and they must therefore be considered cryptic. A variety of lysing and extraction techniques were necessary to detect plasmids in the different C. botulinum groups.

Inhibition of Clostridium botulinum Types A and E Toxin Production by Liquid Smoke and NaCl in Hot-Process Smoke-Flavored Fish.

Liquid smoke in combination with NaCl was an effective inhibitor of outgrowth and toxin production by Clostridium botulinum types A and E spores in hot-processed whitefish, chub and carp stored at an abuse temperature of 25°C for 7 or 14 d. Surface-inoculated type E produced toxin in control samples containing 3.7% water-phase NaCl, but not in liquid smoke-treated samples having less than 2.0% water-phase NaCl. Liquid smoke was less effective when type E spores were injected intramuscularly. Liquid smoke lowered the concentration of NaCl required to inhibit toxin production by surface-inoculated type A from 4.6 to 2.8% in samples stored 7 d. Liquid smoke enhanced the ability of NaCl to prevent toxin production, but should not be considered a substitute for NaCl or refrigerated storage (below 3.3°C).

Inhibition of Clostridium botulinum Types A and E Toxin Formation by Sodium Nitrite and Sodium Chloride in Hot-Process (Smoked) Salmon.

Sodium nitrite and NaCl were evaluated as inhibitors of outgrowth and toxin production by Clostridium botulinum types A and E in abuse-stored (25°C) hot-process salmon. Salmon steaks were brined in NaCl or NaCl plus NaNO2 and inoculated intramuscularly with spores. Steaks were then heated in a simulated hot-smoke process to internal temperatures of 62.8 to 76.7°C (145 to 170°F) for the final 30 min of a 3- to 4-h process, packaged in oxygen-impermeable film and stored at 25°C. During 7 days of storage, toxin production in steaks inoculated with 102 spores per g was inhibited by more than 3.8% water-phase NaCl for type E and 6.1% for type A. Presence of nitrite substantially reduced the salt level required to prevent toxin production. When steaks had more than 100 ppm NaNO2, only 2.5% NaCl inhibited type E toxin production; 150 ppm NaNO2 and 3.5% NaCl inhibited production of type A toxin. When storage time was lengthened to 14 days or the spore inoculum increased to 104 spores per g, more salt and nitrite were required for inhibition. Residual nitrite in samples stored under refrigeration (3.3°C) did not change during 22 days of storage. Under abuse temperature (25°C), residual nitrite decreased to less than 6 ppm by the 14th day in all samples tested regardless of the original nitrite concentration.

[Phage conversion of Clostridium novyi type A (author's transl)]. / Zur Phagenkonversion von Clostridium novyi Typ A.

Clostridium novyi type A, the cause of gas gangrene in man and animal, produces four main toxic components (alpha, gamma, delta, and epsilon). The alpha toxin is a necrotizing toxin produced in lethal amounts by strains of type A. This lethal toxin induces a characteristic subcutaneous colorless gelatineous edema when guinea pigs are infected with C. novyi type A. In this paper we report further evidence on the relationship of bacteriophages to the production of alpha toxin by C. novyi type A. The results showed that five phage-sensitive bacterial strains that did not produce alpha toxin could be isolated from alpha toxin producing stains. These nontoxigenic strains could be infected with specific bacteriophages isolated from the different toxigenic strains of C. novyi type A and converted to alpha toxin production. The toxigenicity of these converted strains depended upon the continued participation of specific bacteriophages. These bacteriophages were shown to be very unstable in culture supernatant fluids.