key: cord-0010123-kw02ygte authors: Drobniewski, F. A. title: The safety of Bacillus species as insect vector control agents date: 2008-03-11 journal: J Appl Bacteriol DOI: 10.1111/j.1365-2672.1994.tb01604.x sha: 6c1fc1369dcea40310d2259af9787ce7e8b1c052 doc_id: 10123 cord_uid: kw02ygte nan One of the success stories of international co-operation in the control of infectious diseases has been the World Health Organization's (WHO) Onchocerciasis Control Programme (OCP) in West Africa; the use of Bacillus thuringienszs (BT) toxins has been an important component of the programme (Rurges 1981; Anon. 1987; Guillet 1990; Webb 1992; Drobniewski 1993a) . Onchocerciasis, or 'river blindness', is a chronic filarial disease caused by the parasitic nematode worm Onchocerca volvulus, and is transmitted by blackflies of the Simulium genus. The WHO estimates that over 90 million people are at risk from acquiring the disease, that there is an overall prevalence of 18 million people and that 1 000 000 cases of blindness have been caused by onchocerciasis (Anon. 1987; Guillet 1990; Webb 1992; Drobniewski 1993) . I t is a significant preventable cause of blindness and two disease control strategies have been pursued by the OCP since its inception in 1974: (1) the identification and treatment of those with onchocerciasis; and ( 2 ) aerial larviciding of rivers to control the vector, Simulium damnosum. The core of vector control has been weekly larviciding with the chemical pesticides temephos, pyraclofos, permethrin, carbosulfan and the bacterial biological control agent, Bacillus thuringiensis var. isruelensas H 14 (BTI). Bacillus thuringiensis (BT) produces crystalline parasporal inclusions during sporulation which are pathogenic to insect larvae, and to dipteran larvae in particular for the var. israelensis strain (Goldberg and Margalit 1977 ; Thomas and Ellar 1983; Ellar et al. 1986; Anon. 1987; Hofte and Whiteley 1989; Ellar 1988a,b, 1989; Guillet et al. 1990; Chilcott et al. 1990; Webb 1992; Aronson 1993) . Ideally, the control strategy would be one of integrated pest management but in practice BTI has been used wherever particular chemical insecticides have become ineffective, due to the development of resistance, or outlawed as a consequence of ecological concerns. Bacillus thuringiensis strains active against lepidopteran crop pests have found widespread use in the developed world, notably in North America, and particularly in ecologically sensitive environments. New strains active against coleopteran and nematode pests have been isolated recently (Krieg et al. 1983; Donovan el al. 1988; Rupar et a/. 1991; Aronson 1993) . It is the dipteran-specific varieties of B. thuringiensis, however, that have captured the imagination internationally because of the benefits obtained through the use of BTI by the OCP and other programmes (Goldberg and Margalit 1987; Anon. 1987; Drobniewski and Ellar 1989; Guillet et a/. 1990; Webb 1992; Drobniewski 1993a) . The OCP has demonstrated the effectiveness of BTI under field conditions, against all the larval stages of Simuliid blackflies and shown it to be environmentally friendly, affecting few non-target species, and exhibiting no crossresistance with the chemical insecticides used by the OCP. It has been a remarkably successful policy with vector transmission interrupted in an area of 600000 km2 and no blindness, reported among the 9 million children born within the boundaries. Approximately 30 million people have been protected from disease and 3.25 million people initially infected by 0. volzdus are now parasite-free (Burges 1981; Anon. 1987; Guillet et al. 1990; Webb 1992; Drobniewski 1993a) . In a world with increasing famine, 15 million hectares of additional cultivatable land have been created. The use of another species producing mosquitocidal toxins, B. sphaericus, may also increase over the next decade (Ilofte and Whiteley 1989; Singer 1990; Davidson and Yousten 1990) but other entomopathogenic species such as B . Ientimorbus, B. popillae and B. larvae have not found widespread use. Within the OCP, between 1981 and 1988, over 2 million litres of BTI were used (Guillet et al. 1990 ) and obviously on this scale an agent must be both effective and completely safe for human use. This paper reviews the experimental evidence for the safety of B. thuringiensis and to a lesser extent, B. sphaericus, the evidence of pathogenicity to man, and established clinical cases. I t will also explore the problems of identifying Bacillus isolates in a clinical context particularly in the developing world where laboratory facilities may be limited and consider how valid species designations are within the genus Bacillus. Bacillus spp. are ubiquitous organisms that successfully occupy a wide variety of ecological niches. Members of the genus are strict aerobes and are genetically heterogenous with the DNA G + C mol % concentration of strains varying from 32 to 69% (Kramer and Gilbert 1989; Turnbull et a/. 1990; Drobniewski 1993b) . Most strains are catalase-positive, motile, with peritrichous flagella, and possess the ability to sporulate in air which differentiates members of the genus from clostridia. The genus is divided into three groups depending on the morphology of the spore and sporangium. Bacillus thuringiensis, B. cereus, B. megaterium, B. anthracis, and B. cereus var. mycoides occupy Group 1, i.e Gram-positive organisms which produce central or terminally-sited ellipsoid or cylindrical spores that do not distend the sporangia. These species are all found in the 'large-cell' subgroup in which the width of the vegetative cells is 2 1 ,urn (Kramer and Gilbert 1989; Turnbull et al. 1990) . Bacillus thuringiensis is therefore morphologically similar to all the medically-important isolates of Bacillus. Group 2 species are Gram-variable and have swollen sporangia with central or terminal ellipsoid spores. The principal members of this group are B. circulans, B. macerans, B . polymyxa, B. popillae, B. larvae, B. lentimorbus, B. alvei, B. stearothermophilus and B. brevis . Bacillus sphaericus (Group 3) is morphologically easier to distinguish from other Bacillus species in that spherical, terminally or sub-terminally sited spores are located within a swollen sporangia. The taxonomic relationship between and within species in Group 1 is not clear. Serological studies have shown significant cross-agglutination between spore antigens of B. cereus, R. anthracis and B. thuringiensis (Lamanna and Eisler 1960; Lamanna and Jones 1961) and the flagellar antigens of B . cereus and B. thuringiensis (Turnbull et al. 1990 ). Enzyme electrophoretic patterns and numerical phenetic analysis have also emphasized the close relationship between these species (Baptist et al. 1978; Priest 1988) . Studies of DNA-DNA hybridization, despite some inconsistency in the overall relatedness of strains and technical difficulties, suggest considerable chromosomal similarity between B. anthracis and some non-anthrax bacilli (Somerville and Jones 1972; Kaneko et al. 1978; Seki et al. 1978) . The development of nucleotide probes has been of value in, for example, veterinary services demonstrating that isolates which were loosely designated 'anthrax-like' were in fact B. anthracis which had lost plasmids (Turnbull 1992 sphaericus is an extremely heterogenous species, and it may in fact be a group of species (Krych et a/. 1980) . In the diagnostic laboratory differentiating between Bacillus species can be difficult although discrimination based on a combination of morphological characters, and the API 20E and API 50 CHB test strips (bio Merieux) can be quite successful (Logan and Berkeley 1984; Bryant et al. 1985; Logan et a/. 1985) . Novel techniques such as pyrolysis mass spectrometry which are available in reference laboratories have been able to discriminate some closely related species; for example, 53 strains of B. subtilis, B. pumilts, B. lichentformis and ' B. amyloliyuefaciens' could be distinguished although the spectra were influenced by media composition, culture maturity and whether the cultures had sporulated (Shute et al. 1984 (Shute et al. , 1988 . Within the developing world (and often, also, in the developed world), the few existing microbiology laboratories cannot justify the expense of investigating an isolate that might be a contaminant. Although a large number of phenotypic tests are used to distinguish between species, in practice sometimes only a single feature is used to separate them, such as the crystalline parasporal inclusion of B. thuringiensis. Not all strains produce crystals, however, and crystalliferous mutants occur readily, as a result of the loss of toxin-encoding plasmids (Gonzalez et al. 1981) , producing organisms that are practically indistinguishable from B . cereus. Indeed, if B . cereus is co-cultured with B . thuringiensis it can be converted to crystal toxin production through plasmid transfer (Gonzalez et al. 1982) . Most BT toxin genes are plasmid encoded as, for example, are the genes for anthrax toxin and other Bacallus virulence determinants (Kramer and Gilbert 1989; Turnbull et al. 1990 ). Extrachromosomal DNA comprises 1&20% of the total potential coding capacity of BT (Aronson 1993) and little is known of its function. Plasmid exchange occurs readily between Bacillus species and this may alter the specificity of non-pathogenic BT strains increasing their potential to cause disease in man. Non-toxic polypeptide factors may also significantly influence toxicity and specificity, for example, BTI encodes a 20 kDa polypeptide which stabilizes the 25 kDa cytolytic toxin (Visick and Whiteley 1991) . Combining these features may create more useful larvicidal agents or synergistic new toxin combinations either naturally or through recombinant DNA techniques; however, the safety of these new combinations cannot be guaranteed. The crystalline toxins exist as protoxins which become solubilized in the alkaline gut contents of dipteran, lepidopteran and coleopteran insect larvae and undergo proteolytic cleavage to form active toxins. The parasporal inclusions of B. sphacricus similarly require solubilization and proteolyic activation. Bacillus thuringiensis toxins interact with mid-gut membranes causing physiological changes which lead to paralysis, cessation of feeding and eventually death (Thomas and Ellar 1983; Ellar et al. 1986; Knowles and Ellar 1986; Anon. 1987; Gill et al. 1987; Wolfersberger et al. 1987; Hofmann et al. 1988; Hofte and Whiteley 1989; Ellar 1988a,b, 1989; Chilcott et al. 1990; Van Rie et al. 1990; Wolfersberger 1990 ). The disruption of the mid-gut epithelium allows mixing of alkaline gut contents with haemolymph, reducing the gut pH and permitting bacterial spore germination in the nutrient-rich mixture. The majority of these toxins have a molecular weight of 65 to 135 kDa and over 40 toxins have been cloned, expressed in other bacteria and plants, and sequenced (recently reviewed by Aronson (1993) and Hofte and Whiteley (1989) ). From sequence and toxicity data a classification scheme was formulated (Hofte and Whiteley 1989) in which there were 13 'CRY' or crystal groups which were homologous but which exhibited toxicity against different target larvae. Zn vitro these activated toxins were cytolytic to cells from the target and related larval species. A fourteenth 'CYT' group was created for a 27 kDa toxin from the var. isruelensis crystal, which by contrast was mosquitocidal, haemolytic and cytolytic to many mammalian and insect cell lines, but which did not share sequence homol-ogy with CRY toxins (Ellar et al. 1985; Hofte and Whiteley 1989) . I t has become apparent that the CYT designation can be applied to a broader group that includes at least five hydrophobic proteins which share the above properties : the 27 kDa protoxins (processed to an active toxin of 25 kDa) of BTI and var. morrisoni PG14 (Ward et al. 1986; Earp and Ellar 1987; Chilcott and Ellar 1988) ; the 28 kDa protoxin of var. darmstadiensis 73-E10-2 which is processed to a 23 kDa toxin (Drobniewski and Ellar 1989) ; the 25 kDa protoxin of var. kyushuensis which forms an active toxin of 23 kDa (Knowles et al. 1992) ; and probably, the 27 kDa toxin of var. jukuokaensis (Yu r't al. 1991) . The var. israelensis and var. morrisoni 27 kDa toxins have been cloned and sequenced and differ by only one nucleotide (Earp and Ellar 1987; Galjart et al. 1987 ). However, a change in only a few key amino acids has a significant effect on the insect specificity of the toxin (Haider and . The CYT toxins of var. darmstadiensis and var. kyushuensis are immunologically related, and in common with the var. isruelensis 27 kDa toxin can be neutralized by, and release entrapped markers from, phospholipid containing liposomes (Gill et al. 1987; Ellar 1988a, 1989; Yu et al. 1991; Knowles et al. 1992) . The mode of action at the molecular level has been facilitated by the purification, or cloning, of individual cytolytic toxins, in place of solubilized crystal mixtures. Activated crystal toxin extracts (Ellar et d. 1986; Knowles and Ellar 1987; Drobniewski and Ellar 1988a) , and more significantly purified toxins (Chilcott and Ellar 1988; Ellar 1988a,b, 1989) bind to insect cell membranes forming transmembrane oligomeric pores of 1 nm radius initially, leading to an influx of electrolytes and water, and colloid-osmotic lysis. Planar lipid bilayer experiments indicate that the toxins form cation-selective channels (Knowles 1992) . The CRY toxins are believed to act in the same way (although this has been fiercely debated) displaying saturable binding to specific membrane receptors, which have been identified on culture cells in uitro and on mid-gut epithelium in vivo Wolfersberger el al. 1987; Hofmann et al. 1988; Wolfersberger 1990; Van Rie et al. 1990; Garczynski et al. 1991) . The crystal structure of a coleopteran specific toxin at 2.5 A resolution has been described (Li et al. 1991) reinforcing the view that the toxins have distinct functional domains, and that conserved hydrophobic regions, and conformational changes in amphipathic helices, are important for toxin insertion and pore formation in membranes (Ellar et al. 1985 Aronson 1993) . Vegetative cells of B. thuringiensis produce several exotoxins and enzymes which may be important in the septicaemia produced in target larvae after the bacterial spores have germinated. They are usually absent from commercial preparations and include : thuringolysin, which like the homologous toxin cereolysin, produced by B. cereus, is a thiol-activated cytolysin; a 'louse factor' which is toxic to lice, an cc-toxin which is toxic to mice as well as lepidopteran larvae; and a heat-stable adenine nucleotide, the pexotoxin which can substitute for ATP in many cellular reactions and has a broad activity spectrum including invertebrates and vertebrates (Holmes and Monro 1965 ; Krieg 1971 ; Forsberg 1976) . Bacillus cereus is a cause of significant wound and ocular infections and produces haemolysins and phospholipases that are probably important virulence determinants. The thiol-activated cytolysins of both B . thuringiensis var. kurstaki and B. cereus have been purified and shown to be biologically, physiochemically and immunologically identical (Honda et al. 1991) . If environmental contamination of wounds by BT increased, thuringolysin and other toxins might be of significance in the establishment of infection. In man, listeriolysin 0, the thiol-activated cytolysin of Listeria monocytogenes, has a role in resisting phagocytic destruction of the organism (Bielecki et al. 1990 ). I t is interesting to speculate whether thuringolysin has a similar role in the establishment of B T septicaemia within insect larvae following disruption of the mid-gut and spore germination. It is conceivable that cereolysin and thuringolysin might play a similar role in any human infections. Several varieties of B . thuringiensis and B . sphaericus have been extensively tested in mammals, and also in human volunteers, and shown to be safe (Burges 1981) . In the 1950s rats fed 2 x 10" B T spores per kg and human volunteers fed 3 x lo9 spores per d for 5 d showed no ill effects (Fisher and Rosner 1959) . This makes biological sense as toxins given per os, or formed by vegetative cells derived from ingested spores, would be degraded in the stomach as they are not acid stable. Only those with a specific absence or reduction of gastric acidity following gastric surgery, antacid or ulcer-healing medication could face any risk. Rats did succumb to B. thuringiensis var. kurstaki and var. isruelensis but only after lo7 to 10' viable organisms were injected intracerebrally (Warren et al. 1984; Siegel and Shadduck 1990a,b) . There was no direct evidence for infection as no replication occurred (Siegel and Shadduck 1990a,b) and the mortality could be explained by the elaboration of bacterial waste metabolites in sensitive areas of the brain. Local abscesses have been noted on subcutaneous injection of BTI but these may have been related simply to persistence of foreign antigen as autoclaved material produced the same effect. These experiments may be relevant in that B. cereus, which is a human pathogen, can produce cerebral and wound abscesses (Turnbull et al. 1990; Drob-niewski 1993b) . Morbidity and mortality were virtually absent from aerosol, oral or intraperitoneal administration of different BTI formulations. BTI spores remained viable in mammalian tissue for lengthy periods of time, however, and their isolation is likely to increase wherever these agents are used (Allen and Wilkinson 1969; Siegel and Shadduck 1990a) . When the BTI b-endotoxin crystal is solubilized in alkaline buffer and administered intravenously or subcutaneously to mice, however, it is lethal at a dose of 5-30 pg g-' body weight (Thomas and Ellar 1983) . The extract is also haemolytic, as are similar extracts of other crystals such as var. darrnstadiensis 73-E10-2 ( Thomas and Ellar 1983; Chilcott and Ellar 1988; Ellar 1988a,b, 1989; Padua et al. 1990 ). I t must be stressed that these experimental conditions mimic the alkaline milieu of insect guts rather than those of the human intestine. Experimental evaluation of B. sphaericus has also shown that the organism is generally a safe one although some formulations can persist after ocular instillation and may act as irritants (Siegel and Shadduck 1990a) . No acute or chronic toxicity was noted when different preparations containing up to 10' viable organisms were administered to mice, rats or guinea pigs via intravenous, intraperitoneal, intracerebral, oral, percutaneous or inhaled routes (De Barjac et al. 1979 , 1987 Siegel and Shadduck 1990a) . Two B. sphaericus isolates did produce lesions when injected intracerebrally or intraocularly, but no bacterial multiplication was observed and these lesions were probably due to the accumulation of metabolites and/or toxins at the inoculum site. Nevertheless, B. sphaericus is a proven human pathogen and some caution must be exercised before its wide-scale use. Whilst the pathogenic potential of B. anthracis is well known, increasingly non-anthrax members of the genus have been recognized as pathogens with isolated reports of severe infections documented to the beginning of this century and probably earlier (Lacorte 1932; Farrar 1963; Pearson 1970; Isaacson et al. 1976; Gordon 1977; Tuazon et al. 1979; Samples and Buettner 1983; Banerjee et al. 1988; Green et al. 1990; Drobniewski 1993b) . Bacillus species are common laboratory contaminants of blood cultures with estimates varying from 0.1 to 0.9% of submitted cultures (Pearson 1970) , and are frequently present in mixed culture, as when isolated from wound specimens for example, making interpretation of their clinical significance difficult. The retrieval of B. thuringiensis var. isruelensis and Acinetobacter calcoaceticus var. anitratus from an infection after an accidental inoculation injury illustrates this point; proteases from the latter organism could have produced toxin activation under what might be very rare conditions (Warren et al. 1984) . Severe systemic illness caused by the closely-related B. cereus is primarily associated with intravenous drug abusers, the immunosuppressed, those with underlying malignancies and neonates. Combining any of these factors potentially increases this risk. Immunosuppression is not uncommon in West Africa because of the high prevalence of chronic parasitological diseases such as malaria, and the prevalence of immunosuppressive diseases, particularly measles, in early infancy. One can speculate that this might predispose individuals to opportunistic B T I infections that would not occur in the immunocompetent. There have been few population-based studies to evaluate post-exposure contamination and infection. One recent prospective study examined routine clinical isolates from individuals living in an area of Oregon aerially sprayed for two successive growing seasons with B. thuringiensis var. kurstakz. T h e population within the area was 80 000 and 55 var. kurstakz isolates were found from a variety of body sites. O f these, 52 were judged to be contaminants but it was argued that in three cases the organism could have been the pathogen responsible for infection (Green et al. 1990) Clinical infections caused by non-anthrax species, such as B. cereus, B. czrculans, B. coagulans, B. subtilzs and B. lzchenzformzs include local infections of burns, wounds and the eye, bacteraemia and septicaemia, meningitis, cerebral abscesses and ventricular shunt-associated infections, pulmonary infection, endocarditis and pericarditis, and toxigenic food-poisoning (Lacorte 1932; Farrar 1963; Pearson 1970; Isaacson et al. 1976; Gordon 1977; Tuazon et al. 1979; Samples and Buettner 1983; Banerjee et al. 1988; Green et al. 1990; Drobniewski 1993b) . Bacillus cereus, for example, is a significant cause of severe ophthalmic lesions including corneal ring abscesses, keratitis, endophthalmitis, and panophthalmitis. Infections are very aggressive and even with prompt antimicrobial therapy, enucleation of the eye and blindness are the usual sequelae (Drobniewski 1993b ). One of the two clinical cases attributed to BT infection involved an isolation from a corneal ulcer but vegetative bacilli were not seen in the material initially taken from the ulcer and subsequent growth on culture medium might have been due to laboratory contamination (Samples and Buettner 1983) . In experiments involving topical ocular instillation in rabbit conjunctivae of B T I and B. sphaericus, organisms continued to be recovered 1 week and 8 weeks later respectively (Siegel and Shadduck 1990a) although there was no evidence for infection. However, some commercial powders can cause minor local irritation and conjunctival discharge which should not be confused with infection (Siegel and Shadduck 1990a) . Bacillus cereus is an acknowledged ocular pathogen and its similarity to B. thuringiensis should necessitate vigilance in monitoring ocular material for the presence of BTI. Bacillus cereus also causes diarrhoea1 and emetic food poisoning syndromes ; BT has not produced any human gastrointestinal illness although fluid production has been demonstrated in ligated rabbit ileal loops as occurs with the enterotoxin of B. cereus (Kramer and Gilbert 1989) . Proven cases of entomopathogens causing clinical disease in mammals including man are extremely rare. Table 1 lists the principal cases of severe illness associated with B. thurinpensis and B. sphaericus infection. Both B. thuringiensis and B. sphaericus are entomopathogens which can cause disease in man. T h e number of reported cases is tiny, although this is probably an underestimate due to inadequate diagnostic laboratory facilities, failure to speciate Bacillus isolates, the mixed microbiological nature of some clinical specimens, and the rejection of clinically significate isolates as contaminants. T h e widespread use of B. rhuringiensis var. isruelensis and the ubiquitous nature of Bacillus species suggest that the risk to public health remains extremely small, particularly in comparison to the benefit accrued to a community. It would nevertheless be prudent for clinical isolates, particularly from ocular lesions in which Bacillus species have a distinct pathogenic role, and which might be missed against the background of for example, onchocerciasis-generated blindness, to be carefully evaluated for the presence of var. isruelensis wherever this agent has been widely used. At present there is no good evidence to discontinue the use of B. thuringiensis in the developed and developing world on grounds of risk to human health. T h e introduction of new varieties and toxin mixtures, such as those derived from recombinant techniques, should not be assumed safe on the basis of previous work and should be carefully evaluated. T h e pathogenic role of B. sphaericus may be a greater hinderance to its more widespread use at the present time. Prospective or retrospective studies would be needed to confirm the safety of any new agent, or combination of agents, so that an adequate assessment of the risk-to-benefit ratio can be obtained. New formulations also contain emulsifiers and dispersal agents which should be assessed for human toxicity as well. The use of any novel entomopathogenic Bacillus toxin needs to weigh the likely environmental and commercial benefit against any potential health risk from the toxin, and where appropriate, to consider the considerable indirect medical advantage accrued through elimination of disease carrying vector organisms. 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