Properties of Cytophaga johnsonae strains causing spoilage of fresh produce at food markets

Two strains of gliding, orange-pigmented bacteria, isolated from fresh bell pepper and watermelon, respectively, showing soft-rot lesions, were identified as Cytophaga johnsonae. They differed from seven type strains of C. johnsonae deposited at the American Type Culture Collection (ATCC) in some properties, such as the ability to utilize glucose, xylose, trehalose, rhamnose, and sucrose. Spherical bodies resembling microcysts of Sporocytophaga sp. in addition to short rods and long filaments were observed in two strains (ATCC 29583 and 29588) throughout the growth cycle and also in aged cultures of other strains. All strains examined were shown to degrade five natural or synthetic polymers (pectin, chitin, starch, protein, and carboxymethyl cellulose). Only six strains (including ATCC 17061, 29587, 29589, and 19366) were able to infect and macerate artificially wounded potato tubers and fruits of pepper, squash, and tomato. The pathogenic strains secreted more pectate lyase in broth medium than the nonpathogenic strains. C. johnsonae, generally known as a soil saprophyte, might occasionally act as an opportunistic pathogen, causing decay of fresh produce in storage or in transit.     

Temperate phages and bacteriocins of the gliding bacterium Cytophaga johnsonae

A collection of 30 independently isolated strains of Cytophaga johnsonae was screened for the presence of temperate bacteriophages. Two strains were found to harbour phages. The newly isolated phages differ in several respects from the 43 previously isolated phages for C. johnsonae. Both phages are polyhedral, approximately 60 nm in diameter, and have no apparent tail structure. They are chloroform sensitive, and plaque formation is inhibited by agar. Both are capable of establishing a stable association with host cells. Twenty-nine of the 30 strains produced diffusible substances that specifically inhibited the growth of other C. johnsonae strains or closely related species and that could not be propagated. These substances appear to be bacteriocins, some of which, like bacteriophages, are active only against motile cells, while other inhibit nonmotile as well as motile cells. One of each of these two types of bacteriocins was partially characterized and both were found to be proteinaceous in nature and bactericidal in effect.     

Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae.

Cytophaga johnsonae displays many features that make it an excellent model of bacterial gliding motility. Unfortunately, genetic analyses of C. johnsonae, or any related gliding bacteria, were not possible because of a complete lack of selectable markers, cloning vectors, transposons, and convenient methods of gene transfer. As a first step toward a molecular analysis of gliding motility of C. johnsonae, we developed these genetic techniques and tools. Common broad-host-range plasmids and transposons did not function in C. johnsonae. We identified one Bacteroides transposon, Tn4351, that could be introduced into C. johnsonae on plasmid R751 by conjugation from Escherichia coli. Tn4351 inserted in the C. johnsonae genome and conferred erythromycin resistance. Tn-4351 insertions resulted in auxotrophic mutations and motility mutations. We constructed novel plasmids and cosmids for genetic analyses of C. johnsonae. These cloning vectors are derived from a small cryptic plasmid (pCP1) that we identified in the fish pathogen Cytophaga psychrophila D12. These plasmids contain the ermF (erythromycin resistance) gene from Tn4351 and a variety of features that facilitate propagation and selection in E. coli and conjugative transfer from E. coli to C. johnsonae.     

Analysis of pectate lyases produced by soft rot bacteria associated with spoilage of vegetables

Isoelectric focusing (IEF) profiles of pectate lyases (PLs) produced by five different groups of soft rot bacteria were analyzed by using the combined techniques of thin-layer polyacrylamide gel IEF and agarose-pectate overlay activity staining. Four strains of soft rot Erwinia spp. produced three or more PL isozymes. All of eight Pseudomonas viridiflava strains examined produced one single PL with a pI of 9.7. All 10 of Pseudomonas fluorescens strains produced two PLs; the major one had a pI of 10.0 and the minor one had a pI of 6.7. A single PL with a pI of greater than or equal to 10.0 was detected in one strain each of Xanthomonas campestris and Cytophaga johnsonae. PLs of six representative strains were purified from culture supernatants by ammonium sulfate precipitation and anion-exchange chromatography. All purified PL samples macerated potato slices, but to different degrees. The Mrs of alkaline PLs produced by P. viridiflava, P. fluorescens, X. campestris, and C. johnsonae were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000, 41,000, 41,500, and 35,000, respectively. IEF profiles of PLs were distinct among the bacterial species. Profiles of non-Erwinia spoilage bacteria were considerably simpler than those of Erwinia spp. The PL with an alkaline pI appeared to be the principal or the sole enzymatic factor involved in tissue maceration caused by most strains of soft rot bacteria.     

Analysis of pectate lyases produced by soft rot bacteria associated with spoilage of vegetables.

Isoelectric focusing (IEF) profiles of pectate lyases (PLs) produced by five different groups of soft rot bacteria were analyzed by using the combined techniques of thin-layer polyacrylamide gel IEF and agarose-pectate overlay activity staining. Four strains of soft rot Erwinia spp. produced three or more PL isozymes. All of eight Pseudomonas viridiflava strains examined produced one single PL with a pI of 9.7. All 10 of Pseudomonas fluorescens strains produced two PLs; the major one had a pI of 10.0 and the minor one had a pI of 6.7. A single PL with a pI of greater than or equal to 10.0 was detected in one strain each of Xanthomonas campestris and Cytophaga johnsonae. PLs of six representative strains were purified from culture supernatants by ammonium sulfate precipitation and anion-exchange chromatography. All purified PL samples macerated potato slices, but to different degrees. The Mrs of alkaline PLs produced by P. viridiflava, P. fluorescens, X. campestris, and C. johnsonae were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000, 41,000, 41,500, and 35,000, respectively. IEF profiles of PLs were distinct among the bacterial species. Profiles of non-Erwinia spoilage bacteria were considerably simpler than those of Erwinia spp. The PL with an alkaline pI appeared to be the principal or the sole enzymatic factor involved in tissue maceration caused by most strains of soft rot bacteria.     

Production of phospholipase C (alpha-toxin), haemolysins and lethal toxins by Clostridium perfringens types A to D.

To obtain high yields of extracellular enzymes and toxins for immunological analysis, type culture collection strains of Clostridium perfringens types A to D and 28 fresh isolates of C. perfringens type A from humans were grown in fermenters under controlled conditions in a pre-reduced proteose peptone medium. The type culture collection strains all showed different characteristics with respect to growth rates and pH optima for growth. Production of phospholipase C (alpha-toxin), haemolysin and lethal activity varied considerably between the different types. Growth and extracellular protein production in fermenters with pH control and static or stirred cultures were compared. Production of all extracellular proteins measured was markedly improved by cultivation in fermenters with pH control. Strain ATCC13124 produced five times more phospholipase C than any of 28 freshly isolated strains of C. perfringens type A, grown under identical conditions. Haemolytic and lethal activities of the ATCC strain were equal or superior to the activities of any of the freshly isolated strains. There were no differences in the bacterial yields and in the production of extracellular toxins between type A strains isolated from clinical cases of gas gangrene and abdominal wounds, and those isolated from faecal samples from healthy persons.     

Comparative studies of the quality of fresh and stored mushrooms of Agaricus bisporus with two tropical Agaricus bitorquis strains

Three white strains of mushroom were grown for quality assessment tests, a commercial Agaricus bisporus strain SOMYCEL U3 currently popular in most major mushroom producing countries and two tropical Agaricus bitorquis strains, ATCC 32675 and AGC W20. Mushrooms were harvested as stage 2 mushrooms (closed buttons with universal veil intact) and stored at 18°C (± 0.5°C) for 5 days during which time colour development, the rate of fruitbody maturation and weight loss were assessed. Throughout the storage period, a reflectance colormeter was used to monitor colour changes on the tops and sides of mushroom fruitbodies. The tops of both A. bitorquis strains were significantly more yellow than the A. bisporus strain, whereas the sides were significantly less yellow. Overall, the A. birorquis strain AGC W20 was clearly the least discoloured and least yellow at the time of harvest. Although all the three strains tested gave similar fresh weight losses during storage, i.e. approximately 10% per day, ATCC 32675 exhibited a very slow maturation rate. Both U3 and AGC W20 matured at a similar much faster rate forming open cups within the 5 day storage period. ATCC 32675 also showed the least increase in the degree of discolouration with time, whether readings were taken from the sides or tops of mushrooms. A breeding programme to combine the most salient features of AGC W20 (an intensely white mushroom at harvest, high yielding with distinct flush pattern) and ATCC 32675 (very slow maturation rate during storage) is suggested.     

Neutral Lipids in the Study of Relationships of Members of the Family Micrococcaceae

The organisms studied were those of the family Micrococcaceae which cannot participate in genetic exchange with Micrococcus luteus and those whose biochemical and physiological characteristics appear to bridge the genera Staphylococcus and Micrococcus. The hydrocarbon compositions of M. luteus ATCC 4698 and Micrococcus sp. ATCC 398 were shown to be similar to those previously reported for many M. luteus strains, consisting of isomers of branched monoolefins in the range C25 to C31. However, Micrococcus sp. ATCC 398 differed somewhat by having almost all C29 isomers (approximately 88% of the hydrocarbon composition). Micrococcus spp. ATCC 401 and ATCC 146 and M. roseus strains ATCC 412, ATCC 416, and ATCC 516 contained the same type of hydrocarbon patterns, but the predominant hydrocarbons were within a lower distribution range (C23 to C27), similar to Micrococcus sp. ATCC 533 previously reported. The chromatographic profile and carbon range of the hydrocarbons of an atypical strain designated M. candicans ATCC 8456 differed significantly from the hydrocarbon pattern presented above. The hydrocarbons were identified as branched and normal olefins in the range C16 to C22. Studies of several different strains of staphylococci revealed that these organisms do not contain readily detectable amounts of aliphatic hydrocarbons. The members of the family Micrococcaceae have been divided into two major groups based on the presence or absence of hydrocarbons. With the exception of M. candicans ATCC 8456, this division corresponded to the separation of these organisms according to their deoxyribonucleic acid compositions.     

Polyamine distributions in the Falvobacterium-Cytophaga-Sphingobacterium complex.

Homospermidine was found as the major polyamine in one newly described species of Flavobacterium (F. indologenes), in three species of Sphingobacterium (S. mizutae, S. multivorium, and S. spiritivorum), and in 10 species of Cytophaga (C. aquatilis, C. arvensicola, C. heparina, C. hutchinsonii, C. johnsonae, "C. keratolytica," C. lytica, C. marinoflava, C. uliginosa, and "C. xantha"). These bacteria also all contain putrescine and agmatine as minor components. Flavobacterium indologenes and C. johnsonae contain an unusual diamine, 2-hydroxyputrescine, as a major polyamine. The polyamine distributions of four other species originally included in Flavobacterium (F. acidurans, "F. dormitator," "F. tirrenicum," and Halomonas halmophila), whose taxonomic positions are or were uncertain, were different from the group mentioned above. They either contain spermidine as the major polyamine or lack any polyamine. These results suggest that homospermidine can serve as a chemotaxonomic marker to delineate true members of the Flavobacterium-Cytophaga-Sphingobacterium complex.     

Virulence of Meloidogyne incognita to expression of N gene in pepper

Four pepper genotypes classified as resistant and four pepper genotypes classified as susceptible to several avirulent populations of M. incognita were compared for their reactions against a population of Meloidogyne incognita (Chitwood) Kofoid and White which had been shown to be virulent to resistant bell pepper (Capsicum annuum) in preliminary tests. The virulent population of M. incognita originated from a commercial bell pepper field in California. The resistant pepper genotypes used in all experiments were the Capsicum annuum cultivars Charleston Belle, Carolina Wonder, and Carolina Cayenne, and the C. chinense cultigen PA-426. The susceptible pepper genotypes used in the experiments were the C. annuum cultivars Keystone Resistant Giant, Yolo Wonder B, California Wonder, and the C. chinense cultigen PA-350. Root gall indices (GI) were ≥ 3.0 for all genotypes in both tests except for PA-426 (GI=2.57) in test 1 and 'Carolina Cayenne' (GI=2.83) in test 2. Numbers of eggs per gram fresh root weight ranged from 20,635 to 141,319 and reproductive indices ranged from 1.20 to 27.2 for the pepper genotypes in both tests, indicating that all eight pepper genotypes tested were susceptible to the M. incognita population used in these tests. The M. incognita population used in these studies overcame resistance conferred by the N gene in all resistant genotypes of both C. annuum and C. chinense.     

Characterization of Porphyrobacter sanguineus sp. nov., an aerobic bacteriochlorophyll-containing bacterium capable of degrading biphenyl and dibenzofuran

Three strains of " Agrobacterium sanguineum", an aerobic marine bacterial species described previously, were re-characterized from phylogenetic and taxonomic viewpoints. 16S rDNA sequence comparisons showed that the " A. sanguineum" strains belong to the alpha-4 subgroup of alpha-Proteobacteria, with members of the genera Erythromicrobium and Porphyrobacter as their closest relatives. DNA-DNA hybridization studies indicated that the " A. sanguineum" strains were distinguishable from any previously known species of these genera. Bacteriochlorophyll a, monosaccharide-type glycosphingolipids, 2-OH fatty acids of C14:0, C15:0, C16:0, and C16:1, and ubiquinone-10 were detected in the " A. sanguineum" strains. The G+C of the DNA was 63.8-64.0 mol%. Two of the " A. sanguineum" strains, IAM 12620 (=ATCC 25659) and ATCC 25661, were able to grow with biphenyl and dibenzofuran as sole carbon source in the presence of 0.05% yeast extract. The medium in these cultures turned yellowish-orange at the exponential phase of growth due to the release of soluble chromogenic metabolites. The remaining " A. sanguineum" strain, ATCC 25660, and all test strains of Erythromicrobium and Porphyrobacter neither grew nor produced yellow-orange pigment with biphenyl or dibenzofuran. In PCR experiments, bphA1 gene, coding for the large subunit protein of biphenyl dioxygenase, was detected in " A. sanguineum" IAM 12620 and ATCC 25661. Based on these results, we propose classifying " A. sanguineum" IAM 12620 and ATCC 25661 as a new species of the genus Porphyrobacter with the name Porphyrobacter sanguineus sp. nov.     

Clostridia from Grana Cheese

Forty strains of proteolytic and saccharolytic clostridia isolated from Grana cheese were re-identified by DNA-DNA homology. Thirty culture collection strains were also examined for comparison. Organisms of the species Clostridium tyrobutyricum which present variability in phenotypic characteristics were found to constitute a homogeneous group, genetically unrelated to all the reference or competitor strains used. The proteolytic clostridia from cheese show a high level of homology with the reference strains C.sporogenes ATCC 319 and C. sporogenes ATCC 3584 and negligible homology with C. bifermentans ATCC 19299. Three strains with phenotypic characters very similar to those of the species C. butyricum present a level of DNA homology with C. butyricum ATCC 19398 ranging from 61–71%. In the light of the results obtained some taxonomic conclusions have been drawn.     

Agglutination,Toxigenicity and Sorbitol Fermentation of Clostridium difficile

A total of 79 Clostridium difficile strains from different sources (50 strains from the fecal specimens of healthy adults, 13 from patients receiving antibiotics without gastrointestinal complications, 13 from antibiotic-associated pseudomembranous colitis (PMC) or diarrhea patients, and three strains from ATCC) were investigated for agglutinability, using formol-treated cells as antigen, in relation to toxigenicity. C. difficile strains tested were divided into four serovars, I, II, III, and IV, by the cross-agglutination test. The agglutinin absorption test revealed that strains of serovar I, agglutinable with high titers (5,120–10,240) to antiserum prepared against a highly toxigenic C. difficile strain, ATCC 17859, possessed the serovar-specific antigen. All of the strains of serovar I were highly toxigenic and all 13 strains isolated from the fecal specimens of antibiotic-associated PMC or diarrhea patients belonged to this serovar, whereas 19 (38%) out of 50 strains from healthy adults and four (30.8%) out of 13 strains from patients receiving antibiotics without gastrointestinal complications possessed this antigen. None of the strains of other clostridial species than C. difficile were agglutinated by the three reference antisera used. Further study on the sugar fermentation test disclosed that the sorbitol-fermenting property of C. difficile is very closely related to the toxigenicity and agglutinability.     

Plasmid localization of a type E botulinal neurotoxin gene homologue in toxigenic Clostridium butyricum strains, and absence of this gene in non-toxigenic C. butyricum strains

It has been shown recently that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755) contain a botulinal neurotoxin type E (BoNT/E) gene closely related to that of C. botulinum type E. In this study, we show that this gene is located on a large plasmid in the two toxigenic C. butyricum strains and is absent in 18 non-toxigenic C. butyricum and C. beijerinckii strains. Interestingly, the 230 bp upstream and the 1260 bp downstream of the neurotoxin coding sequence are not present in either the non-toxigenic C. butyricum or C. beijerinckii strains. Our data suggest a BoNT/E gene transfer from C. botulinum E to originally non-toxigenic C. butyricum strains.     

Effect of turbulent-flow pasteurization on survival of Mycobacterium avium subsp. paratuberculosis added to raw milk

A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity of Mycobacterium avium subsp. paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72 degrees C in duplicate trials. No strains survived at 72 degrees C for 15 s; and only one strain survived at 69 degrees C. Means of pooled D values (decimal reduction times) at 63 and 66 degrees C were 15.0 +/- 2.8 s (95% confidence interval) and 5.9 +/- 0.7 s (95% confidence interval), respectively. The mean extrapolated D72 degrees C was <2.03 s. This was equivalent to a >7 log10 kill at 72 degrees C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6 degrees C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72 degrees C for 15 s, the minimum M. avium subsp. paratuberculosis kill was >4 log10. Properly maintained and operated equipment should ensure the absence of viable M. avium subsp. paratuberculosis in retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2 degrees above 72 degrees C.     

Serological properties and immunobiological activities of lipopolysaccharides from black-pigmented and related oral Bacteroides species

Lipopolysaccharides (LPS) from five species of oral Bacteroides, B. gingivalis strains 381 and ATCC 33277, B. oralis ATCC 33269, B. loescheii ATCC 15930, B. intermedius ATCC 25611 and B. corporis ATCC 33547, were extracted from whole cells by the phenol/water procedure, and subsequently purified by treatment with nuclease and ultracentrifugation. The LPS were composed of hexoses, glucosamine, fatty acids and phosphorus. Heptose and 2-keto-3-deoxyoctonate were not detected. The LPS preparations from B. gingivalis strains 381 and ATCC 33277 presented very similar SDS-polyacrylamide gel electrophoresis patterns when stained with ammoniacal silver. They produced a fused precipitin band against an antiserum to B. gingivalis 381 LPS in immunodiffusion tests. Antisera raised against the LPS from B. loescheii and B. intermedius reacted with the LPS prepared from all the oral Bacteroides strains except those of B. gingivalis. All the LPS preparations were mitogenic for spleen cells of BALB/c (nu/nu) mice, but not for thymus cells from C3H/HeN mice. The LPS induced marked mitogenic responses and polyclonal B cell activation for spleen cells of not only C3H/HeN (LPS responder) mice, but also C3H/HeJ (LPS nonresponder) mice. The mitogenic responses were not suppressed significantly upon addition of polymyxin B to the reaction mixture. These LPS also enhanced interleukin-1 production by murine peritoneal macrophages and mouse cell line J744. 1 macrophages. Hydrolysis of B. gingivalis ATCC 33277 LPS in 1 m-HCl at 100 degrees C for 1 h yielded lipid and polysaccharide. The lipid portion was largely composed of fatty acids and glucosamine, and was mitogenic for spleen cells from C3H/HeJ as well as C3H/HeN mice, while the polysaccharide portion induced no significant mitogenic responses under similar experimental conditions.     

Biochemical properties of Fusobacterium naviforme and phenotypically similar isolates

Three strains which resemble the type strain of Fusobacterium naviforme (ATCC 25832) by morphological and physiological criteria were isolated from human clinical specimens. All were non-fermentative, produced indole and, in common with other members of the genus Fusobacterium , butyrate was a major end-product of metabolism. Glutamate dehydrogenase and 2-oxoglutarate reductase were present in both taxa, but the enzymes of the test strains migrated to only about half the distance of that of strain ATCC 25832. The latter contained meso -diaminopimelic acid as its peptidoglycan dibasic amino acid whereas the test strains possessed meso -lanthionine. The wide divergence in DNA base composition between strain ATCC 25832 (49 mol% G + C) and the clinical isolates ( ca 30–31 mol% G + C) was reflected in their low DNA-DNA homology ( ca 5–15%). The present study therefore revealed major differences between F. naviforme (ATCC 25832) and the new isolates and indicate that the latter may belong to a hitherto undescribed taxon within the genus Fusobacterium.     

Ornibactin production and transport properties in strains of Burkholderia vietnamiensis and Burkholderia cepacia (formerly Pseudomonas cepacia)

Several strains of Burkholderia vietnamiensis, isolated from the rhizosphere of rice plants, and four strains formerly known as Pseudomonas cepacia including two collection strains and two clinical isolates were compared for siderophore production and iron uptake. The B. vietnamiensis (TVV strains) as well as the B. cepacia strains (ATCC 25416 and ATCC 17759) and the clinical isolates K132 and LMG 6999 were all found to produce ornibactins under iron starvation. The two ATCC strains of B. cepacia additionally produced the previously described siderophores, pyochelin and cepabactin. Analysis of the ratio of isolated ornibactins (C4, C6 and C8) by HPLC revealed nearly identical profiles. Supplementation of the production medium with ornithine (20 mm) resulted in a 2.5-fold increase in ornibactin synthesis. Ornibactin-mediated iron uptake was independent of the length of the acyl side chain and was observed with all strains of B. vietnamiensis and B. cepacia, but was absent with strains of Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas stutzeri, known to produce pyoverdines or desferriferrioxamines as siderophores. These results suggest that ornibactin production is a common feature of all Burkholderia strains and that these strains develop an ornibactin-specific iron transport system which is distinct from the pyoverdine-specific transport in Pseudomonas strains.     

Biofilm formation by Escherichia coli O157:H7 on stainless steel: effect of exopolysaccharide and Curli production on its resistance to chlorine

The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log(10) CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 microg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12 degrees C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.