Microbial contamination of pharmaceuticals has been posing severe challenges for the researchers & pharmaceutical manufacturers throughout the generations. They lead to greater problems both medically and economically. Contaminations can result in the batch failures as the pathogens breakdown the formulations, affecting the potency, stability and efficacy of the drug. From the medical aspect, sterility issues with presence of many pathogenic organisms can cause serious health threats to consumers, especially those who are already ill or in a weakened state.
The common pharmaceutical contaminants include bacteria and in rare cases fungi. While there are many culprits to name, this article mainly focuses on some of the most troublesome gram-positive bacteria.
Product contamination with gram-positive bacteria implicates human intervention as many of them are a part of the human skin flora. In the present culture, large parts of the pharmaceutical processes are handled by the machinery, but still there are many points where the human operator has to come in contact with the machinery, materials and packaging. These contact points later become the source of bacterial contamination if the operator exhibits inadequate personal cleanliness.
According to a report by the National sanitation foundation of USA, people pose the greatest risk of contamination in clean rooms. Even the cleanest of environments can become contaminated by something as minor as an unwashed hand.
In addition, there are other sources of gram-positive bacteria that causes contamination of the raw material, equipment, surfaces or packaging material, eventually leading to bacterial spoilage of the finished pharma product. These sources include the soil, dust, environment and material transfer.
However, not all contaminations lead to product spoilage and health hazards as many of the contaminants don’t get to survive within the pharmaceutical material due to low water activity. The moisture level available for microbial proliferation determines the type of bacteria that can most probably survive in the product. Most gram-positive bacteria, such as Enterococcus spp., Staphylococcus aureus or Streptococcus pyogenes require relatively low moisture content and therefore can survive for a long time within the material. Therefore, there are acceptable microbial limits set by the several pharmacopoeias that should be strictly considered by companies before releasing any pharma product.
Consequences of microbial contamination
Contamination in a pharma product by any gram-positive bacteria can lead to product spoilage to various degrees. The presence of microbes can affect the safety, quality and activity of the drug along with causing stability loss. The microorganisms usually either metabolizes the active ingredient of the drug or it acts on the excipients/ formulation additives. The spoilage is usually categorized in three forms- Physico-chemical spoilage, Chemical spoilage and Biological spoilage.
- In physicochemical spoilage, the contamination leads to physical changes in the drug whether it is the colour, odour, taste, texture or presence of gas production. For example, in syrups, some microbes metabolise the sugar molecules and cause concentration changes which eventually leads to the product becoming unstable and unsuitable for use.
- In chemical spoilage, the several metabolic activities performed by the microbial cells lead to unwanted chemical reactions within the drug. For example, Bacillus & Clostridium spp. may lead to the hydrolysis of gelatin capsules, thus rendering the drug unstable. Some microorganisms may also cause acetylation of the drug using their cellular enzyme which leads to loss of drug activity. The best example for this spoilage is the acetylation of Chloramphenicol by Staphylococci & Streptococci spp using Chloramphenicol acetyl-transferase. Few other types of spoilage in this section includes depolymerization of the polymers (eg. depolymerization of starch by amylase) or degradation of the active ingredient (Eg. Degradation of penicillin by beta-lactamase containing bacteria).
- After introduction into a product, bacterial cells utilize various compounds of the formulation to perform their metabolic activities. These activities account for proliferation but simultaneously may cause production of metabolites/ toxins that are released by the cells into the pharmaceutical preparations. Such metabolites and toxins give rise to biological spoilage and toxicity within the product. Most common example for this kind of spoilage is the production of exotoxins by Clostridium botulinum that renders the product toxic to human consumption.
Which are the main culprits to blame?
In March 2019, the company Kingston Pharma LLC had to recall its cough syrup bottles only because it showed presence of Bacillus cereus & B.circulans. The major pernicious microorganisms from the group of gram-positives are Enterococcus spp. (including Vancomycin resistant enterococci), Staphylococcus aureus (including MRSA), Clostridium spp., Streptococcus pyogenes, Bacillus & Micrococcus spp.
VRE & MRSA being multi-drug resistance are especially aggressive against immunocompromised patients. Infections with these bacteria can sometimes lead to sepsis in the patient’s body and eventually death for serious cases.
Therefore, it becomes crucially important for the pharmaceutical manufacturers to keep these microorganisms away by practicing proper quality assessment and right contamination control measures.
The sterility check of pharmaceutical products involves identifying the bacterial contaminants using conventional pharmacopoeial methods, as well as simplified, commercially available biochemical test kits. Every FDA approved plant follows the USP guidelines (Chapter 61 & 62) for the routine tests.
Detailed insight into the tests performed.
The routine investigation of pharmaceutical products includes the total plate count test followed by tests for the specified microorganisms. Bacterial isolates showing inconclusive or contradictory results are further subjected to molecular technologies like PCR-based assays. These tests help in identifying the contaminants on the basis of colony and cellular morphology, gram reactions, and key enzyme activities. However, the level of identification required in monitoring programs depends on the type of the operation and the location from which the isolate was recovered. For example, for non-sterile manufacturing areas, it may be sufficient to identify isolates to genus level but microbes isolated from aseptic processing areas often need characterization up to the strain level.
Microbial enumeration test of the product is ideally performed by first preparing a 1:10 dilution of the sample in a suitable diluent and then pour plating it for calculating the total aerobic microbial count (TAMC). The two major culture media used in this step are Soybean Casein Digest Agar & Sabouraud Dextrose Agar.
Isolation of the contaminants and their identification is primarily done by cultivating the pathogens in sterile Tryptic Soy broth (TSB) and then streaking them onto the selective agar medium. The isolates are identified by performing the gram-stain reaction, to differentiate the gram-positives from the gram-negative bacteria. Further examination involves performing confirmatory biochemical and serological tests.
The gram-positive cocci are streaked onto Mannitol Salt Agar and then identified by subjecting to confirmatory tests specific for Staphylococcus spp. On the other hand, sporulating, rod-shaped gram-positive isolates are subjected to spore staining to confirm the presence of Bacillus spp. Some of the main biochemical tests specified for the gram-positive bacteria are-
TM Media offers a comprehensive range of products required for the identification of main gram-positive bacterial contaminants in pharma. The chart mentioned below gives a summarised view of all its solution
* Products mentioned are apart from Carbohydrate consumption broth and H2O2 (For catalase).
Product recalls due to microbial contamination can have a huge impact on the company. Beyond the recalls, ceasing the production for identifying the sources of contamination can also have huge consequence on a company’s financial health. Therefore, proper prevention policies should be followed in the facilities to mitigate the chances of sterility issues. The professionals should also implement the modern strategies for contamination control like routine environmental monitoring. Only with correct measures, we can control the consequences of bacterial contamination, both economical and medical.