Unique appendages associated with spores of Bacillus cereus isolates

Electron microscopic observations revealed the presence of a new type of large appendage on the spores of two Bacillus cereus strains isolated from phylloplanes. The appendages were thin and sword-like in shape, having the sizes of 1.5 to 2.8 μm in length and 0.03 to 0.6 μm in width. There were no core or sheath structures in these appendages. The number of appendages on a spore ranged from three to more than twenty, radiating from the swelling on one end of the exosporium. These appendages gave a unique octopus- or jellyfish-like feature to the spores.     

Characterization of Bacillus cereus isolates associated with fatal pneumonias: strains are closely related to Bacillus anthracis and harbor B. anthracis virulence genes

Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are associated with food-borne illnesses, periodontal diseases, and other more serious infections. In one such infection, B. cereus G9241 was identified as the causative agent of a severe pneumonia in a Louisiana welder in 1994. This isolate was found to harbor most of the B. anthracis virulence plasmid pXO1 (13). Here we report the characterization of two clinical and one environmental B. cereus isolate collected during an investigation of two fatal pneumonia cases in Texas metal workers. Molecular subtyping revealed that the two cases were not caused by the same strain. However, one of the three isolates was indistinguishable from B. cereus G9241. PCR analysis demonstrated that both clinical isolates contained B. anthracis pXO1 toxin genes. One clinical isolate and the environmental isolate collected from that victim's worksite contained the cap A, B, and C genes required for capsule biosynthesis in B. anthracis. Both clinical isolates expressed a capsule; however, neither was composed of poly-D-glutamic acid. Although most B. cereus isolates are not opportunistic pathogens and only a limited number cause food-borne illnesses, these results demonstrate that some B. cereus strains can cause severe and even fatal infections in patients who appear to be otherwise healthy.     

Identification of a novel enterotoxigenic activity associated with Bacillus cereus.

A strain of Bacillus cereus isolated from a food poisoning outbreak characterized by vomiting has been shown to be capable of causing vomiting when cultures grown on rice, but not other media, were fed to Rhesus monkeys. In contrast, a strain isolated from a diarrhoeal outbreak produced diarrhoea, but not vomiting, when grown on various media in similar feeding trials. Furthermore, culture filtrates from the diarrhoeal strain caused fluid accumulation in ligated rabbit ileal loops whereas those from the vomiting strain did not. It is proposed that at least two enterotoxins are involved, one responsible for the vomiting and one for the diarrhoeal symptoms.     

A severe necrotic enterotoxin produced by certain food, food poisoning and other clinical isolates of Bacillus cereus.

The ability of certain strains of Bacillus cereus consistently to elaborate a filterable non-dialysable toxin capable of causing severe disruption and necrosis of the intestinal mucosa and submucosa is confirmed. This property is not universal to all B. cereus strains and different degrees of production of this toxin are exhibited by the different strains which produce it. The necrotic effect is produced by whole-cell cultures of the toxin producing strains in broth and in boiled rice. Some characteristics of this necrotic toxin are described and its relationship with the diarrhoeal and other known B. cereus toxins is discussed.     

Roseomonas, a new genus associated with bacteremia and other human infections.

In the 1980s, a pink bacterium different from species of the genus Methylobacterium was implicated in human infection. Using biochemical tests and DNA hybridization, we examined 42 strains of pink-pigmented, gram-negative bacteria that were not members of the genus Methylobacterium. The isolates included 6 strains each of CDC "pink coccoid" groups I, II, III, and IV; 10 isolates from Gilardi's "unnamed taxon"; and 8 blood isolates from ill, debilitated, or immunosuppressed patients. The DNA hybridization studies supported the creation of six genomospecies encompassing the 42 strains. Reactions for esculin hydrolysis, glycerol oxidation, and D-mannose oxidation enabled separation of genomospecies 1 through 4. These tests, as well as motility, nitrate reduction, citrate utilization, and oxidation of L-arabinose, D-galactose, and D-xylose, differentiated genomospecies 5 and 6 from each other and from genomospecies 1 through 4. These organisms were susceptible in vitro to the aminoglycosides, tetracycline, and imipenem and generally susceptible to the quinolones. We propose the new genus, Roseomonas, for these bacteria to include three named species, Roseomonas gilardii sp. nov., Roseomonas cervicalis sp. nov., and Roseomonas fauriae sp. nov., and three unnamed genomospecies.     

Severe clinical conditions associated with Bacillus cereus and the apparent involvement of exotoxins.

Twenty-one cases of infection with Bacillus cereus are summarised. The histories supplied showed that at least 15 of these were associated with severe or potentially severe symptoms including two deaths. Analysis of the production of exotoxins, including haemolysin and phospholipase, by these strains is given, and the relevance of these metabolites to the severity of the condition is discussed. Three incidents of bovine mastitis resulting from B. cereus and involving three deaths are also included. The observations presented here together with those of previous reports which are reviewed indicate that B. cereus may be of clinical importance, not just an opportunist but also as an agent of potentially severe infections in its own right.     

Genetic structure of Aspergillus flavus populations in human and avian isolates

Aspergillus flavus is the second leading cause of allergic, invasive, and colonizing fungal diseases in humans, and also the second most frequent organism associated with avian infections. Currently, it is not known whether there is a link between the environmental isolates and/or human isolates of A. flavus and those responsible for aspergillosis in birds. Microsatellite typing was used to analyze 29 A. flavus clinical and environmental avian isolates and 63 human clinical isolates collected from patients with a variety of aspergillosis diseases. The combination of all six markers yielded 77 different genotypes with a 0.98 D value. A. flavus genotypes obtained from avian isolates were compared with those obtained from human clinical and environmental samples. The standardized indices of association I _A and rBarD were significantly different from zero (p?<?0.01), suggesting a prevailing clonal reproduction. There was high genetic diversity between the hospital and poultry environments of A. flavus isolates. The human environmental population was significantly differentiated from environmental and clinical avian populations (F _st?>?0.25). The avian clinical subpopulation exchanged few strains with the environmental human (N _m?=?7.24) and avian (N _m?=?6.60) populations. The minimum spanning tree analysis identified three A. flavus genotype clusters that were highly structured according to the isolation source (p?<?10^?4).     

Sudden death of a young adult associated with Bacillus cereus food poisoning

A lethal intoxication case, which occurred in Brussels, Belgium, is described. A 20-year-old man died following the ingestion of pasta contaminated with Bacillus cereus. Emetic strains of B. cereus were isolated, and high levels of cereulide (14.8 μg/g) were found in the spaghetti meal.     

Genetic population structure, clonal phylogeny, and pathogenicity of Salmonella paratyphi B.

Genetic diversity and relationships among 123 strains of Salmonella paratyphi B (serotype 1,4,[5],12:b:[1,2]) were estimated from an assessment of electrophoretically demonstrable allelic variation at 24 chromosomal enzyme gene loci. Fourteen electrophoretic types, marking clones, were distinguished, the phylogeny of the clonal lineages was reconstructed, and biotype and other phenotypic characters were mapped onto this structure. Most d-tartrate-negative strains are members of an abundant, globally distributed clone (Pb 1) that is polymorphic for many biotype characters (including d-tartrate utilization), bacteriophage type, rRNA pattern, and colicin M and phage ES18 sensitivity. This clone is largely responsible for S. paratyphi B enteric fever in humans. In contrast, d-tartrate-positive strains (formerly known as S. java) occurred in all seven of the clonal lineages identified by population genetic analysis, although most d-tartrate-positive isolates belong to only two clones (Pb 3 and Pb 4), which vary in frequency geographically. Monophasic strains represent four closely related clones forming a distinctive phylogenetic lineage. The Kauffmann hypothesis of convergence in serotype among distantly related cell lineages through recombination (via phage transduction or other means) may account for the considerable genotypic diversity among clones of S. paratyphi B. Pb 4, Pb 6, and Pb 7 are more closely allied with clones of S. typhimurium and S. saintpaul than with other clones of S. paratyphi B. Sensitivity or resistance to colicin M and phage ES18 and the electrophoretic pattern of the rRNA, which were incorporated into a recently proposed scheme for the identification of types of S. paratyphi B, individually or in combination fail to mark clones or other meaningful phylogenetic subdivisions.     

Characterization of Bacillus thuringiensis isolates and their differential toxicity against Helicoverpa armigera populations

Bacillus thuringiensis isolates were characterized in rhizospheric cotton soils by using acetate selection process from eight different locations in South India. The fact that B. thuringiensis indices were higher in proportion in soil samples taken from Tamil Nadu and Karnataka than from Andhra Pradesh, indicates the abundance of B. thuringiensis populations in the cotton rhizosphere. Biochemical typing of the isolates designated eight local isolates (BtNg13, BtCo1, BtHyb7, BtAm2, BtRm5, BtWr3, BtPl 4, BtN 9), which belong to subspecies kurstaki, the most prevalent subspecies. Toxicity assays on American boll worm larval (F(1) ) populations collected from the Andhra Pradesh, Bangalore and Coimbatore regions, with a susceptible insect strain against different isolates of B. thuringiensis kurstaki spore-crystal mix, revealed distinct susceptibility patterns and specificity. The highest susceptibility was observed in the F(1) populations of Coimbatore, followed by Bangalore and Hyderabad populations, in comparison with the susceptible insect strain. Significant differences were observed (p < 0.0005 and CD = 5.3975) among Btk local isolates, H. armigera biotypes, Btk spore-crystal mix concentration and their interactions, through the Multifactorial ANOVA analysis. The toxicity of local B. thuringiensis isolates was higher than that of HD-1 (reference B.t.k strain). Indigenous Btk isolates have an enormous potential for the management of H. armigera in terms of development of resistance to HD-1. The present study would serve as a baseline data for future resistance monitoring of B. thuringiensis strains in H. armigera in Southern India.     

Genetic diversity of Bacillus thuringiensis serovars revealed by RFLP using random DNA probes.

EcoRI and HindIII restriction fragment length polymorphism (RFLP) profiles using 2 random DNA probes, named 104 and 106, were generated for 85 B. thuringiensis strains. These include 80 serovars, 4 intra-serovar strains: kurstaki HD-1, dendrolimus, tenebrionis and sandiego, and a non-serotypeable strain B. thuringiensis var. wuhanensis. A total of 47 EcoRI and 65 HindIII restriction patterns were generated when hybridization results from both probes were combined. Seventy-seven B. thuringiensis strains showed distinctive hybridization profiles. The dendrogram resulting from the numerical analysis of the distance matrix revealed fourteen distinct phylogenetic groups at the 96% banding patterns similarity. The intra-serovar strains showed higher similarity with their respective type serovars. However, different serovars from a common H-serotype did not always cluster in the same phylogenetic group. Alternatively, several mosquitocidal serovars clustered in a single phylogenetic group. The correlation between serotyping and banding pattern similarity is discussed.     

Association of hepatitis B and human immunodeficiency virus infections in Tanzanian population groups

A study was performed to determine whether there is a correlation between hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infection in population groups in the Dar es Salaam area in Tanzania where HBV infection is endemic. A panel of 460 sera from army recruits, health personnel and pregnant women was tested. In the whole group seromarkers of HBV infection were found in 61.9 % of 134 HIV positive subjects versus 51.5 % of 326 seronegative subjects, a difference which was not statistically significant (p>0.05). In the group of pregnant women, however, 66.7 % of 120 HIV positive subjects had markers of HBV infection versus 49.0 % of the 157 HIV seronegative subjects (p<0.01). This shows that a considerable proportion of young females are also exposed to HBV at the time they acquire HIV infection.     

Synergy between toxins of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus

Synergistic interactions among the multiple endotoxins of Bacillus thuringiensis subsp. israelensis de Barjac play an important role in its high toxicity to mosquito larvae and the absence of insecticide resistance in populations treated with this bacterium. A lack of toxin complexity and synergism are the apparent causes of resistance to Bacillus sphaericus Neide in particular Culex field populations. To identify endotoxin combinations of the two Bacillus species that might improve insecticidal activity and manage mosquito resistance to B. sphaericus, we tested their toxins alone and in combination. Most combinations of B. sphaericus and B. t. subsp. israelensis toxins were synergistic and enhanced toxicity relative to B. sphaericus, particularly against Culex quinquefasciatus Say larvae resistant to B. sphaericus and Aedes aegypti (L.), a species poorly susceptible to B. sphaericus. Toxicity also improved against susceptible Cx. quinquefasciatus. For example, when the CytlAa toxin from B. t. subsp. israelensis was added to Bin and Cry toxins, or when native B. t. subsp. israelensis was combined with B. sphaericus, synergism values as high as 883-fold were observed and combinations were 4-59,000-fold more active than B. sphaericus. These data, and previous studies using cytolytic toxins, validate proposed strategies for improving bacterial larvicides by combining B. sphaericus with B. t. subsp. israelensis or by engineering recombinant bacteria that express endotoxins from both strains. These combinations increase both endotoxin complexity and synergistic interactions and thereby enhance activity and help avoid insecticide resistance.     

Unusual envelopes associated with parasporal inclusions of a mosquitocidal Bacillus thuringiensis serovar fukuokaensis isolate

A mosquitocidal soil isolate of Bacillus thuringiensis serovar fukuokaensis (H3ade) produced spherical parasporal inclusions measuring 0.6-0.7 microm in diameter. Inclusion matrix was homogeneous substance surrounded by a thick, highly electron-dense envelope 30-50 nm in thickness. The envelopes were associated with both intracellular and extracellular inclusions. Densely woven network was the inner structure of the envelope. Often, inclusions had round-shaped, enveloped small protrusions on the surface.     

Pneumonia and empyema infection associated with a Bacillus species that resembles B. alvei.

An organism resembling Bacillus alvei was isolated from the lung and pleural fluid of an immunocompetent patient. The isolate differed from the type strain of B. alvei in its ability to reduce nitrate and its inability to produce dihydroxyacetone and acetylmethylcarbinol. The isolate was resistant to ciprofloxacin and showed intermediate susceptibility to vancomycin.     

MICs of selected antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a range of clinical and environmental sources as determined by the Etest

This paper presents Etest determinations of MICs of selected antimicrobial agents for 76 isolates of Bacillus anthracis chosen for their diverse histories and 67, 12, and 4 cultures, respectively, of its close relatives B. cereus, B. thuringiensis, and B. mycoides derived from a range of clinical and environmental sources. NCCLS breakpoints are now available for B. anthracis and ciprofloxacin, penicillin, and tetracycline; based on these breakpoints, the B. anthracis isolates were all fully susceptible to ciprofloxacin and tetracycline, and all except four cultures, three of which had a known history of penicillin resistance and were thought to originate from the same original parent, were susceptible to penicillin. Based on NCCLS interpretive standards for gram-positive and/or aerobic bacteria, all cultures were susceptible to amoxicillin-clavulanic acid and gentamicin and 99% (one with intermediate sensitivity) of cultures were susceptible to vancomycin. No group trends were apparent among the different categories of B. cereus (isolates from food poisoning incidents and nongastrointestinal infections and food and environmental specimens not associated with illness). Differences between B. anthracis and the other species were as expected for amoxicillin and penicillin, with all B. anthracis cultures, apart from the four referred to above, being susceptible versus high proportions of resistant isolates for the other three species. Four of the B. cereus and one of the B. thuringiensis cultures were resistant to tetracycline and a further six B. cereus and one B. thuringiensis cultures fell into the intermediate category. There was a slightly higher resistance to azithromycin among the B. anthracis strains than for the other species. The proportion of B. anthracis strains fully susceptible to erythromycin was also substantially lower than for the other species, although just a single B. cereus strain was fully resistant. The Etest compared favorably with agar dilution in a subsidiary test set up to test the readings, and it compared with other published studies utilizing a variety of test methods.     

Characterization of multidrug-resistant Escherichia coli isolates associated with nosocomial infections in dogs

Multidrug-resistant opportunistic pathogens have become endemic to the veterinary hospital environment. Escherichia coli isolates resistant to 12 antibiotics were isolated from two dogs that were housed in the intensive care unit at The University of Georgia Veterinary Teaching Hospital within 48 h of each other. Review of 21 retrospective and prospective hospital-acquired E. coli infections revealed that the isolates had similar antibiotic resistance profiles, characterized by resistance to most cephalosporins, beta-lactams, and the beta-lactamase inhibitor clavulanic acid as well as resistance to tetracycline, spectinomycin, sulfonamides, chloramphenicol, and gentamicin. E. coli isolates with similar resistance profiles were also isolated from the environment in the intensive care unit and surgery wards. Multiple E. coli genetic types were endemic to the hospital environment, with the pulsed-field gel electrophoresis fingerprint identified among E. coli isolates from diseased animals and the hospital environment matching. The extended-spectrum cephalosporin resistance in these nosocomial E. coli isolates was attributed to the cephamycinase-encoding gene, bla(CMY2). Chloramphenicol resistance was due in part to the dissemination of the florfenicol resistance gene, flo, among these isolates. Resistance encoded by both genes was self-transmissible. Although bla(CMY2) and flo were common to the polyclonal, nosocomial E. coli isolates, there was considerable diversity in the genetic compositions of class 1 integrons, especially among isolates belonging to the same genetic type. Two or more integrons were generally present in these isolates. The gene cassettes present within each integron ranged in size from 0.6 to 2.4 kb, although a 1.7-kb gene cassette was the most prevalent. The 1.7-kb gene cassette contained spectinomycin resistance gene aadA5 and trimethoprim resistance gene dfrA17.     

Comparison of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates associated with food intoxication with isolates from human nasal carriers and human infections

Staphylococcus aureus represents an organism of striking versatility. While asymptomatic nasal colonization is widespread, it can also cause serious infections, toxinoses and life-threatening illnesses in humans and animals. Staphylococcal food poisoning (SFP), one of the most prevalent causes of foodborne intoxication worldwide, results from oral intake of staphylococcal enterotoxins leading to violent vomiting, diarrhea and cramps shortly upon ingestion. The aim of the present study was to compare isolates associated with SFP to isolates collected from cases of human nasal colonization and clinical infections in order to investigate the role of S. aureus colonizing and infecting humans as a possible source of SFP. Spa typing and DNA microarray profiling were used to characterize a total of 120 isolates, comprising 50 isolates collected from the anterior nares of healthy donors, 50 isolates obtained from cases of clinical infections in humans and 20 isolates related to outbreaks of staphylococcal food poisoning. Several common spa types were found among isolates of all three sources (t015, t018, t056, t084). DNA microarray results showed highly similar virulence gene profiles for isolates from all tested sources. These results suggest contamination of foodstuff with S. aureus colonizing and infecting food handlers to represent a source of SFP.