Bacillus cereus contamination can lead to serious consequences…

By Jennifer Johnson

A couple of recent incidents have brought Bacillus cereus back into focus. In the UK for example, liquid baby formula contaminated with the bacteria killed one premature baby and was responsible for sickening 14 others in six different hospitals. Then, in China, 139 people become sick after consuming fermented
black beans
(douchi) in Yunan. After studies were done on vomit samples and the unprepared douchi, three Bacillus cereus strains were isolated.

Bacillus cereus is an aerobic and facultatively anaerobic, motile, spore-forming gram-positive rod.  B. cereus exists in two forms: spores and vegetative cells.  Because B. cereus is found in soil, so raw plant foods such as rice, potatoes, peas, beans and spices, are common sources of the organism. The presence of the organism in processed foods results from the contamination of the raw materials and the subsequent resistance of the spores to thermal and other manufacturing processes. During food’s cooling process, spores may germinate, enabling B. cereus to multiply and produce high levels of the emetic toxin cereulide.

After consumption of contaminated foods, B. cereus causes two types of gastroenteritis. One is an emetic syndrome with a relatively short incubation period of 1-6 hours that results in nausea and vomiting. The other is a diarrheal syndrome with an incubation period of 6-24 hours causing watery diarrhea, abdominal cramps, and pain.

This quick onset of symptoms can pose a serious problem for passengers on an
international flight for example, where many people can become sick in a short period of time. While foodborne illness associated with air travel is quite uncommon, foodborne outbreaks have been recorded by the airline industry as a result of improper testing or handling of food. When this does occur, it can have serious implications for passengers and crew members and means quality, safe, in-flight catering must rely on high standards of food preparation and storage; this applies at the airport kitchens (or at subcontractors’ facilities) and on the aircraft.

Bacillus cereus has also been recognized as a cause of potentially fatal systemic infections outside the GI tract, particularly in high-risk patients, such as infants, immune-compromised individuals, and chronically debilitated or elderly patients.  These infections include bacteremia, septicemia, meningitis, cerebral abscess, pneumonia, and endocarditis.

The risk of illness after ingestion of vegetative B. cereus cells is influenced by the strain, composition of the food, the liquid nature of the food and the age of the individual. Young children are particularly susceptible and may be more severely affected by a B. cereus contamination resulting in severe complications and even death. Because liquid foods, such as infant formula, are transported faster to the small intestine they are protected from the influence of gastric conditions, providing more opportunity for the survival of the pathogen.

BioLumix B. cereus Vial:

Detecting B. cereus as quickly and easily as possible is of paramount importance when corrective action(s) can minimize public exposure. BioLumix has an assay that detects B. cereus in 24 hours. The B. cereus vial’s sensor detects production of CO2. The disposable vial contains a transparent solid sensor located at the bottom which changes its optical properties whenever CO2 diffuses into it. Only gases can penetrate the sensor; blocking liquids, microorganisms, and particulate matter. Consequently, the optical readings are not masked by the sample. CO2 generated by B. cereus metabolism in the liquid medium placed above the sensor, diffuses into the sensor and interacts with an indicator reagent to provide an indication of the presence of the carbon dioxide. The medium contains growth promoting ingredients and inhibitors to gram-negative and non-Bacillus cereus gram-positive organisms.  The method used is very simple; just pipette the diluted sample into the vial and your results can be obtained in 24 hours or less.

Advantages of BioLumix Vials:

  • Vials can be stored at room temperature
  • Prefilled ready-to-use vials come with a Certificate of Analysis
  • Automated same day test results
  • Vials are available for a variety of tests, each with organism-specific enrichment medium
  • A single organism can be detected in a vial, if capable of growth in the medium
  • Liquid culture from a vial can be used to perform further tests

 

Rapid Testing for Gram negative Bacteria- the BioLumix Approach

Gram Negative Bacteria- Cell Wall

Gram negative cell walls are more complex than Gram positive cell walls, both structurally and chemically. Structurally, a Gram negative cell wall contains two layers external to the cytoplasmic membrane. Immediately external to the cytoplasmic membrane is a thin peptidoglycan layer, which accounts for only 5% to 10% of the Gram negative cell wall by weight. External to the peptidoglycan layer is the outer membrane, which is unique to Gram negative bacteria. For Gram negative species, many of the lytic virulence factors such as collagenases, hyaluronidases, proteases, and beta-lactamase are located within the periplasmic space.

The outer layer of the Gram negative bacterial cell wall is made up of lipopolysaccharide and proteins. This outer layer protects a thin layer of peptidoglycan. For the Gram positive bacteria, the outer layer of the cell wall is the peptidoglycan layer and does not contain lipoproteins. Below the outer layer of lipopolysaccharide, there exist layers of periplasmic space and the plasma membrane.

The pathogenic capability of Gram-negative bacteria is often associated with their cell walls, especially, the lipopolysaccharide layer (also known as LPS or endotoxin layer). In humans, LPS can triggers an immune response characterized by cytokine production and immune system activation.

Gram Negative Bacteria- Who are they?

Important gram negative bacteria include: the Enterobacteriaceae family, Pseudomonads, Aeromonas, Plesiomonas, Xahothomonas, Burkholderia, Haemophilus.

The Enterobacteriaceae family contains a number of pathogens including E. coli, Salmonella, Shigella, Klebsiella pneumoniae, Pasteurella, and Yersinia. Pseudomonas aeruginosa is an opportunistic pathogen and can cause urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, etc. Pseudomonas aeruginosa is found in many natural and domestic environments including plants, soils and surface water, especially warm moist environments containing organic material or contaminated by human or animal waste.

Members of the genus Aeromonas and Plesiomonas are involved in human intestinal disease. The species Hemophilus influenzae is a cause of meningitis in children, while Pasteurella. multocida causes cholera in fowl. Burkholderia cepacia, an important pathogen of pulmonary infections in people with cystic fibrosis.

Current methodology

In USP <61-62> the test for Bile-tolerant Gram-negative Bacteria (BTGNB) replaces the “Test for Enterobacteriaceae and Certain Other Gram-Negative Bacteria”. In older USP protocols. USP <62> does require testing for absence of P. aeruginosa and Burkholderia cepacia.

The method usually involves a 1:10 dilution using Soybean–Casein Digest Broth (TSB) as the diluent, and incubation at 20 to 25 C for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 hours but not more than 5 hours). Thereafter the appropriate amount is sub-cultured on Violet Red Bile Glucose Agar (VRBGA) and incubates at 30 to 35 C for 18 to 24 hours. For the testing of lower numbers of BTGNB, the MPN method can be used. For P. aeruginosa the TSB is pre-incubated for at 30 to 35 C for 18 to 24 hours. Growth is sub-cultured onto a plate of Cetrimide Agar and incubate at 30 to 35 C for 18 to 72 hours

The BioLumix approach

The BioLumix system offers two approaches for the detection of gram negative bacteria:

  1. The Two Vial Approach: An approach similar to the USP procedure where two separate vials are used. One vial detects Bile-tolerant Gram-negative Bacteria and another vial detects P. aeruginosa.
  2. The Single vial approach: An approach where a single vial is used to detect all gram negative bacteria.

A 1:10 dilution of the product is made in TSB. For specified levels of >10 cfu/g, a 1.0 ml sample of this dilution is added to the ENT (Enterobacteriaceae) vial and to a PSE (Pseudomonas) vial. The system automatically monitors the vials and will determine in if the samples are clean or contaminated. When the specified level of absent in 1.0 gram or 10 grams is required, the TSB is pre-incubated overnight as an enrichment step, and then 0.1 ml of the sample is added to the vial.

Single Vial Approach

The GN (Gram Negative) vial has excellent inclusivity for gram negative organisms and does not detect most of the gram positive bacteria. After the first dilution in TSB, with or without pre-incubation depending on the specified level, 0.1 ml of the TSB is added to a single GN vial. This is a very simple and fast procedure to access the absence or presence of gram negative bacteria. This single vial can replace the two vials listed above with better coverage of gram negative bacteria.

ENT Vial: The Enterobacteriaceae vial monitors a change in color due to a pH shift as Enterobacteriaceae organisms ferment glucose in the presence of selective media. The vial medium selectivity is similar to VRBGA.

PSE Vials: The Pseudomonas vial uses the same CO2 sensor as the BioLumnix Total Aerobic Count vial, but with a selective medium. The vial medium selectivity is similar to Centrimide agar.

GN Vial: The hydrolysis of fluorogenic synthetic substrates by bacterial enzymes causes an increase in fluorescence. A fluorogenic synthetic enzyme substrate containing 4-methylumbelliferon, common to all gram negatives, is used in the presence of selective media that inhibits the growth of gram-positive bacteria. The GN vial detects the presence of all Enterobacteriaceae, Pseudomonas, Burkholderia and many other gram negative bacteria. The curves below show the growth of quadruplicate samples for P. aeruginosa (figure on the right: green, lt. blue, red, and purple) and for E. coli (figure on the left: green, lt. blue, red, and purple). A negative control (Bacillus species) is shown in each figure and is represented along the baseline in dark blue.

Advantages: The results of all these assays are available overnight (24 hours), the system is fully automated including achieving of data, data maintenance and report generation, it can be used to create a paperless laboratory. The system is unaffected by product interference, delivering accurate results with faster product release. These assays are simpler to perform than the standard methods saving time, labor, and money.

References

United States Pharmacopeia (2009) Chapter <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests. The National Formulary. Rockville, MD, The United States Pharmaceopeial Convention.

United States Pharmacopeia (2009) Chapter <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms. The National Formulary. Rockville, MD, The United States Pharmaceopeial Convention.

New Bacillus Cereus Vial for BioLumix

Bacillus cereus…

Bacillus cereus is a Gram-positive, facultatively aerobic spore former whose cells are large rods and with spores that do not swell the sporangium.  B. cereus spores are more resistant to heat and chemical treatments than vegetative pathogens such as Salmonella, and E. coli.

Bacillus cereus is responsible for 5% of foodborne illnesses worldwide, causing severe nausea, vomiting and diarrhea. Detecting potential B. cereus as quickly and easily as possible is of paramount importance. B. cereus food poisoning has two recognized types of illness are caused by two distinct metabolites. The diarrheal type of illness is caused by a large molecular weight protein, while the vomiting (emetic) type of illness is believed to be caused by a low molecular weight, heat-stable peptide.

The symptoms of B. cereus diarrheal type food poisoning include watery diarrhea, abdominal cramps, and pain occurs 6-15 hours after consumption of contaminated food. Nausea may accompany diarrhea, but vomiting rarely occurs. Symptoms persist for 24 hours in most instances. The emetic type of food poisoning is characterized by nausea and vomiting within 0.5 to 6 h after consumption of contaminated foods. Occasionally, abdominal cramps and/or diarrhea may also occur. Duration of symptoms is generally less than 24 h.

A wide variety of foods including meats, milk, vegetables, and fish have been associated with the diarrheal type food poisoning. The vomiting-type outbreaks have generally been associated with rice products; however, other starchy foods such as potato, pasta and cheese products have also been implicated. Food mixtures such as sauces, puddings, soups, casseroles, pastries, and salads have frequently been incriminated in food poisoning outbreaks.

Two different methods are described in BAM1 for the detection of B. cereus: (i) plate count method with Mannitol–Egg Yolk–Polymyxin (MYP) agar and (ii) a Most Probable-Number (MPN) method with Tryptic Soy Broth (TSB) supplemented with 0.1% Polymyxin Sulfate.  The MPN method allowed for better recovery and therefore for higher counts than MYP2, probably due to the better ability of the broth to recover injured organisms.

BioLumix Vial:

A new vial (BCP) was developed specifically for B. cereus detection in the BioLumix system.  It uses the CO2 sensor at the bottom of the vial.  The medium contains growth promoting ingredients peptones, casein digest and Yeast Extract. Sodium Pyruvate is added to facilitate the resuscitation of damaged Bacillus cells. Manganese Chloride is added to aid the germination of spores.  Inhibitors include Cyclohexamide to prevent growth of YM; LiCl, Cyclohexamide and Polymixin B are used as inhibitors.

                                    ATCC 10876- Dark Blue; ATCC 11778- Green; ATCC 14549- Light blue; and ATCC13061-Red

 Twenty five cultures of gram negative organisms and Twenty one cultures of gram positive organisms were tested in the BC vial.  None of the gram negative organisms grew in the vial and all gram positive organisms did not grow except one strain of Enterococcus feacalis (ATCC 19433). The new BC vial recovers well vegetative cells as well as spores. The method used is very simple, just pipette the diluted sample into the vial.  The results are obtained in 24 hours or less.  This vial is ready for free product trials.

References:

  1. E. Jeffery Rhodehamel and Stanley M. Harmon (2001). Bacteriological Analytical Manual, Chapter 14: Bacillus cereus (2001). US Food and Drug Administration.
  2. N. M. Harper, K.. K. Getty, K. A. Schmidt, A. L. Nutsch, and R. H. Linton. Comparing the Mannitol–Egg Yolk–Polymyxin Agar Plating Method with the Three-Tube Most-Probable-Number Method for Enumeration of Bacillus cereus Spores in Raw and High-Temperature, Short-Time Pasteurized Milk. J. Food Prot.71:473–478.