Tuesday, December 23, 2014

The Risk of Bacillus cereus to Pharmaceutical Manufacturing

A review of warning letters U.S. Food and Drug Administration indicates that contamination events associated with Bacillus species represents a relatively high proportion of microbiological related citations. During the period March 2013 to August 2014, 7 warning letters relating to contamination from spore-forming bacteria were issued. Extending the review back to 2007, it is noteworthy that the most common microorganism associated with contamination is Bacillus cereus.
This article considers some of the inspectorate findings relating to Bacillus contamination and goes on to consider the implications for the control of pharmaceutical products manufacture that arise from these regulatory observations. The discussion has a focus on Bacillus cereus, given its relative ubiquity, and extends to the general risks that arise from spore-forming microorganisms and the risk-mitigating actions that can be taken.

Bacillus risks to pharmaceutical manufacturing

The foremost reason for the detection of species of Bacillus in pharmaceutical products, according to FDA warning letters, relates to inadequate cleaning and disinfection regimes. This includes a failure to include a sporicidal agent as part of the disinfection regime or, where such a sporicide is used, a failure to qualify it in a way that demonstrates that the disinfectant can kill or inactivate spores.
A secondary reason is due to insufficient material control and in not ensuring that materials are of a suitable cleanliness (or sterile, where appropriate) for entry into pharmaceutical controlled environments. The third most common reason is attributable to problems with air handling systems.
With cleaning, disinfection, materials, and clean air supply being the points of origin, a robust environmental monitoring program should be capable of detecting contamination concerns. However, here, FDA inspectors have also been critical. The reasons for the incidences of Bacillus bacteria in several facilities remaining undetected was attributed to a failure to develop a contamination control strategy. The FDA noted that some organizations had not identified where contamination could potentially occur and had not taken steps to orientate environmental monitoring towards appropriate critical control points. Even where environmental monitoring was undertaken, one company was criticized for accepting a level of spore formers as part of the standard environmental flora1; whilst another firm was found not to be using a sporicidal disinfectant, which the Agency felt was an insufficient especially given that a media fill failure had occurred and been linked to a Bacillus contaminant.2 Another related failure was where an environmental monitoring program adequately detected Bacillus organisms, but the trending was imperfect and an upward trajectory in incidents was not notified.3

Bacillus cereus

It is notable that one of the most commonly isolated species of Bacillus is Bacillus cereus. Bacillus cereus is an endemic, soil-dwelling, Gram-positive, rod-shaped, beta hemolytic bacterium.4  B. cereus bacteria are facultative anaerobes. Under certain conditions the bacteria can produce endospores that enable them to survive in inhospitable environments (in the case of B. cereus: central or terminally sited ellipsoid or cylindrical spores). B. cereus can grow over a temperature range from 5°C to 50°C, with an optimum growth of between 28°C to 35°C; a pH range 5 to 9; and at a water activity (aw) greater than 0.94.5
The organism has long been associated with foodborne illnesses (it is most notoriously linked with “Fried Rice Syndrome,” since the bacterium can be contracted from fried rice dishes that have been sitting at room temperature for several hours6). B. cereus is also known to cause chronic skin infections (keratitis).7
Identification of B. cereus can sometimes be difficult, as the FDA Bad Bug Book points out.8  B. cereus is not easily differentiated from B. cereus var. mycoides, B. thuringiensis, and B. anthracis (DNA-DNA hybridization studies suggest that considerable chromosomal similarity exists between these species). Differentiation of these organisms depends upon determination of motility (most B. cereus are motile), presence of toxin crystals (B. thuringiensis), hemolytic activity (B. cereus and others are beta hemolytic whereas B. anthracis is usually non-hemolytic), and rhizoid growth which is characteristic of B. cereus var. mycoides.9  Modern biochemical test kits, or the use of genotypic microbial methods, are normally able to correctly identify the organism from other species of Bacillus.

Is Bacillus cereus an objectionable microorganism?

With sterile products, all microorganisms are a risk because the product is intended to be free of contamination and any contamination has the potential of causing serious harm to patients, especially given that most of these types of medicines are quite often used with the immunocompromised. In relation to non-sterile products, weighing up whether or not Bacillus cereus an objectionable microorganism is a difficult question, for what is and what is not an ‘objectionable’ microorganism requires risk assessment based on a number of objective criteria, including the risk to the intended patient population and the number of organisms present at a given time. So here the answer is simply “it depends.” Perhaps it is better to phrase the question: is Bacillus cereus a significant concern to pharmaceutical manufacturing?
Here the answer is a tentative “yes,” given the ubiquity of the microorganism in the environment. However, the organism is probably not found in greater numbers than several other species of environmental Bacillus. In this sense, any mesophilic, aerobic-growing Bacillus species found in high numbers is a potential risk. This arises as the spores of most Bacillus species are resistant to environmental stressors (such as desiccation) and to sterilization processes (such as heat or radiation); moreover, spores are difficult, when present, to eliminate from medical and pharmaceutical materials.
However, of the species of Bacillus most likely to be found, Bacillus cereus is one of the most virulent. It is both interesting and concerning that Bacillus cereus has been associated with some other recent examples of pharmaceutical contamination. In a review by Sutton,10 it is noted that there have been several cases of B. cereus contamination in relation to alcohol wipes. This includes the widespread distribution, and serious impact, of wipes produced by H&P Industries (under the brand name Triad) in 2012.
In addition, several recent FDA 483 observations and Warning Letters demonstrate deficiencies in addressing bacterial spore control. To avoid such regulatory difficulties, control measures to minimize the risk of bacterial spores are required. For this, an examination of the contributory factors that could lead to the introduction of such spores into the facility needs to be undertaken together with an effective remediation strategy. The factors to examine include facility design, personnel compliance, and cleaning and disinfection practices.

Risk Considerations in Relation to Bacillus Species

The issues arising from FDA Warning Letters highlight the need to be mindful of the risks from spore forming microorganisms as part of a contamination control strategy. Endospore forming microorganisms can make up between 5% and 10% of the microflora found in a standard cleanroom.11 This final part of the article considers some of the areas to focus upon as part of a spore reduction contamination control strategy.

Incoming Materials

Incoming raw materials can sometimes contain bacterial spores, especially with drier substances. In general, synthetic materials have a low bacterial count, however, they can contain spores. Dry powders of natural origin are more likely to carry high numbers of spore forming organisms. Hence purchasing such materials from an approved supplier and carrying out appropriate indicator organism testing (for certain risk materials, it may be appropriate to add a Bacillus species to complement the standard USP specified microorganisms).
In addition to chemicals, packaging—especially cardboard, can contain Bacillus species. The introduction of cardboard into cleanroom facilities should be avoided. In contrast, laminates, metal foils, and blister-pack materials all have smooth impervious surfaces with a high-temperature stage employed in their manufacture and, therefore, have low surface microbial counts.12
Controls should be introduced where in-coming materials for pharmaceutical processing are held before transfer to the process areas. One key aspect is with storage, and in ensuring that sufficient air can circulate around the storage containers. Air movement will allow outer packaging to dry out if it has become damp. On movement into core production, a risk of cross-contamination from the outer packaging remains a possibility. This can be minimized through effective air handling systems.12

Environmental Control

Control of the pharmaceutical environment is important in relation to microbiological control in general. With Bacillus spores in particular, control should be centered on good cleanroom design, with a focus on minimizing airborne contamination. Airborne contaminants are mainly spore-formers, in addition to other organisms shed by individuals via the deposition of skin flakes. Air control is achieved through having air filtration and ensuring good turbulent airflow within cleanrooms (and unidirectional airflow as required in cabinets). Rooms of different cleanliness classes should be at different pressure differentials in order to prevent air from the “dirtier” area trailing into the “cleaner” area.
Spores can also be carried into cleanrooms by staff via their clothing. Adequate changing facilities should be provided to enable staff to change effectively on entry into the cleanroom and to change quickly at the end of working sessions. Where cleanroom clothing may be re-worn (such as with non-sterile manufacturing) changing practices should be defined so that personnel do not contaminate their factory clothing.
With control of spore-formers, inadequate facility design can lead to areas where spore formers remain undiscovered, presenting a contamination risk in the future. Of particular concern here is damage to surfaces which can create niches in which spores can settle and remain. Surfaces should be designed to minimize contamination and to enable them to be easily cleaned and disinfected. For example, the use of coving and designing chemically resistant surfaces.
Repair and maintenance should extend to all areas of the facility, including cold areas. All species of Bacillus will survive in cold temperatures and some are capable of growth. For example, psychrotrophic B. cereus strains can grow at commercial refrigeration temperatures (2°C to 8°C).13

Non-sterile Processing

Different types of pharmaceutical products will be at a greater or lesser risk to Bacillus contamination than others (and the extent to which this becomes a serious risk requires an assessment of the organism as objectionable, as discussed above). Whilst taking care not to over generalize, in a manufacturing facility dealing with dry powder mixing, granulation and drying, and final sacheting or tabletting, contamination risks to the product from the environment will predominantly be Bacillus, along with mold spores. Such contamination arises from the environment dust, together with anything shed by the operators. With such processes, good handling and ventilation control can keep cross-contamination to a minimum.
Risks to dry products, such as tablets, can be re-presented at later manufacturing stages. Aqueous granulation and drying can become a problem if drying is not carried out immediately or if temperature tray drying is carried out over an extended time. Proliferation of microbiota originating from the raw materials can occur during the tray drying stage. These microorganisms may die through the process of drying as the available water activity is reduced.12
Further in terms of considering points of risk, it should be noted that the mechanical forces, together with the application of heat, involved in pressing tablets is often sufficient for the destruction of mold spores and vegetative bacteria. However, the concern is that Bacillus spores can survive this process.12

Repairs and Maintenance

Bacterial spores are often introduced into pharmaceutical areas through dust contamination. Based on this, any structural alterations to buildings or uncontrolled sweeping may give rise to these contaminants remaining in the vicinity. Thus strict controls should be in place during facility downtime.

Environmental Monitoring

The adequacy of controls in place in manufacturing environment is assessed through environmental monitoring. Here the importance is not only with undertaking monitoring at a sufficiently high frequency and with orientating monitoring locations to points of risk towards the product. There is also a need to understand and to characterize the microorganisms recovered.
In relation to the types of microorganisms recovered, environmental contamination of dry surfaces such as floors and walls comprise mainly Gram-positive rods, cocci, and fungal spores. Here species of Bacillus will be found (these organisms are less common in ‘wet areas’ like washbays, where Gram-negative bacteria will predominate).
In establishing an environmental monitoring regime, the agars used must demonstrate that they can recover a wide-range of microorganisms, including Bacillus species.

Cleaning and Disinfection Practices

Even with other control measures in place, spore contamination remains a risk. To guard against this, cleaning and disinfection practices need to be robust. Bacillus endospores in particular are resistant to hostile physical and chemical conditions; this means that they can be difficult to remove if a sanitization regime is not effective.14
This leads to some points that are worth considering in relation to cleaning and disinfection15:
  • The importance of detergents: are detergents used prior to the application of a disinfectant? This is a necessary step since many disinfectants have poor penetrative ability and require the removal of the soil barrier in order to reach microorganisms.
  • The efficacy of cleaning practices is important. Both detergents and disinfectants need to be wiped onto surfaces in order for them to be effective.
  • The preparation of detergents and disinfectants. It is important to check if “clean” water is being used as not to add a high bioburden to the cleaning solution. Furthermore, the mops and buckets should be suitable for cleanroom use (and with an aseptic facility, the cleanroom items should be sterile).
  • Arguably the most important point when considering the risks posed by spore forming microorganisms is whether a sporicidial disinfectant is being used routinely. The periodic use of a sporicide is important because standard disinfectants, such as alcohols, quaternary ammonium compounds, phenols and amphoteric surfactants, whilst effective against bacteria in their vegetative state, are ineffective against spores. Although the spore state itself is not capable of causing infection, under suitable conditions, germination of Bacillus spores may take place within minutes (the rate and the extent of spore germination is dependent on strains, treatments, and environmental factors).
  • Biocides that have sporicidal activity include hypochlorites and hydrogen peroxide/peracetic acid blends.
  • The disinfectants used for the routine sanitization program need to be qualified to show that they can reduce a known population of microorganisms from a surface. Such an evaluation should be undertaken using an appropriate method.
The robust adherence to good cleaning and disinfection practices is important, for spores of Bacillus are very adhesive to surfaces of equipment due to 3 characteristics: 1) their relatively high hydrophobicity, 2) low spore surface charge, and 3) unique morphology.16


This article has focused on one recent tragic incident, where a breakdown with environmental controls appears to be linked to a serious contamination incident. The article has gone on to consider the risks that endospore forming microorganisms present to pharmaceutical processes, particularly in relation to cleaning and disinfection. Here the most appropriate activity is the regular use of a sporicidal disinfectant.
Another inference from the article relates to the need to consider which microorganisms present in a facility are “objectionable.” With a sterile product any microorganism found in the product is objectionable. That said, to what extent does this apply to the microorganisms isolated through environmental monitoring? How concerned should microbiologists be with repeated detection of certain microorganisms? If the organisms have a resistance to the standard disinfectants then such concerns should be acute. Concern with objectionable microorganisms runs through most aspects of manufacture.
Screening for objectionable microorganisms should form part of an organization’s contamination control strategy—whether in relation to what is in the product, what is found in the environment, or both. The organization should draw up its own list of types of organisms of concern and consider measures that need to be put in place to minimize the risk to the product and patient. In most scenarios, this will include occasions when repeated incidents of Bacillus spores occur.


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