MOLECULAR PATHOGENESIS OF BACILLUS SPP., WITH EMPHASIS ON THE DAIRY INDUSTRY ALYSSA A. GRUTSCH, PIERRE S. NIMMER, RACHEL H. PITTSLEY, KATHERINE G. KORNILOW, & JOHN L. MCKILLIP DEPARTMENT OF BIOLOGY & BIOTECHNOLOGY CERTIFICATE PROGRAM, BALL STATE UNIVERSITY, MUNCIE, IN Copyright 2018, Fine Focus all rights reserved MANUSCRIPT RECEIVED 07 JUNE 2018; ACCEPTED 10 NOVEMBER 2018 203 The bacterial species Bacillus cereus accounts for 1.4-12% of foodborne illness outbreaks worldwide, a statistic that is certainly an underestimate. This bacterial genus is capable of contaminating a wide range of food products, including rice, chicken, vegetables, spices, and dairy products. B. cereus endospores are partially resistant to pasteurization, dehydration, gamma radiation, and other physical stresses used in food processing, and their adhesive characteristics promote biofilm-forming capability on a variety of substrates in dairy operations. B. cereus and other closely-related species produce several types of exotoxins, including at least four hemolysins, three phospholipases, a heat/acid stable emetic toxin called cereulide, and three well-studied heat-labile enterotoxins that all cause gastroenteritis following ingestion. While a great deal of information on virulence gene presence and expression is known in B. cereus, very little has been done to explore the virulence potential of thermoduric spore-formers that may be found in ultrahigh temperature (UHT) pasteurized milk, and their ability to produce biofilms. Biofilm production is understood to be under similar regulation as toxins and other extracellular virulence determinants. This chapter describes the current status of knowledge with Bacillus spp. relevant to the dairy industry, virulence potential, and biofilm production from the perspective of food safety. ABSTRACT CORRESPONDING Katherine G. Kornilow kgkornilow@bsu.edu KEYWORDS • Bacillus spp. • enterotoxins • food pathogen Bacillus spp. bacteria show a wide range of characteristics that allow them to live in most natural environments (Griffiths, 2010). Bacillus comprise a large group of ubiquitous Gram-positive, rod- shaped, aerobic-to-facultatively anaerobic endospore-forming saprophytes (Weber & Rutala, 1988). Although the majority of Bacillus spp. are nonpathogenic, a few (Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis) opportunistically infect animal hosts (mammals and insects) (Vilian et al., 2006). Bacillus microscopic morphology may be individual or as long chains in primary isolates from soil or water samples (Weber & Rutala, 1988). The size BACILLUS – GENERAL INFORMATION 204 • FINE FOCUS, VOL. 4 (2) BACILLUS SPP. VIRULENCE • 205 of an individual rod can range from 0.5 x 1.2 um to 2.5 x 10 um. Spores produced by Bacillus spp. are resistant to heat (including to some extent, pasteurization conditions), cold, ionizing radiation, dehydration, and many disinfectants (Griffiths, 2010). The endospores are oval or cylinder shaped and are found centrally, sub-terminally or terminally. Over 30 species of Bacillus spp. are recognized, and are divided into two groups based mostly on the 16S rRNA/DNA sequences: the Bacillus subtilis group and the Bacillus cereus group. Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus are mesophilic, have ellipse shaped spores, and are the most common members of the B. subtilis group. B cereus, B. anthracis, B. thuringiensis, Bacillus weihenstephanensis, and Bacillus mycoides do not ferment mannitol, produce lecithinase, and comprise the B. cereus group. The colony morphology of Bacillus spp. is diverse across species. Bacillus spp. grow on nutrient agar or peptone media and exhibit ideal growth at a pH 7; however some Bacillus spp. grow at a pH of 9, while other species can endure pH 2. Bacillus spp. grow best within a temperature range of 30°-45°C, but thermophilic variants grow optimally at 65°C. All Bacillus spp. metabolize organic substrates such as amino acids, organic acids and sugars by aerobic respiration, anaerobic respiration, or fermentation, depending on species and environment. The enzymatic processes and metabolic characteristics are typically the criteria for Bacillus species differentiation. Bacillaceae family members demonstrate a wide range of characteristics, including the ability to produce a battery of enzymes, antibiotics, and other secondary metabolites (Schallmey et al., 2004). For example, Bacillus spp. have unique abilities to synthesize and/or secrete many substances which are beneficial and show great success in agriculture and industry. Many Bacillus species exhibit antibacterial and antifungal activity against phytopathogens through secretory products (Yu et al., 2002), a logical evolutionary strategy since Bacillus spp. are soilborne or are found in epiphytes (plant that grows non-parasitically on another plant) and/or endophytes (living within a plant host)(Fravel, 2005). Many antimicrobial compounds are well recognized in the biotechnology and biopharmaceutical industries for their surfactant properties are derived from B. subtilis (Jacques, 2007). A surfactant lowers surface tension between two liquids or a solid and a liquid (Singhal, 2007). Surfactants are used for foam creation and stabilization in food processing, household products (paint, detergent, fabric softener), solubilization of agrochemicals, oil recovery, crude oil drilling lubricants, and bioremediation of water insoluble pollutants. Phenotypically, the genus is difficult to delineate into species, but using genotypic methods, determination of relatedness has been revisited in recent years (Sneath, 1986). The mole % G + C content of the DNA is a well-regarded metric by which organisms may be compared genetically. The Bacillus genus is diverse and has a G + C content from 33-69% (Winn et al., 2006). Sequencing of16S rRNA genes and DNA- DNA hybridization methods have been used to assign species names (Goto et al., 2000). 16S rDNA has a hypervariant region (HV region) on the 5’ end. This HV region is highly specific to each Bacillus spp. and is a good genotyping target. Overall, much emphasis has been placed in recent years on defining criteria for species determination within the genus Bacillus, although no single accepted system or approach has been established yet. BACILLUS IN CLINICAL SETTINGS Through biofilm production, B. cereus has been implicated in contaminating intravenous catheters (Hernaiz et al., 2003) resulting in B. cereus-mediated sepsis (Kuroki et al., 2009; Ozkocaman et al., 2006). The formation of biofilms also allows the release of planktonic bacteria that produce additional biofilms increasing the severity of the infection (Costerton et al., 1999). In addition to catheter contamination, B. cereus and its endospores have been shown to contaminate air filtration and ventilation equipment (Bryce et al., 1993), fiber optic bronchoscopy equipment (Goldstein & Abrutyn, 1985; Richardson et al., 1986), linens (Barrie et al., 1994), gloves (York, 1990), specimen collection tubes and balloons used in manual ventilation (VanDerZwet et al., 2000), alcohol-based hand wash solutions (Hsueh et al., 1999), plaster- impregnated gauze (Rutala et al., 1986), and many antiseptics such as chlorhexidine and povodone iodine (Dubuoix et al., 2005). The most common types of infections B. cereus causes, other than foodborne illness, include fulminant bacteremia, central nervous system (CNS) involvement (meningitis and brain abscesses), pneumonia, gas gangrene-like cutaneous infections and endophthalmitis. Other molecular techniques are also used to identify bacterial species including Bacillus spp. The following molecular techniques are used to confirm identify of Bacillus and other bacterial species: (1) polymerase chain reaction (PCR)(Adzitey et al., 2013); (2) pulsed field gel electrophoresis (PFGE); (3) random amplified polymorphism deoxyribonucleic acid (RAPD); and (4) matrix assisted laser desorption/ionization time of flight (MALDI-TOF)(Murray, 2012). PCR is a DNA replication process that amplifies small portions of DNA (amplicons) exponentially with the help of oligonucleotide primers and DNA polymerase. PCR has many different variations, including real time PCR (qPCR). qPCR is a powerful approach wherein template bacterial DNA is amplified and quantified at the same time using a standard curve-based comparison of type strain standards. PFGE is an agarose gel electrophoresis method that separates large pieces of genomic DNA. This separation of DNA is done by applying an electrical current that periodically changes between three different directions, providing a means to accurately resolve small differences in genomic sequences for bacterial community analyses. RAPD is a PCR based method that uses arbitrary primers to randomly amplify segments of target DNA, essentially acting as a DNA fingerprinting system for bacterial species. MALDI-TOF is a simple and rapid technique. Bacterial colonies are removed from the plate, mixed with a UV absorbing matrix (saturated solution of α-cyano-4- hydroxy-cinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid) and dried on a target plate. The target plates are exposed to laser pulses that develop an energy transfer from the matrix to the nonvolatile analyte molecules. The analyte is removed in the form of gas. The molecules are enhanced in a flight tube to the mass spectrometer. MALDI-TOF is accurate, rapid, and after initial purchase, inexpensive. These characteristics perhaps explain why MALDI-TOF is being used in hospitals for quick identification of bacterial infections. 206 • FINE FOCUS, VOL. 4 (2) BACILLUS SPP. VIRULENCE • 207 B. CEREUS-MEDIATED ENDOPHTHALMITIS B. cereus is not only capable of causing food- associated toxicoinfections, but can cause endophthalmitis as well (Davey & Tauber, 1987; Hermandy et al., 1990; Ullman et al., 1987). B. cereus is not the only pathogen capable of causing endophthalmitis, but is considered one of the most aggressive pathogens causing this condition. Because there is a limited immune response when a pathogen enters the eye, a wide spectrum of pathogens can enter and elicit a wide array of effects. Symptoms can range from a relatively painless anterior chamber inflammation (Aaberg et al., 1998), to an explosive ocular and periorbital infection caused by B. cereus (Schemmer & Drebe, 1987). Specific toxin production by a particular microorganism is theorized to account for the difference in symptoms. B. cereus induced endophthalmitis is characterized by a corneal ring abscess followed by increased pain, chemosis, proptosis, retinal hemorrhage, and perivasculitis (Callegan et al., 1999). Fever, leukocytosis, and general malaise often appear as the systemic manifestations of this condition (Martinez et al., 2007). B. cereus induced endophthalmitis can be divided into two categories: exogenous and endogenous. An exogenous source is due to blunt trauma that penetrates the eye, which may occur due to occupation (for example, metal workers), in an agricultural setting (David et al., 1994) or infection resulting from unsterile instruments during cataract surgery. In one example in Rome, an ophthalmologist had four of his cataract patients lose vision in their treated eye one day after their cataract surgery (Simini, 1998). B. cereus is ranked second behind Staphylococcus aureus which is responsible for about 70% of post-cataract surgery endophthalmitis (Han et al., 1996). The three main risk factors surgeons need to be aware of to reduce posttraumatic endophthalmitis are the presence of an intraocular foreign body, delay in closure of the globe, and the location/extent of the laceration of the globe. Endogenous sources represent about 2-8% of all endophthalmitis cases (Romero et al., 1999) and are due to bacteria entering the posterior segment of the eye. The most common pathogen to enter the posterior segment of the eye is Candida albicans but other common pathogens include S. aureus, B. cereus, Escherichia coli, Neisseria meningitides and Klebsiella spp. B. cereus can accomplish this route of entry through blood transfusion, contaminated needles/illicit drug injection paraphernalia (Grossniklaus et al., 1985), or by iatrogenic administration of medications such as B vitamins or insulin (Motoi et al., 1997). Moyer et al (2009) demonstrated that B. cereus is capable of disrupting tight junctions between endothelial cells and the basement membrane of retinal capillaries and retinal pericytes as early as 4h post-infection. Such changes are hypothesized to be responsible for causing the loss of retinal structure and function (Kopel et al., 2008; Moyer et al., 2009). The exact toxins from B. cereus responsible for causing this breakdown of the blood retinal barrier are unknown but are theorized to consist of the following molecules that may be working individually or in concert to achieve this effect: the Hbl enterotoxin, the Nhe enterotoxin, a crude exotoxin (CET) derived from cell-free B. cereus culture filtrates, phosphatidylcholine- preferring phospholipase C (PC-PLC), collagenase, cereolysin O (Shany et al., 1974), or cereolysin AB (Scott et al., 1996). However, only the Hbl enterotoxin protein has been identified for its role in endophthalmitis (Callegan et al., 1999a). 208 • FINE FOCUS, VOL. 4 (2) Hbl enterotoxin has been shown to cause irreversible tissue damage to the photoreceptors of the retina in less than 12- 24h causing blindness in the infected eye (Beecher et al., 1995; Davey & Tauber, 1987). B. cereus is capable of disrupting the blood retinal barrier as early as 4h in retinal tissues, 6h post-infection in aqueous humor, and in all other ocular tissues 12h post- infection (Callegan et al., 1999b). B. cereus has been shown to be a more rapid and virulent endophthalmitis pathogen compared to S. aureus and Enterococcus faecalis. Additionally, B. cereus seems to exhibit an almost immediate inflammatory response despite low numbers of the organism present at the early stages of infection. Limited research exists addressing the exact role the immune system plays in endophthalmitis, but the eye is known to be an immunoprivileged site as was first described by Medawar in 1948 (Cunha-Vaz, 1997). The eye restricts both the adaptive and innate immune systems in such a way to balance the challenge of pathogen infection against inflammation-induced vision loss (Streilien, 2003). In most instances of B. cereus induced endophthalmitis, vision loss occurs regardless of the type of therapeutic or surgical intervention utilized because the severity of the disease has progressed to such a condition, that too many toxins have been released by B. cereus and many bacteria will have migrated in the eye out of the reach of antibiotics (Callegan et al., 2006). Thus within a 12-18h time frame, massive tissue destruction occurs to the retina and surrounding ocular tissues resulting in antibiotics no longer being maximally effective (Callegan et al., 2002). In addition, the inflammatory response inside the eye is so aggressive that even if the antibiotics control B. cereus, the inflammation produced causes damage to surrounding ocular structures thus making it difficult to manage ocular infections. BACILLUS IN FOOD Foodborne illness from a variety of microorganisms affects on average 76 million individuals in the U.S. each year resulting in some 5,000 deaths (Mead et al., 1999). Worldwide statistics on Bacillus cereus foodborne illness are underestimated due to a variety of factors, including emetic symptoms similar to Staphylococcus aureus intoxication and diarrheal symptoms similar to those elicited by Clostridium perfringens type A. Most affected individuals do not seek medical attention due to the short duration of signs and symptoms. B. cereus seems to account for between 1.4-12% of foodborne illness outbreaks worldwide (Stenfors et al., 2008). Bacillus spp. are capable of contaminating a wide range of food products, including rice, chicken, vegetables, spices, and dairy products. Contamination in the dairy industry may occur when B. cereus spores come in contact with the udders of cows (Andersson et al. 1995), if the spores colonize feed or bedding, or if the spores survive pasteurization (Claus & Berkley, 1986; Sneath, 1986). This is a serious problem in the food industry because B. cereus endospores are in many instances partially resistant to the BACILLUS SPP. VIRULENCE • 209 heat of pasteurization, dehydration, gamma radiation, and other physical stresses. This resistance is due to the ultrastructure of the endospore of course, but also in part to the hydrophobic nature of the spores that allows them to adhere strongly to surfaces and develop biofilm-like properties (Mattson et al., 2000; Ronner et al., 1990). For example, an irradiation dose of 1.25-4 kGy needs to be administered to reduce spores by 90% (De Lara et al., 2002). Also, pasteurization may result in the activation and germination of spores (Hanson et al., 2005). In addition, B. cereus endospores germinate in response to particular nutrients such as glycine or in response to physical stress such as temperature (spore germination can occur over 5-50°C in cooked rice) (Granum, 1994) and high pressures (i.e. 500 MPa). Thus foods need to be cooked at least at a temperature of 100°C (212°F) or above to kill most of the endospores (Griffiths & Shraft, 2002). Thermoduric sporeformers have many important reasons to be the subject of great interest within the dairy industry (Burgess et al., 2010). Thermophilic bacilli produce heat-resistant (80-100°C) and highly heat- resistant (>106°C) endospores in UHT treated products, which can lay dormant for years. Heat, chemicals, and pH levels can activate a spore for germination and outgrowth. This is particularly important in the dairy industry because heat is used as a preservation mechanism. B. subtilis has a low activation temperature of 65-70°C. Once the spores are activated, germination is elicited by nutrients that bind to germination receptors. A nutrient mixture of asparagine, glucose, fructose and K+ (AGFK) triggers B. subtilis spore germination (Setlow, 2003). Many people consider B. anthracis, B. thuringensis, and B. cereus to be the same species (Helgason et al., 2000). B. anthracis is found in the soil and infects primarily herbivorous animals, causing human disease (Winn et al., 2006; Kolsto et al., 2009). This disease may be contracted by local infections of skin lesions, through the gastrointestinal (GI) route, or by inhalation. Respiratory and GI-acquired routes are highly lethal forms of anthrax. B. anthracis virulence mechanisms easily allows for the spread of the bacteria to the lymph nodes. Once in the lymph nodes, the bacteria disseminate via the bloodstream and internal organs. B. anthracis spores are highly resistant to adverse environmental conditions and it is difficult to be certain that the organism has been fully eradicated from endemic areas (Winn et al., 2006). The endospores are maintained in soil and stay dormant indefinitely. The virulence determinants produced by B. anthracis are composed of three proteins: a protective antigen (PA), an edema factor (EF), and the lethal factor (LF). Virulent strains are also typically capsule-producers. Toxin expression and production is enhanced by elevated CO2 and growth temperatures of 35-37˚C. B. anthracis strains harbor two large plasmids, pXO1 and pXO2 (Kolsto et al., 2009). These plasmids are needed for full virulence. pXO1 contains the coding for the PA (pag), EF (cya), and LF (lef). pXO2 contains a five-gene operon for the biosynthesis of a polyglutamate capsule. This capsule is important for the ability to escape the host immune system, by protecting the vegetative cells from phagocytosis. B. thuringensis classification has been accomplished by H serotyping, which utilizes bacterial flagellar antigens (Sneath, 1986). This species has unique insecticidal properties demonstrating activity against several insect orders, as well as nematodes, mites and protozoa. B. thuringensis produces protoxins during sporulation (Aronson et al., 1986). These toxins are either parasporal inclusions or found on the spore surface. B. 210 • FINE FOCUS, VOL. 4 (2) thuringensis produces parasporal crystals during sporulation, which are inclusions of insecticidal toxins. The midgut of the larvae have proteases that convert protoxins to toxins, activating the toxin to bind to receptors on columnar midgut cells. This binding event results in pore formation of the midgut epithelium, and susceptible insects die from this extensive damage and pH changes as midgut contents mix with the hemocoel cavity. Three common subspecies variants have been recognized and well characterized over the last 40 years: (1) B. thuringiensis subsp. kurstaki; (2) B. thuringiensis subsp. israelensis; and (3) B. thuringiensis subsp. japanensis. Each produces crystalline endotoxin specific for a unique order of insect for selectively toxic biological control. Interestingly, each is also extremely genetically similar to the type strain pathogen in this family, B. cereus. B. cereus and other Bacillus spp. are a major cause of foodborne illness globally and a major cause of endophthalmitis (Weber & Rutala, 1988; Stenfors et al., 2008; Moyer et al., 2008). B. cereus has an optimum growth temperature of 30°-40°C, although psychotrophic members can grow in temperatures as low as 4°C. B. cereus can grow in a pH of 5.0-8.8 with optimal pH of 6.0-7.0. Food poisoning due to B. cereus is underreported because it is short-term and self-limiting. In 2005, Bacillus spp. were responsible for 1.4% of foodborne illness in Europe. In the Netherlands 12% of foodborne illness was caused by B. cereus between 1993-1998. In 2006, an average of 63,400 (0.4%) people were domestically affected with B. cereus food poisoning (Scallan et al., 2011). Reports of B. cereus induced food poisoning has increased in industrialized countries, however reporting and testing is variable. In the US passive surveillance is usually performed due to low hospitalization of B. cereus food poisoning. Foods frequently contaminated by B. cereus include milk, dairy products, dry foods, rice, egg products and legumes. Two types of food-related illnesses are caused by B. cereus: (1) Type 1: short-incubation “emetic” and (2) Type 2: long incubation “diarrheal.” Type 1 has an incubation time of 2 hours and lasts approximately 9 hours. Type 2 has an incubation time of 9 hours and lasts 24 hours. Type 1 is mostly associated with contaminated rice and type 2 is associated to contaminated meat or vegetables. The main virulence factor for type 1 food poisoning caused by B. cereus is cereulide (Cueppens et al., 2011). Cereulide is a small molecular weight heat stable exotoxin that can withstand treatment at 121°C for 2 hours at a pH of 7.0. This stability means the toxin can withstand frying, roasting, and microwave exposure, eliciting a foodborne emetic intoxication in susceptible individuals. The main causes of type 2 foodborne illness are hemolysin B (Hbl) and non-hemolytic enterotoxin (Nhe), both comprised of three components encoded by separate operons (Fig. 1) – typical AB toxin architecture. Hbl is made of the cytolytic subunits HblC and HblD, and the protein B binding domains. The Hbl operon also has a fourth member, the hblB gene. However, hblB is not transcribed and is likely a pseudogene. Nhe is made of the cytolytic protein NheA, and the protein B binding sections NheB and NheC. In recent research, 7.5% of reported emetic symptoms have been linked to Hbl and Nhe. These toxins are a product of aerobic, spore forming B. cereus. Aerobic spore formers in food are ubiqui- tous. This ubiquity makes it impossible to prevent aerobic spore formers from being present in many fresh foods. Spore counts in raw milk vary throughout the year, but are highest in winter when dairy cows are primarily indoors. Pasteurization is effective Figure 1. Bacillus spp. HBL and NHE operons. The plcR region is the regulator gene as de- scribed in the text [24].” plcR plcR hblC hblD hblA nheB nheCnheA hblB in inactivating vegetative cells in raw milk, but fails to kill many spores. The spores have no competition from vegetative cells, so they proliferate rapidly if the product is mishan- dled or improperly stored. The sporulated Bacillus, upon germination, can adhere to pipelines and equipment, causing biofilm formation. These spores and vegetative cells in equipment and raw milk may be toler- ant to sterilization. Biofilm extrapolymeric substances (EPS) offer a significant survival strategy to established populations of bacte- ria. These counteractive techniques include ultra-high temperature (UHT) processing, previously known to inactivate all living material, however spores are now known to survive UHT-processing. UHT-processing is achieved by treating fluid milk at 135-150°C for 1-8 seconds. The milk flows continuously during this process and is packaged into pre-sterilized contain- ers (aseptic packaging). The UHT process is designed to kill almost all organisms includ- ing spores. The concern is that some spores still survive and there is no competition for these spores, giving them an ideal environ- ment to proliferate. The growing concern for psychrotolerant spore formers is that they show potential to induce foodborne illness and produce spoilage defects caused by enzymatic activity. These concerns are due to a combination of the following reasons: (1) longer refrigeration storage pre-pasteur- ization; (2) higher temperatures used for pasteurization; (3) prolonged shelf life; and (4) pasteurization activates the germination of spores. A combination of these “advantag- es” are beneficial for B. cereus endospores to form from vegetative cells or vegetative cells to form endospores. Production length of milk treatment has been reduced to 6-8 hours to help reduce thermophile growth (Burgess et al., 2010). Once a production cycle is complete, a clean- ing-in-place (CIP) method is performed on the equipment. CIP consists of the following steps: (1) a warm water rinse; (2) a 1.5% caus- tic wash at 75°C for 30 minutes; (3) a water rinse; (4) a 0.5% nitric acid wash at 70°C for 20 minutes; and (5) a second water rinse. These steps have helped with growth within the equipment, but not within the milk itself. Table 1 indicates the time and temperature requirements laid out by the Food and Drug administration for pasteurization regimes, including UHT pasteurization. BACILLUS SPP. VIRULENCE • 211 BACILLUS SPP. BIOFILMS Adherence of microbial biofilms to dairy production surfaces makes sanitization more difficult, and increases cost via labor and chemical usage along with lost production time. FDA involvement and subsequent product recalls can also occur causing fur- ther financial problems for dairies. Araújo et al. (2009) have proposed a basic mechanism for biofilm adhesion based on six general stages. First, the biofilm surface must be primed for adhesion with the existence of food deposits. The biofilm-producing microorganism must then come into contact with the primed surface. Positive and neg- ative biochemical forces including van der Waals forces and other electrostatic forces then allow the biofilm to make a non-per- manent attachment to the surface when microorganism are between 20 and 50 nm away. Irreversible adhesion results within 1.5 nm when extracellular polysaccharide production, ionic bonds, and hydrophobic forces occur. The fourth stage is described by the multiplication of bacterial cells and an increase in secreted polysaccharides and the fifth stage involves strong metabolism in the biofilm. Lastly, microorganisms begin to be released from the biofilm during the sixth stage, shedding bacteria to generate new biofilms elsewhere. Several authors have identified a variety of mesophilic Bacillus subspecies capable of surviving ultra-high temperature pasteur- ization via endospore formation (Araújo et al. 2009; Lindsay et al. 2002; Scheldeman et al. 2006; Sutyak et al. 2008). Using bacterial cultures sampled from dairies, 16s rRNA, and PCR amplification some of the most prevalent and potentially problematic species, in regards to biofilm production, have been characterized. These species include B. cereus, B. amyloliquefaciens, and several others. The level of virulence activity in B. cereus cells is due to a number of different envi- ronmental factors, including temperature, pH, oxygen tension, glucose concentrations, and specific antimicrobial chemical com- pounds (Glatz and Goepfeort, 1976; Suther- land and Limond, 1993). Biofilm production is understood to be under similar regulation as toxins and other extracellular virulence determinants, which suggests that subin- hibitory stress may have great influence on overall potential for Bacillus spp. to become problematic in dairy microbiology settings. 212 • FINE FOCUS, VOL. 4 (2) QUORUM SENSING Quorum sensing is a regulatory system where the bacterium (B. cereus) recognizes an extracellular signal caused by an autoin- ducer (AI) to sense the density of B. cereus in the immediate environment. Quorum sensing is used to govern cell density, and the corresponding regulation of relevant gene expression that would enhance sur- vival during the log-to-stationary phase transition in dense cultures, or in a natural environment such as soil, food, or within a host [28]. Quorum sensing mechanisms control many processes in the bacterial cell, including sporulation, biofilm production, and virulence factor secretion [27]. Quorum sensing involves direct or indirect activa- tion of a related receptor protein by the AI (Graumann, 2012). This activation results in up- or down-regulation of specific genes. All quorum sensing routines are dependent on three principles: (1) the bacterial species produces AIs; (2) AIs are detected by mem- brane or cytoplasm receptors; and (3) AIs produce a positive feedback loop (Rutherford et al., 2012). Gram-positive bacteria use small, post-trans- lationally modified peptides as same species AIs, called Autoinducing Peptides (AIPs) (Graumann, 2012). AIPs are expressed as large, precursor peptides and processed into smaller, cyclic, thiolactone-containing pep- tides that are transported across the mem- brane. This transportation can happen in two ways: (1) two-component signaling (Fig. 2) or (2) AIP-binding transcription factor signal- ing (Fig. 3). In the two-component signal- ing method, once the AIPs are transported outside of the cell they are too hydrophilic to cross the membrane without help. The AIPs remain in the extracellular matrix. The bacteria sense the AIP as it binds to the receptor protein (histidine kinase) located in the neighboring bacterial cell surface. This binding induces phosphorylation of the kinase. The phosphoryl group is then trans- ferred to an aspartate residue of the response regulator. Then this binds to the promoter region of target genes, which activates or represses transcription. Figure 2. Two-component quorum sensing of Gram-positive bacteria. AI synthase is used to process and transport the pro-AIP out of the cell. Once the concentration of AIP outside the cell is high, AIP binds to histidine kinase receptors. This binding activates the kinase activity of the receptor, inducing autophosphorylation. The phosphoryl group binds to the response regulator and activates transcription of the quorum sensing system genes [27]. AIP AIP AIP AIP AIPAIP AIP AI Synthase b Pro-AIP b Cytoplasmic Response Regulator -Histidine-Kinase Receptor Phosphoryl Quorum Sensing Regulation Ins ideOutsid e BACILLUS SPP. VIRULENCE • 213 QUORUM SENSING & BACILLUS SPP. PATHOGENESIS Quorum sensing in B. cereus is dependent on a protein PlcR. PlcR is a pleiotropic regulator of most virulence factors specific to the B. cereus group (Nhe and Hbl) (Rutherford et al., 2012). The activity of PlcR depends on binding to the AIP that is produced from the PapR protein. PapR is a small signaling pep- tide that acts as a quorum sensing effector (Slamti & Lereclus, 2005) (Fig. 3). PapR is 48 amino acids long and is encoded by an open reading frame located downstream from plcR. PapR is secreted from the cell forming a PapR pro-AIP. PapR pro-AIP is processed by neutral protease B (NprB) to form the active AIP. The AIP is transported back into the cell by an oligopeptide permease system (Opp). AIP then binds to the transcription factor PlcR, activating the protein. This PlcR-AIP complex regulates the production of virulence factors and a positive feedback loop for papR. It has been shown that PlcR expression is positively regulated by CodY expression (Frenzel et al., 2012). CodY is a global transcriptional regulator that facilitates advantageous changes in re- sponse to variations of available nutrients in Gram-positive bacteria (Sonenshein, 2005). CodY is a GTP and isoleucine binding pro- tein that also initiates endosporulation. The binding of GTP and isoleucine act as co-re- pressors of the transcription of many genes. Figure 3. AIP binding PlcR (transcription factor) quorum sensing signaling in B. cereus. A high population density outside the cell activates PapR (pro-AIP) and then the PapR (pro- AIP) is secreted outside of the cell. PapR (pro-AIP) is processed by the protease NprB to become a heptapeptide AIP. AIP is transported back into the cell using an Opp. Once AIP is inside the cell, it binds and activates PlcR. This PlcR-AIP complex regulates virulence factors and also produces a positive feedback loop for PapR secretion [27]. 214 • FINE FOCUS, VOL. 4 (2) AIP Ins ideOutsid e b Pclr-AIP Opp Processed by NprB PapR (Pro-AIP) High-Population Density PapR (Pro-AIP) A ctivates Virulence Factors Nhe, Hbl Figure 4. CodY regulates PapR and PlcR expression in B. cereus. Nutrient availability for the cell regulates the expression of CodY [27, 30]. Endosporulation happens when there is a response to bacterial starvation by limited levels of carbon, nitrogen or phosphorous. Endosporulation is a defense mechanism of Gram-positive bacteria, like a turtle hiding in its shell, to protect its genome. Nucleotide synthesis is dependent on carbon, nitro- gen, and phosphorus. This spore keeps the genome dormant until the environment is favorable enough to replicate. CodY is a transcriptional activator of the plcR gene [30] (Fig. 4). In a ΔcodY B. cereus strain, PlcR expression was strong in exponential, late exponential, and stationary phases of growth. In contrast, a wildtype B. cereus strain showed expression of PlcR in only the stationary phase of growth. CodY was first found in B. subtilis to control expression of more than 100 stationary phase genes. Thus, it is generally accepted that (like plcR), codY is widely conserved among Bacillaceae family members. B. amyloliquefaciens belongs to the B. subtilis group (Priest et al., 1987). Members of this group exhibit similar behaviors physiolog- ically, although B. amyloliquefaciens is not a subspecies of B. subtilis due to the difference in β-amylase production. B. amyloliquefa- ciens has been found to share less than 5% homology at the DNA level with B. subtilis. B. subtilis has been shown to express CodY (Serror & Sonenshein, 2002; Ratnayake-Le- camwasam et al., 2001). Phelps and McKillip (2002), using DNA PCR, found that hblC, hblD, hblA, nheA, and nheB genes or gene homologues were present in a different strain of B. amyloliquefaciens obtained from a Louisiana creamery, although expression of these genes was not measured. Thus, the po- tential for this species to harbor and express these or other virulence factors (via global effectors CodY and/or PlcR) is a realistic possibility, despite this species being placed (at least currently) in the B. subtilis group rather than the B. cereus group. Nutrient Availability CodY PapR PlcR Virulence Factors High Population Density BACILLUS SPP. VIRULENCE • 215 The debate over proper identification and understanding of Bacillus virulence has been ongoing for over 50 years (Rasko et al., 2005). Recent public awareness of potential bioterrorism using the anthrax toxin produced by B. anthracis has lead government agencies to fund multiple studies aimed at rapidly differentiating B. anthracis from other closely related Bacillus species such as B. cereus and B. thuringiensis, since B. anthracis produces the anthrax toxin encoded by two plasmid-based operons, pXO1 and pXO2. The anthrax toxin primarily kills herbivore mammals, but can also kill humans. Not to be underestimated, B. cereus can cause severe food poisoning through its production of emetic and diarrheal toxins. While heavily used as an insecticidal agent in crops with its Cry crystalline toxins, B. thuringiensis has also recently been demonstrated to cause food poisoning symptoms in humans similar to B. cereus. Ironically, species like Bacillus coagulans, which has been found to harbor the nheA gene, are readily used as probiotics in human health. Bacillus spp. were originally differentiated into species at a time when biologists did not possess the molecular tools to delve deeper than biochemical tests and phenotypical observations. While this strategy worked well for other genera, 16S rRNA analysis of differences among B. cereus, B. thuringiensis, and B. anthracis have shown these species to have a nucleotide sequence difference of less than 1%. Recent advances in molecular biology have allowed scientists to scrutinize the genetic properties of these three “species”. After exhaustive studies using DNA-DNA hybridization, 16S and 23S rRNA comparative analyses, multilocus sequence typing (MLST), fluorescent SUMMATION & FUTURE WORK amplified fragment length polymorphism analysis, rep-PCR, and small nucleotide polymorphism (SNP) analyses, scientists have been unable to reliably differentiate these three Bacillus species. While many methods have been pursued, most results have suggested that B. cereus, B. thuringiensis, and B. anthracis should be considered the same species due to highly conserved nucleoidal genetic sequences. Due to the easily identifiable symptoms of B. anthracis and B. cereus, there is recent concern among biologists that the “B. anthracis” species may in fact be an oversampled subset of B. cereus. Other scientists speculate that B. anthracis may have only recently evolved to the point to be considered distinct from B. cereus. Unfortunately, recent literature is contradictory when discussing how similar two separate Bacillus genomes need to be in order to be considered the same species. There are claims that B. thuringiensis, B. cereus, and B. anthracis should be considered one species on the basis of genetic evidence. Alternatively, other scientists claim that current taxonomy has not divided Bacillus strains enough, suggesting that more species or subspecies than currently listed in literature exist. No commonly accepted definition that separates these species on genetic evidence has been found. There are three nhe genes that are encoded on the nheABC operon, and have been shown to remain conserved as a cluster during genetic recombination. It can reasonably be assumed that the presence of the most proximal subunit of nhe indicates the presence of the other two genes. In the literature, all genes encoding the Nhe and Hbl enterotoxins have been readily located downstream in both B. cereus and B. 216 • FINE FOCUS, VOL. 4 (2) thuringiensis (Phelps & McKillip, 2002). The presence of the nheABC operon does not necessarily indicate a virulent strain, but has a very high likelihood of expressing these genes in a host environment or in food under permissive conditions. Thus, future work to determine the pathogenicity of nheA positive samples could include the use of a Tecra VIA to detect enterotoxin proteins. Without this step, the virulence of nheA positive samples cannot be definitively determined. A large degree of genetic variation exists in nhe sequences among Bacillus spp., giving rise to false negative results in PCR-based detection assays. Strains negative for nheA in real-time PCR have been found to produce the enterotoxin Nhe as determined using a Tecra VIA kit. The nheABC operon is mobile among Bacillus spp. through horizontal gene transfer (HGT). Indeed, HGT has been observed among Bacillus spp. and can serve as a mechanism explaining the incidence of non-B. cereus samples positive for nheA. While no data has been found to suggest that this gene transfer mechanism uses an integron, the anthrax-like operon pXO16 found in B. thuringiensis is part of a conjugative plasmid. It is reasonable conjecture that other Bacillus species may also harbor conjugative plasmids that aid in HGT. Within Bacillus, most virulence factors are encoded on plasmids (55), which have been demonstrated to readily transfer between differing species. Indeed, a recent study indicated that the virulence genes associated with B. cereus infection undergo frequent rearrangement both within the bacterial nucleoid and between species. Thus, a better method than traditional biochemical tests to detect pathogenic Bacillus strains is to screen for virulence operons present in plasmids or in nucleoidal DNA. Bacillus genomes that have been sequenced display a high level of genetic synteny in their gene order. Two genes that encode for bacterial ribosomes, 16S and 23S rDNA, contain genetic sequences that are less than 1% different when compared between B. cereus, B. thuringiensis, and B. anthracis (12). A dissimilarity of 3% between 16S or 23S rDNA sequences is the minimal “cut off” between two strains to be considered as distinct species. Additionally, the gyrB gene sequence shared among these species is very homologous. Because these genes are shared among different species within the Bacillus genus, they cannot be used to differentiate species. However, 16S and 23S rRNA can be used to differentiate between different strains of B. anthracis. Interestingly, there are a number of mechanisms that facilitate the movement of genes between different members of the Bacillus genus. One such mechanism is through the natural action of bacteriophage. After lysing its host cell, the bacteriophage will insert its genes into Bacillus genomes. While normally either lytic or lysogenic, it is possible for prophage to undergo random mutation, which renders it unable to enter the lysogenic cycle. In this way, genes from one species of bacteria can be transferred to Bacillus spp. As previously mentioned, Bacillus operons may be on conjugative plasmids. Additionally, Bacillus spp. are naturally competent, allowing these microbes to naturally take up random DNA in their vicinity. The virulence genes for Nhe are present in more strains of Bacillus than is currently accepted within the scientific community. This research identified several “species” of Bacillus that were not previously known to harbor the Nhe enterotoxin operon. Given BACILLUS SPP. VIRULENCE • 217 218 • FINE FOCUS, VOL. 4 (2) that a debate is currently underway about the very identity of B. cereus and other strains, it is improper for food safety experts to screen food products only for B. cereus. Phenotypic- based classification techniques have failed to accurately differentiate Bacillus species. Additionally, no molecular-based approach can accurately differentiate Bacillus. The bottom line is the determination of species within Bacillus does not even matter when concerned with food safety. Molecular techniques should instead screen for virulence determinants in microbes instead of identifying said microbes. Since endospore formation enables Bacillus spp. to be ubiquitous in the environment and on food, all foods should be examined in this way. This is the only true way to determine whether food products are safe for human consumption. REFERENCES 1. Aaberg, T.M., Flynn, H.W., Schiffman, J., & Newton, J. 1998. Nosocomial acute-onset postoperative endophthalmitis survey: a 10 year review of incidence and outcomes. Ophthalmology 105:1004-1010. 2. Adminstration, U.S.F.a.D., Grade A “Pasteurized Milk Ordinance”, P.H.S. Department of Health and Human Services, Food and Drug Adminstration, Editor. 2001: Washington, D.C. 3. Adzitey, F., Huda, N., & Ali, G. R. R. 2013. Molecular techniques for detecting and typing of bacteria, advantages and application to foodborne pathogens isolated from ducks. 3 Biotech: p. 97–107. 4. Andersson, A., Ronner, U., & Granum, P.E. 1995. What problems does the food industry have with the spore-forming pathogens Bacillus cereus and Clostridium perfringens? Int. J. Food Microbiol. 28:145- 155. 5. Aronson, A. I., Beckman, W., & Dunn, P. 1986. Bacillus thuringiensis and related insect pathogens. Microbiological Reviews, 50(1), 1–24. 6. Barrie, D., Hoffman, P. N., Wilson, J. A., & Kramer, J. M. 1994. Contamination of hospital linen by Bacillus cereus. Epidemiology and Infection, 113(2), 297–306. 7. Beecher, D. J., Pulido, J. S., Barney, N. P., & Wong, A. C. 1995. Extracellular virulence factors in Bacillus cereus endophthalmitis: methods and implication of involvement of hemolysin BL. Infection and Immunity, 63(2), 632–639. 8. Bryce, E., Smith, J., Tweeddale, M., Andruschak, B., & Maxwell, M. 1993. Dissemination of Bacillus cereus in an Intensive Care Unit. Infection Control and Hospital Epidemiology, 14(8), 459-462. 9. Burgess, S. A., Lindsay, D., & Flint, S. H. 2010. Thermophilic bacilli and their importance in dairy processing. International Journal of Food Microbiology, 144(2), 215-225. 10. Callegan, M. C., Booth, M. C., Jett, B. D., & Gilmore, M. S. 1999. Pathogenesis of gram-positive bacterial endophthalmitis. Infection and Immunity, 67(7), 3348- 3356. 11. Callegan, M. C., Engelbert, M., Parke, D. W., Jett, B. D., & Gilmore, M. S. 2002. Bacterial Endophthalmitis: Epidemiology, Therapeutics, and Bacterium-Host Interactions. Clinical Microbiology Reviews, 15(1), 111–24 12. Callegan, M. C., Jett, B. D., Hancock, L. E., & Gilmore, M. S. 1999. Role of Hemolysin BL in the Pathogenesis of Extraintestinal Bacillus cereus Infection Assessed in an Endophthalmitis Model. Infection and Immunity, 67(7), 3357–3366. 13. Callegan, M. C., Novosad, B. D., Ramirez, R., Ghelardi, E., & Senesi, S. 2006. Role of swarming migration in the pathogenesis of Bacillus endophthalmitis. Investigative Ophthalmology & Visual Science, 47(10), 4461-4467. 14. Ceuppens, S., Rajkovic, A., Heyndrickx, M., Tsilia, V., Van De Wiele, T., Boon, N., & Uyttendaele, M. 2011. Regulation of toxin production by Bacillus cereus and its food safety implications. Critical Reviews in Microbiology, 37(3), 188-213. 15. Chapman, K. W., & Boor, K. J. 2001. Acceptance of 2% ultra-pasteurized milk by consumers, 6 to 11 years old. Journal of Dairy Science, 84(4), 951-954. 16. Claus, D. & Berkley, R.C.W. 1986. Genus Bacillus Cohn 1872, 174. In P.H.A Sneath, N.S. Mair, M.E. Sharpe, & J.G. Holt (Ed.), Bergey’s Manual of Systematic Bacteriology.( pp. 1105-1137) Williams and Wilkins, Baltimore. 17. Costerton, J. W., Stewart, P. S., & Greenberg, E. P. (1999). Bacterial biofilms: a common cause of persistent infections. Science, 284(5418), 1318-1322. 18. Cunha-Vaz, J. G. 1997. The blood-ocular barriers: past, present, and future. Documenta Ophthalmologica, 93(1-2), 149-157. 19. Davey Jr, R. T., & Tauber, W. B. 1987. Posttraumatic endophthalmitis: the emerging role of Bacillus cereus infection. Clinical Infectious Diseases, 9(1), 110-123. 20. David, D. B., Kirkby, G. R., & Noble, B. A. 1994. Bacillus cereus endophthalmitis. The British Journal of ophthalmology, 78(7), 577. 21. De Lara, J., Fernández, P. S., Periago, P. M., & Palop, A. 2002. Irradiation of spores of Bacillus cereus and Bacillus subtilis with electron beams. Innovative Food Science & Emerging Technologies, 3(4), 379-384. 22. Dubouix, A., Bonnet, E., Alvarez, M., Bensafi, H., Archambaud, M., Chaminade, B., ... & Marty, N. 2005. Bacillus cereus infections in traumatology– orthopaedics department: retrospective investigation and improvement of healthcare practices. Journal BACILLUS SPP. VIRULENCE • 219 220 • FINE FOCUS, VOL. 4 (2) ofIinfection, 50(1), 22-30. 23. Priest, F. G., Goodfellow, M., Shute, L. A., & Berkeley, R. C. W. 1987. Bacillus amyloliquefaciens sp. nov., nom. rev. International Journal of Systematic and Evolutionary Microbiology, 37(1), 69-71. 24. Fravel, D. R. 2005. Commercialization and implementation of biocontrol 1. Annu. Rev. Phytopathol., 43, 337-359. 25. Frenzel, E., et al., 2012. CodY orchestrates the expression of virulence determinants in emetic Bacillus cereus by impacting key regulatory circuits. Molecular Microbiology, 85(1), 67-88. 26. Goldstein, B., & Abrutyn, E. 1985. Pseudo-outbreak of Bacillus species: related to fibreoptic bronchoscopy. Journal of Hospital Infection, 6(2), 194-200. 27. Goto, K., Omura, T., Hara, Y., & Sadaie, Y. 2000. Application of the partial 16S rDNA sequence as an index for rapid identification of species in the genus Bacillus. The Journal of General and Applied Microbiology, 46(1), 1-8. 28. Gracias, K. S., & McKillip, J. L. 2011. Triplex PCR- based detection of enterotoxigenic Bacillus cereus ATCC 14579 in nonfat dry milk. Journal of Basic Microbiology, 51(2), 147-152. 29. Granum, P. E. 1994. Bacillus cereus and its toxins. Journal of Applied Microbiology, 76(S23). 30. Graumann, P. (Ed.). 2012. Bacillus: cellular and molecular biology. Horizon Scientific Press. 31. Griffiths, M. W. 2010. Bacillus cereus and Other Bacillus spp. Pathogens and Toxins in Foods,1-19. 32. Griffiths, M. W., & Schraft, H. 2002. Bacillus cereus food poisoning. Foodborne Diseases, 2, 261-270. 33. Grossnikiaus, H., Bruner, W. E., Frank, K. E., & Purnell, E. W. 1985. Bacillus cereus panophthalmitis appearing as acute glaucoma in a drug addict. American Journal of Ophthalmology, 100(2), 334-335. 34. Yu, G. Y., Sinclair, J. B., Hartman, G. L., & Bertagnolli, B. L. 2002. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biology and Biochemistry, 34(7), 955-963. 35. Han, D. P., et al., 1996. Spectrum and susceptibilities of microbiologic isolates in the Endophthalmitis Vitrectomy Study. American Journal of Ophthalmology, 122(1), 1-17. 36. Hanson, M. L., Wendorff, W. L., & Houck, K. B. 2005. Effect of heat treatment of milk on activation of Bacillus spores. Journal of Food Protection, 68(7), 1484- 1486. 37. Helgason, E., et al., 2000. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—One Species on the Basis of Genetic Evidence. Applied and Environmental Microbiology, 66(6), 2627–2630. 38. Hemady, R., Zaltas, M., Paton, B., Foster, C. S., & Baker, A. S. 1990. Bacillus-induced endophthalmitis: new series of 10 cases and review of the literature. The British Journal of Ophthalmology, 74(1), 26–29. 39. Hernaiz, C., Picardo, A., Alos, J. I., & Gomez Garces, J. L. 2003. Nosocomial bacteremia and catheter infection by Bacillus cereus in an immunocompetent patient. Clinical Microbiology and Infection, 9(9), 973-975. 40. Hsueh, P.-R., et al., 1999. Nosocomial Pseudoepidemic Caused by Bacillus cereus Traced to Contaminated Ethyl Alcohol from a Liquor Factory. Journal of Clinical Microbiology, 37(7), 2280–2284. 41. Ongena, M., & Jacques, P. 2008. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology, 16(3), 115-125. 42. Kolstø, A. B., Tourasse, N. J., & Økstad, O. A. 2009. What sets Bacillus anthracis apart from other Bacillus species? Annual Review of Microbiology, 63, 451-476. 43. Kopel, A.C., Carvounis, P.E., & Holz, E.R. 2008. Bacillus cereus endophthalmitis following invitreous bevacizumab injection. Ophthalmic Surg. Lasers Imaging 39:153-154. 44. Kuroki, R., et al., 2009. Nosocomial bacteremia caused by biofilm-forming Bacillus cereus and Bacillus thuringiensis. Internal Medicine, 48(10), 791-796. 45. Martinez, M. F., Haines, T., Waller, M., Tingey, D., & Gomez, W. 2007. Probable occupational endophthalmitis from Bacillus cereus. Archives of Environmental & Occupational Health, 62(3), 157-160. 46. Mattson, M. P., Culmsee, C., Yu, Z., & Camandola, S. 2000. Roles of nuclear factor B in neuronal survival and plasticity. Journal of Neurochemistry, 74(2), 443-456. 47. Mead, P. S., et al., 1999. Food-related illness and death in the United States. Emerging Infectious Diseases, 5(5), 607–625. 48. Motoi, N., et al., 1997. Necrotizing Bacillus cereus infection of the meninges without inflammatory reaction in a patient with acute myelogenous leukemia: a case report. Acta Neuropathologica, 93(3), 301-305. 49. Moyer, A. L., et al., 2008. Bacillus cereus induces permeability of an in vitro blood-retina barrier. Infection and Immunity, 76(4), 1358-1367. 50. Moyer, A. L., Ramadan, R. T., Novosad, B. D., Astley, R., & Callegan, M. C. 2009. Bacillus cereus– induced permeability of the blood–ocular barrier during experimental endophthalmitis. Investigative Ophthalmology & Visual Science, 50(8), 3783-3793. 51. Murray, P. R. 2012. What Is New in Clinical Microbiology—Microbial Identification by MALDI-TOF Mass Spectrometry: A Paper from the 2011 William Beaumont Hospital Symposium on Molecular Pathology. The Journal of Molecular Diagnostics : JMD, 14(5), 419– 423. 52. Ozkocaman, V., et al., 2006. Bacillus spp. among hospitalized patients with haematological malignancies: clinical features, epidemics and outcomes. Journal of Hospital Infection, 64(2), 169-176. 53. Phelps, R. J., & McKillip, J. L. 2002. Enterotoxin production in natural isolates of Bacillaceae outside the Bacillus cereus group. Applied and Environmental Microbiology, 68(6), 3147-3151. 54. Pomerantsev, A. P., Pomerantseva, O. M., & Leppla, S. H. 2004. A Spontaneous Translational Fusion of Bacillus cereus PlcR and PapR Activates Transcription of PlcR-Dependent Genes in Bacillus anthracis via Binding with a Specific Palindromic Sequence. Infection and Immunity, 72(10), 5814–5823. 55. Rasko, D. A., Altherr, M. R., Han, C. S., & Ravel, J. 2005. Genomics of the Bacillus cereus group of organisms. FEMS Microbiology Reviews, 29(2), 303-329. 56. Ratnayake-Lecamwasam, M., Serror, P., Wong, K. W., & Sonenshein, A. L. 2001. Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels. Genes & Development, 15(9), 1093-1103. 57. Richardson, A. J., Rothburn, M. M., & Roberts, C. 1986. Pseudo-outbreak of Bacillus species: related to fibreoptic bronchoscopy. Journal of Hospital Infection, 7(2), 208-210. 58. Romero, C. F., Rai, M. K., Lowder, C. Y., & Adal, K. A. 1999. Endogenous endophthalmitis: case report and brief review. American Family Physician, 60, 510-523. 59. Rönner, U., Husmark, U., & Henriksson, A. 1990. Adhesion of Bacillus spores in relation to hydrophobicity. Journal of Applied Microbiology, 69(4), 550-556. 60. Rutherford, S. T., & Bassler, B. L. 2012. Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harbor Perspectives in Medicine, 2(11), a012427. 61. Scallan, E., et al., 2011. Foodborne illness acquired in the United States—major pathogens. Emerging Infectious Diseases, 17(1), 7. 62. Schallmey, M., Singh, A., & Ward, O. P. 2004. Developments in the use of Bacillus species for industrial production. Canadian Journal of Microbiology, 50(1), 1-17. 63. Scheldeman, P., Herman, L., Foster, S., & Heyndrickx, M. 2006. Bacillus sporothermodurans and other highly heat-resistant spore formers in milk. Journal of Applied Microbiology, 101(3), 542-555. 64. Schemmer, G. B., & Driebe, W. T. 1987. Posttraumatic Bacillus cereus endophthalmitis. Archives of Ophthalmology, 105(3), 342-344. 65. Scott, I. U., et al., 1996. Endophthalmitis associated with microbial keratitis. Ophthalmology, 103(11), 1864- 1870. 66. Vilain, S., Luo, Y., Hildreth, M. B., & Brözel, V. S. 2006. Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil. Applied and Environmental Microbiology, 72(7), 4970-4977. 67. Serror, P., & Sonenshein, A. L. 1996. CodY is required for nutritional repression of Bacillus subtilis genetic competence. Journal of Bacteriology, 178(20), 5910-5915. 68. Setlow, P. 2003. Spore germination. Current Opinion in Microbiology, 6(6), 550-556. 69. Shany, S., Bernheimer, A. W., Grushoff, P. S., & Kim, K. S. 1974. Evidence for membrane cholesterol as the common binding site for cereolysin, streptolysin O and saponin. Molecular and Cellular Biochemistry, 3(3), 179-186. 70. Shaligram, N. S., & Singhal, R. S. 2010. Surfactin- A Review on Biosynthesis, Fermentation, Purification and Applications. Food Technology and Biotechnology, 48(2), 119-134. 71. Simini, B. (1998). Outbreak of Bacillus cereus endophthalmitis in Rome. BACILLUS SPP. VIRULENCE • 221 72. Slamti, L., & Lereclus, D. 2005. Specificity and polymorphism of the PlcR-PapR quorum-sensing system in the Bacillus cereus group. Journal of Bacteriology, 187(3), 1182-1187. 73. Sneath, P., Bergey’s Manual of Systematic Bacteriology. Vol. 2. 1986, Baltimore, MD: Wiliam and Wilkins Co.in Liquid Soil Extract and in Soil. Applied and Environmental Microbiology, 2006. 72: p. 4970- 4977. 74. Sonenshein, A. L. 2005. CodY, a global regulator of stationary phase and virulence in Gram-positive bacteria. Current Opinion in Microbiology, 8(2), 203-207. 75. Stenfors Arnesen, L. P., Fagerlund, A., & Granum, P. E. 2008. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiology Reviews, 32(4), 579-606. 76. Stenfors Arnesen, L. P., Fagerlund, A., & Granum, P. E. 2008. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiology Reviews, 32(4), 579-606. 77. Streilein, J. W. 2003. Ocular immune privilege: therapeutic opportunities from an experiment of nature. Nature reviews. Immunology, 3(11), 879. 78. Ullman, S., Pflugfelder, S. C., Hughes, R., & Forster, R. K. 1987. Bacillus cereus panophthalmitis manifesting as an orbital cellulitis. American Journal of Ophthalmology, 103(1), 105-106. 79. Van Der Zwet., et al., 2000. Outbreak of Bacillus cereus infections in a neonatal intensive care unit traced to balloons used in manual ventilation. Journal of Clinical Microbiology, 38(11), 4131-4136. 80. Weber, D., & Rutala, W. 1988. Bacillus Species. Infection Control & Hospital Epidemiology, 9(8), 368-373. 81. Wilson, G. S. 1943. The pasteurization of milk. British Medical Journal, 1(4286), 261. 82. Winn, W. C. 2006. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. Lippincott williams & wilkins. 83. York, M. K. 1990. Bacillus species pseudobacteremia traced to contaminated gloves used in collection of blood from patients with acquired immunodeficiency syndrome. Journal of Clinical Microbiology, 28(9), 2114- 2116. 222 • FINE FOCUS, VOL. 4 (2)