Differentiation of Pathogenic Clinical Isolates and Laboratory Strains of Bacillus cereus by NMR-Based Metabolomics Analysis (2023)

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FEMS Microbiology Letters

Volume 242 Number 1,

January 1, 2005

, pp. 127-136

Edited by Palen

Author link opens overlay panel, , , ,


six differentBacillus cereusStrains were selected from two differenteco-type: (1) three commonly used laboratory strains considered avirulent, and (2) three clinical isolates from meningitis patients. Screening of genomic DNA for genes encoding known toxins yielded no candidate genes that could clearly distinguish the two groups. However, the application of multivariate pattern recognition methods to metabolite profiles derived from different strains1HNMR spectroscopy(Metabolomics) The ability to classify different profiles. These two distinct ecotypes are clearly separated based on their metabolite profiles, suggesting that metabolomics approaches can be used to classify pathogens according to their expressed physiology, even if direct mechanistic links to specific pathogens cannot be inferredVirulence factors.This metabolomic approach can also be widely applied in general microbiology and microbial ecology to differentiate and identify different functional/physiological ecotypes.Strainor species.


Bacillus cereusConsidered an opportunistic human pathogen that can cause infections of varying severity. Most commonly associated with self-limited gastrointestinal infections caused by enterotoxin production,Bacillus cereusIt can also cause a range of non-gastrointestinal illnesses ranging from severe ocular infections to potentially fatal bacteremia and meningitis [1], [2]. The genetic backgrounds of these pathogenic strains are extremely similar. It has been suggested that this bacterium could be considered the same species as the other two members.Bacillus cereusGroup: Agriculturally importantBacillus thuringiensisand mammalian pathogensBacillus anthracis(causative agent of anthrax), which differ only by the possession of virulence genes transmitted by plasmids [3], [4], [5]. Therefore, it is important to be able to distinguish harmless or low-risk stains from strains that may be dangerous pathogens [6].

There are several possible approaches for untargeted analysis of the ecophysiological state or "pathotype" of bacterial strains. However, there are several advantages to using metabolomic analysis in this application. Metabolomics is based on the comprehensive analysis of the low molecular weight metabolite complement of biological samples. Since metabolites are downstream of gene transcription and enzymatic activity, metabolomics has the potential to more accurately map or describe the actual physiological state of one or more cells [7]. Metabolomics has been used to study bacterial responses to different physiological states and stressors [8], [9], [10], and some recent studies have shown that NMR-based analysis can be used for differentiation and classification of microbial species, including yeast In vivo analysis of strains and magic angle rotation analysis of marine unicellular algae [11], [12]. It was also shown that individual strainsSaccharomyces cerevisiaeStrains with single-gene deletions can be differentiated by metabolomics, and strains with gene deletions from closely related metabolic regions cluster together in metabolic space [13], [14]. It can therefore be inferred that metabolomics might be used as an adjunct to the analysis and possibly classification of microbial strains into groups of functional/clinical significance. Our aim in this study was to test whether metabolomic analysis could prove a useful addition to existing methods for profiling and typing membershipBacillus cereusGroup, in particular, if it is possible to differentiate strains based on their pathogenic potential. Previous bacterial metabolomic studies have used model organisms and well-defined stress or growth effects [8], [9], [10]. Thus, this study is the first example of metabolomic profiling of diverse bacteria to address important physiological/clinical questions.

We chose six differentBacillus cereusStrains from two different pathogenic types—three laboratory strains generally considered non-pathogenic, and three clinical isolates from meningitis patients—were analyzed for the presence of known virulence genes, And NMR-based untargeted metabolomics analysis. Therefore, the aim of this experiment was to collect metabolite profiles from different strains and then apply a multivariate pattern recognition method to the spectral data to determine whether (1) individual bacterial strains segregate in metabolic space, i.e., can be identified on the basis of their metabolic signature, and (2) whether the different pathotypes are clustered according to their metabolic differences. If these differences are found, they can form the basis of a predictive model for strain taxonomy. This can be formally stated that the null hypothesis is that there is no metabolite pattern characteristic of the differentBacillus cereusPathological type.

partial fragment

bacterial strain

The bacterial strains used in this study belonged to one of two groups, either "non-pathogenic" laboratory strains or clinical isolates. The laboratory strain isBacillus cereusType strain ATCC 14579 (referred to as TS in this paper for brevity),Bacillus cereus569 andBacillus cereusT. The causative strain isBacillus cereusSJ4,Bacillus cereusSJ10 andBacillus cereusSJ12. The pathogenic strains were all isolated from immunocompromised acute lymphoblastic leukemia patients in the same hospital (St. Jude)

(Video) Direct from Sample Metabolomics Using Rapid Evaporative Ionisation Mass Spectrometry

growth characteristics

Monitoring the growth curves of the different strains revealed that five of the six strains showed no statistically significant differences in growth rates in rich media (one-way ANOVA,P= 0.63), the average doubling time was 35.9±1.2 minutes. The mean doubling time of the sixth strain, SJ4, was significantly longer at 39.1±0.6 minutes (P=0.019)。

Genomic Screening for Suspected Virulence Factors

We screened for the presence/absence of a total of 11 different genes encoding known/putative virulence factors; the complete results are

genetic profile

There are no candidate genes that can be used to distinguish laboratory strains from clinical isolates. In fact, some laboratory strains shared the same genetic profile as some clinical isolates (TS and T, compared to SJ12). Also, these genes are all potential virulence genes, so clinical isolates possess fewer of these genes than laboratory strains differ (e.g. TS and T are positive for all genes screened, while 569 is positive)

in conclusion

Screening of genomic DNA for 11 different suspected virulence genes fails to distinguish common laboratory strainsBacillus cereus, generally considered nontoxic, fromBacillus cereusStrains isolated from separate meningitis outbreaks. In contrast, NMR-based multivariate metabolite profiling (metabolomics) revealed that two distinct groups of bacteria were separated based on their biochemical properties. Appropriate metabolomic analysis can be used for screening

thank you

JGB and TLW are funded by the UK Biotechnology and Biological Sciences Research Council. thisBacillus cereusSJ4, SJ10, and SJ12 strains were a gift from Dr. J.L. Shenep, St. Jude Children's Research Hospital, Memphis, TN, USA.


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    Copyright © 2004 European Federation of Microbiological Societies. Published by Elsevier B.V. all rights reserved.


    How do you identify Bacillus cereus in the laboratory? ›

    cereus have a pink–purple color, surrounded by a characteristic halo formed of pink precipitation, which permits their identification. Finally, a hemolysis test is performed on bacterial colonies to confirm for B. cereus strains. This method confirms only presumptive B.

    What are the different strains of Bacillus cereus? ›

    pseudomycoides, B. thuringiensis, B. toyonensis, and B. weihenstephanensis.

    What is the pathogenicity of Bacillus cereus? ›

    cereus, whether intestinal or nonintestinal, is intimately associated with the production of tissue-destructive exoenzymes. Among these secreted toxins are four hemolysins, three distinct phospholipases, an emesis-inducing toxin, and proteases.

    What is the metabolism of Bacillus cereus? ›

    Metabolism. Bacillus cereus has mechanisms for both aerobic and anaerobic respiration, making it a facultative anaerobe. Its aerobic pathway consists of three terminal oxidases: cytochrome aa3, cytochrome caa3, and cytochrome bd, the use of each dependent on the amount of oxygen present in the environment.

    What are the clinical findings of Bacillus cereus? ›

    Bacillus cereus is a facultatively anaerobic, toxin-producing gram-positive bacterium found in soil, vegetation, and food. It commonly causes intestinal illnesses with nausea, vomiting, and diarrhea.

    What is the laboratory diagnosis of Bacillus species? ›

    Diagnosis is confirmed by observation of characteristic encapsulated bacilli in polychrome methylene blue-stained smears of blood, exudate, lymph, cerebrospinal fluid, etc., and/or by culture. Other Bacillus infections are diagnosed by culture of the bacteria.

    What is biochemical test for Bacillus cereus? ›

    Biochemical Test of Bacillus cereus
    Basic CharacteristicsProperties (Bacillus cereus)
    Gelatin HydrolysisNegative (-ve)
    Gram StainingPositive (+ve)
    Growth in KCNPositive (+ve)
    HemolysisPositive (+ve)
    47 more rows
    Apr 4, 2022

    How to differentiate Bacillus cereus from other Bacillus species? ›

    B. anthracis is non-hemolytic on sheep blood agar, susceptible to penicillin, lysed by the gamma phage, and non-motile. Conversely, B. cereus is hemolytic on sheep blood agar, resistant to penicillin, resistant to lysis by the gamma phage, and motile.

    What are the morphological characteristics of Bacillus cereus? ›

    Summary: Bacillus cereus is a Gram-positive aerobic or facultatively anaerobic, motile, spore-forming, rod-shaped bacterium that is widely distributed environmentally.

    Where is Bacillus cereus most commonly found? ›

    Bacillus cereus is an aerobic spore-forming bacterium that is commonly found in soil, on vegetables, and in many raw and processed foods.

    Where is Bacillus cereus found in the body? ›

    In the enterotoxin form of the disease, you produce the toxin in your small intestine. This happens after you eat food with the bacteria or the cells they produce (spores). This is the most common type of B. cereus in the U.S. and Europe.

    What is the main source of Bacillus cereus? ›

    cereus is found in soil, raw plant foods such as rice, potatoes, peas, beans and spices are common sources of B. cereus. The presence of B. cereus in processed foods results from contamination of raw materials and the subsequent resistance of spores to thermal and other manufacturing processes.

    What kills Bacillus cereus? ›

    cereus: Steaming under pressure, roasting, frying, and grilling foods will destroy the vegetative cells and spores if temperatures within foods are ≥ 145ºF (63ºC).

    What antibiotics are used to treat Bacillus cereus? ›

    Severe non-anthrax Bacillus cereus infections are commonly treated with vancomycin, gentamicin, linezolid, levofloxacin, and clindamycin antibiotics (18–21).

    What are the symptoms of the Bacillus cereus infection? ›

    The symptoms of B. cereus diarrheal type food poisoning include abdominal pain, watery diarrhea, rectal tenesmus, moderate nausea that may accompany diarrhea, seldom vomiting and no fever.

    Why test for Bacillus cereus? ›

    Bacillus Cereus Testing

    It is isolated from foods relatively frequently, making it an important environmental indicator organism for the food industry. Foods bearing contamination risk include meat and milk products, vegetables, soups, spices and especially baby food.

    What are some interesting facts about Bacillus cereus? ›

    Interesting Facts:

    Vegetative cells of this bacterium can be killed with heat but its spores may survive. Some strains of this bacterium can be beneficial as probiotics for animals. B. cereus has been found to be one of the most common contaminants in pharmaceutical manufacturing facilities.

    What are the benefits of Bacillus cereus? ›

    Plants benefit from the presence of B. cereus since it is capable of inhibiting plant disease caused by protest pathogens and also enhances plant growth [23].

    How do you treat a Bacillus infection? ›

    Antibiotic therapy with vancomycin or clindamycin has achieved high cure rates in Bacillus endocarditis. Intravenous drug abusers with endocarditis caused by Bacillus species have responded well to clindamycin.

    How are Bacillus species identified and isolated? ›

    Bacillus species were identified by their phenotypic and biochemical characteristics. The antimicrobial effects of Bacillus extract against the target bacteria including Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shigella dysenteriae and Corynebacterium diphtheriae were examined.

    What are the medically important Bacillus species? ›

    Of the 34 species of the genus Bacillus, the two of greatest medical importance are B. anthracis, the causative agent of anthrax, and B. cereus, which causes food poisoning.

    How long does Bacillus cereus testing take? ›

    Test Frequency and Turnaround Time (TAT)

    The Bacillus cereus count is performed Monday to Saturday. Turnaround time is up to 7 days.

    What specimen was tested to isolate B. cereus? ›

    Various selective solid media such as MYP (mannitol-egg yolk-phenol red-polymyxin-agar) and PEMBA (polymyxin-pyruvate-egg yolk-mannitol-bromthymol blue-agar) were used for the isolation and detection of B. cereus from food.

    What enzymes are produced by Bacillus cereus? ›

    Most of the isolated B. cereus/B. thuringiensis strains were found to produce extracellular enzymes: lecithinase, gelatinase, lipase and protease. These enzymatic activities are very important for dairy industry.

    What other tests can be used to isolate differentiate and enumerate Bacillus cereus from food samples? ›

    Mannitol-egg yolk-polymyxin (MYP) agar was used as a presumptive test for the presence of B. cereus in the food samples. Various biochemical tests for the confirmation of suspicious colonies appearing on MYP agar were evaluated.

    What distinguishing features does Bacillus bacteria have? ›

    Bacillus anthracis — Key Characteristics
    • Large, box-car shaped, gram-positive rod in short or long chains.
    • Non-swelling, oval spores formed when grown on culture media. ...
    • Encapsulated rods may be seen in clinical specimens.
    • Ground-glass appearance of colonies.
    • Nonhemolytic on sheep blood agar.
    • Nonmotile.

    What are the characteristics of Bacillus cereus under microscope? ›

    Bacillus cereus is gram-positive rod-shaped bacilli with square ends. Occasionally may appear gram variable or even gram-negative with age. They are single rod-shaped or appear in short chains. Clear cut junctions between the members of chains are easily visible.

    What are the biochemical properties of Bacillus cereus? ›

    Biochemical Test and Identification of Bacillus cereus
    Basic CharacteristicsProperties (Bacillus cereus)
    Gelatin HydrolysisNegative (-ve)
    Gram StainingPositive (+ve)
    Growth in KCNPositive (+ve)
    HemolysisPositive (+ve)
    47 more rows
    Aug 9, 2022

    What are the virulence characteristics of Bacillus cereus? ›

    Several virulence factors of B. cereus are responsible for causing diarrheal symptoms following infection and include the tripartite toxins hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE) as well as cytotoxin K (CytK, also known as hemolysin IV).

    Is Bacillus cereus urease positive or negative? ›

    Bacillus cereus is a gram-positive, spore-forming rod known to be present in various environments.

    What is the simple stain of Bacillus cereus? ›

    B. cereus is the Gram-positive, aerobic or facultative anaerobic, motile and spore-forming bacteria. They are generally found in soil, vegetation, and in many raw foods. Based on their morphological structure, they are classified as Gram-positive large rods, which appear purple on Gram staining procedure.

    What do Staphylococcus aureus and Bacillus cereus have in common? ›

    Bacillus cereus sensu lato species, as well as Staphylococcus aureus, are important pathogenic bacteria which can cause foodborne illness through the production of enterotoxins.

    How does Bacillus cereus spread? ›

    MODE OF TRANSMISSION: The primary mode of transmission is via the ingestion of B. cereus contaminated food 1 2: emetic type of food poisoning has been largely associated with the consumption of rice and pasta, while the diarrheal type is transmitted mostly by milk products, vegetables and meat.

    What are three toxins produced by Bacillus cereus? ›

    cereus food poisoning is probably under reported. Among the enterotoxins produced by B. cereus, the hemolytic enterotoxin hemolysin BL (HBL), the non-hemolytic enterotoxin (Nhe), and cytotoxin K (CytK) are claimed to play a major role in diarrheal disease and will be addressed in this review.

    Does all rice have Bacillus cereus? ›

    Uncooked rice can contain spores of Bacillus cereus, bacteria that can cause food poisoning. The spores can survive when rice is cooked. If rice is left standing at room temperature, the spores can grow into bacteria. These bacteria will multiply and may produce toxins (poisons) that cause vomiting or diarrhoea.

    Is Bacillus cereus fatal? ›

    cereus is uncommon though well known1 2; however, it is rarely fatal in immunocompetent individuals. It usually results in a self-limited gastroenteritis, requiring supportive treatment. 3–5 B. cereus causes two distinct syndromes: emetic and diarrhoeal.

    Can Bacillus cereus go away on its own? ›

    Most people who become sick due to Bacillus cereus will get better on their own. People with diar- rhea and vomiting must drink plenty of fluids to prevent dehydration. Antibiotics cannot be used to treat this illness.

    What is the best way to prevent Bacillus cereus? ›

    cereus you should aim to eat your food as soon as possible after it is cooked. If you can't do that, then hot foods should be kept above 60˚C and cold foods, below 5˚C. Meats and vegetables should be cooked to an internal temperature of 60˚C and kept there for at least 15 seconds.

    Can Bacillus cereus be treated? ›

    In contrast, patients with invasive disease require antibiotic therapy and prompt removal of any potentially infected foreign bodies, such as catheters or implants. Bacillus cereus is usually susceptible in vitro to vancomycin, clindamycin, ciprofloxacin, imipenem, and meropenem.

    How do you test for Bacillus cereus? ›

    On MYP medium, colonies of B. cereus have a pink–purple color, surrounded by a characteristic halo formed of pink precipitation, which permits their identification. Finally, a hemolysis test is performed on bacterial colonies to confirm for B. cereus strains.

    How long do symptoms of Bacillus cereus poisoning usually last? ›

    Some Bacillus cereus bacteria may produce poison that causes fever and diarrhoea. The symptoms, which may be serious, usually start six to 24 hours after eating and can last for one or two days.

    Can Bacillus cereus cause sepsis? ›

    cereus also induces a multitude of other serious infections such as fulminant sepsis and devastating central nervous system infections [9, 19]. In hospital, B. cereus is however usually regarded by the physicians as an environmental contaminant. Thus, despite positive blood samples, B.

    How would you describe Bacillus cereus under a microscope? ›

    Bacillus cereus is gram-positive rod-shaped bacilli with square ends. Occasionally may appear gram variable or even gram-negative with age. They are single rod-shaped or appear in short chains. Clear cut junctions between the members of chains are easily visible.

    What does Bacillus cereus look like on agar? ›

    Colony Morphology. When grown under aerobic conditions on 5% sheep blood agar at 37°C, B. cereus colonies are dull gray and opaque with a rough matted surface (Fig.

    What stain would be used to identify Bacillus? ›

    How do you identify Bacillus cereus? B. cereus is a Gram-positive bacterium, and it can be identified by Gram staining technique. The selective medium which is used to identify this species is MYP agar, in which a characteristic halo with pink precipitation surrounding the pink-purple colonies act as identification.

    What is the identification test for Bacillus and Clostridium? ›

    Starch hydrolysis test

    This test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes a-amylase and oligo-1,6-glucosidase. Often used to differentiate species from the genera Clostridium and Bacillus.

    What does Bacillus bacteria look like under a microscope? ›

    Bacillus subtilis is a typical germ, which is rod-shaped and Gram-positive. When cultured on ordinary nutrient agar, the morphology circular colony of this bacteria is rough, opaque, fuzzy white or slightly yellow with jagged edges [1, 7].

    What do you call the stain used to visualize Bacillus cereus? ›

    The endospore stain is a differential stain used to visualize bacterial endospores. Endospores are formed by a few genera of bacteria, such as Bacillus .

    What is the preferred growth media for Bacillus cereus? ›

    The MYP agar has been the standard media for plating B. cereus, but it has little selectivity so background flora is not inhibited and can mask the presence of B. cereus. Bacara is a chromogenic selective and differential agar that promotes the growth and identification of B.

    What is the morphological characteristic of Bacillus cereus? ›

    B. cereus are facultative anaerobes that are motile and able to form endospores, have colonial morphology of about 2-7 mm in diameter, and have a white granular texture 3. B. cereus grows above 10-20°C and below 35-45°C with an optimum temperature of about 37°C 1 2.

    Is Bacillus cereus hemolytic on blood agar? ›

    Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in blood agar.

    What are the identifying features of Bacillus bacteria? ›

    Bacillus anthracis — Key Characteristics
    • Large, box-car shaped, gram-positive rod in short or long chains.
    • Non-swelling, oval spores formed when grown on culture media. ...
    • Encapsulated rods may be seen in clinical specimens.
    • Ground-glass appearance of colonies.
    • Nonhemolytic on sheep blood agar.
    • Nonmotile.

    What techniques can be used to identify and differentiate species of bacteria? ›

    Bacteria are identified routinely by morphological and biochemical tests, supplemented as needed by specialized tests such as serotyping and antibiotic inhibition patterns. Newer molecular techniques permit species to be identified by their genetic sequences, sometimes directly from the clinical specimen.

    What is Bacillus cereus testing? ›

    Tests and diagnosis

    Food poisoning caused by B. cereus can be confirmed by isolation of this bacterium in food, stool or vomit samples. Different media are available to culture this pathogen. Commercial kits are also available to detect the diarrhoeal enterotoxin, but this is not the case for emetic toxin.

    Which stain is most helpful in identifying species of Bacillus and Clostridium? ›

    Gram stain is used to identify the organism causing a disease or pathological condition. Examples of gram-positive cocci include Staphylococcus and Streptococcus species. Gram-positive bacilli include Corynebacterium, Clostridium, and Listeria species.


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