What Superbugs Are Lurking in Your Body?

Estimates from the World Health Organizations suggest that the usage of antibiotics over the past 70 years has added about 20 years to the average human’s lifespan. When you take an antibiotic, you expect it to do its job and attack the pathogen that’s making you ill – but that’s not always the case.

Bacteria, fungi, viruses, and other live organisms evolve over the course of time and become immune to human medications. Part of the problem is that doctors frequently over-prescribe antibiotics due to improper information received from patients or simply due to human error. As human reliance on medication increases, so does microbial resistance. Why?

The Crucial Role of Microbiology Testing in the Expanding Pharmaceutical Industry

Over the years, the U.S. has remained the world’s biggest market for pharmaceutical products and the global leader in biopharmaceutical research. In fact, the massive U.S. pharmaceutical industry accounts for about 80 percent of all research and development in biotechnology in the world. The United States National Institute of Health claims that there are approximately 140,000 clinical trials underway for pharmaceutical products awaiting FDA approval, which the Pharmaceutical Research and Manufacture of America (PhRMA) claims to have an annual value of over $67 billion.  

Microbiology Instruments Appropriate for Enzyme Testing

Enzymes are intricate, stable, globular proteins that serve as catalysts in chemical reactions. These reagents reduce the activation energy in order to increase the rate of the reaction at the same temperature. Their stability prevents them from being consumed like other reagents in the reaction. While they can accelerate chemical reactions by more than 1,000 times, the effect of enzymes can either be enhanced by promoters, or reduced by inhibitors. Most industrial enzymes are obtained from microorganisms like fungi and bacteria, though they can also be obtained from animal and plant sources, unless they are activated.

The Pharmaceutical Industry and Its Dependence on Microbiology

Put simply, microbiology entails the study of microorganisms that one can't see with the naked eye. These are from bacteria, fungi, protozoa and such similar organisms. There is an intricate association between microbes to specific diseases. This has kept scientists at the front on research concerning them for centuries. The role of microbiology on advances within the healthcare industry, and especially the pharmaceutical industry, has led to great discoveries ranging from medicines and vaccines to devices.

Understanding Microbiological Quality Control and Assurance Solutions

In the ever-changing pharmaceutical industry, competition is rife when it comes to the speedy development of products. Microbiological testing is essential to ensure safe products are made without any environmental hazards and at a reduced cost. This has led to an increased use of rapid detection kits that cut costs and time in detection of pathogens in any given sample. 

Regulations and Requirements for Pharmaceutical Microbiology Labs

In microbiology, quality assurance stands for the measures put in place to ensure quality control, as well as quality improvement. Quality control stands for the constant observation of work practices, paraphernalia and chemical substances so as to spot and correct defects. Once there are proper quality control measures in place, then quality improvement comes into focus as a way of making existing systems more efficient.

BioLumix Microbial Limit Vial (MC)

Introduction:

The Microbial Limit vial is used to test primarily Personal Care, Cosmetic and over the counter Pharmaceutical (OTC) products for microbial content (contamination). Each of these types of products may have preservatives in their composition and the Microbial Limit vial helps to neutralize the inhibition of microbial growth that many preservatives provide. Neutralization of the preservative allows for a proper evaluation of whetheror not the product has contaminants. Often the contaminating bacteria in the product while in the presence of the preservative remain “injured” and unable to replicate. Thelack of replication might be interpreted as the lack of contamination.

How It Works

The Microbial Limit vial’s sensor detects production of CO2 by microorganisms, based upon the principle that CO2 is a universal metabolite produced by all microorganisms. 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 bacterial metabolism in the liquid medium diffuses into the sensor and interacts with an indicator reagent to provide an indication of the presence of the carbon dioxide.

Applications:

The Microbial Limit vial is used to test primarily Personal Care, Cosmetic and over the counter Pharmaceutical (OTC) products for microbial content (contamination). Each of these types of products may have preservatives in their composition and the Microbial Limit vial helps to neutralize the inhibition of microbial growth that many preservatives provide. Neutralization of the preservative allows for a proper evaluation of whether or not the product has contaminants. Often the contaminating bacteria in the product while in the presence of the preservative remain “injured” and unable to replicate. The lack of replication might be interpreted as the lack of contamination.

The first step of the assay is to perform a 1:10 dilution of the product in neutralizing broth such as D/E (Dey/Engley) broth, Letheen Broth, or TAT (Tryptone-Azolectin-Tween) Broth. There after 1.0-0.1 ml of the sample is added to the Microbial Limit vial. The Microbial Limit vial contains the neutralizers that inhibit many common preservatives and this neutralization event helps the customer to correctly measure the presence of contaminating organisms.

Examples of Growth Curves Using the Microbial Limit Vial:

In the curves shown below in the Figure, there is an example of both a positive curve and a negative curve. The bacterium used was Pseudomonas aeruginosa.

The BioLumix Microbial Limit vial was specifically designed to be used in complying with USP. Due to the fact the Microbial Limit vial has both Lecithin and Tween in its media composition helps allow for neutralization of the preservative in the sample to be maintained during the assay for viable organisms. Thus, this vial is useful to the customer that has already determined the amount of neutralizing buffer and its content of neutralizer to be used when the product sample is first prepared in diluent. Together the use of the correct neutralizer and the use of the BioLumix Microbial Limit vial helps ensure an accurate assay for the replicating organisms.

Table 1 summarizes the types of Products that customers test in the BioLumix Microbial Limit vial to measure the presence of organisms.

Summary:

The versatility of the BioLumix Microbial Limit vial includes the ability to support growth of most aerobic bacteria, many yeast and some mold organisms. In most cases YMC vial is used for the detection of yeast and molds. The BioLumix Microbial Limit vial can be used for determination of microbial content (contamination), for use in suitability studies that test whether a product can support growth of microorganisms, and in Preservative Efficacy Studies (PET analysis) that is used for cosmetic products. The BioLumix Microbial Limit vial can also be used by customers whose products include Dietary Supplements and Nutraceutical products for which preservatives (natural or chemical) are also added. Supplement products with natural preservatives also need to be neutralized and tested for their ability to support microbial growth.

Rapid Microbiological Testing of E. coli with the BioLumix Vial

Escherichia coli are Gram negative rod-shaped organisms found naturally in the lower intestines of warm blooded organisms. Most serotypes of this organism are relatively harmless, making up a small percentage of bacterial colonization in the gut. These serotypes prevent the establishment of pathogenic strains.

E. coli is one of the most common bacteria found in the gut of animals. This includes humans. There are other animal species that contain this organism in the gut including reptiles and fish. E. coli colonizes the gut and can cause infection in the urinary tract and brain stem (meningitis) as well as intestinal diseases referred to as gastroenteritis. There are five classes of E. coli that produce disease. The most serious disease is the Enterohemorrhagic (EHEC) class. These organisms can cause diarrhea distinct from some others (including Shigella) in that there is copious bloody discharge and no fever. The life threatening situation is its toxic effects on the kidneys (hemolytic uremia).

Why Test for the Presence of E. coli?

E. coli is often used as indicator organisms to test the effectiveness of effluent disinfection in a wastewater treatment plant, on animal products as well as in nutraceutical and pharmaceutical products. While these organisms are generally harmless, they do live under the same conditions that human pathogens live. Since we cannot test for every pathogen, we test for easily detectable indicator organisms. The assumption is that if we kill the indicator organisms then we most likely kill the pathogens during effluent disinfection. E. coli has reemerged as an indicator, partly facilitated by the introduction of newer methods that can rapidly identify E. coli.

Current Methodology

The current methodology can take anywhere from 3-7 days and includes multiple broths, agars, transfers and temperatures. Different methodologies are utilized by the various industries. Below are some examples:

Nutraceutical and Dietary supplements: The protocol described in USP <2022> requires absence of the organisms in 10 grams of product. A 1:10 dilution of product is made into TSB or another enrichment medium. This mixture is incubated for 24 to 48 hours at 30°C to 35°C, and then 1.0 mL is transferred to 10 mL of MacConkey broth. This mixture is incubated for 24 to 48 hours at 42°C to 44°C. A loop from the MacConkey broth is transferred to MacConkey agar and the plate is incubated for 18 to 24 hours at 30°C to 35°C. If typical colonies appear, these colonies are then transferred to Levine Eosin Methylene Blue agar and incubated 24 to 48 hours at 30°C to 35°C. If none of the colonies exhibit green metallic sheen under reflected light or if none of the colonies exhibit a blue-black appearance under transmitted light, the sample meets the requirement for the absence of Escherichia coli. Because results can sometimes be misread due to interpretation of plates, identification may be run on the sample(s) adding another 2-3 days of testing.

Pharmaceutical: The protocol described in USP <62> again requires the absence of E. coli in 10 grams of product. A 1:10 dilution is made and incubated for 18 to 24 hours at 30°C to 35°C. From this mixture, 1.0 mL is transferred to 100 mL of MacConkey broth and incubated 24 to 48 hours at 42°C to 44°C. A loop is transferred to MacConkey agar and this plate is incubated 18 to 72 hours at 30°C to 35°C. Growth of typical colonies indicates the presence of E. coli which is confirmed with an identification test.

Food Testing: The food industry follows Bacteriological Analytical Manual (BAM). In most cases, the Most Probable Number (MPN) method is utilized. This is labor intensive, multi-step assay consists of presumptive (in LST tubes), confirmed (in BGLB tubes), and completed phases (in EC tubes). In the assay, serial dilutions of a sample are inoculated into broth media. Analysts score the number of gas positive (fermentation of lactose) tubes, from which the other 2 phases of the assay are performed and then uses the combinations of positive results to consult a statistical table. From this table, the analyst is able to estimate the number of organisms present. The 3-tube MPN test is used for testing most foods. The 5-tube MPN is used for water, shellfish and shellfish harvest water testing, and there is also a 10-tube MPN method that is used to test bottled water or samples that are not expected to be highly contaminated. Positive EC tubes must be transferred to L-EMB agar and if typical colonies are observed, they must be further identified.

The BioLumix E. coli Vial

The E. coli vial, or EC vial, is a membrane vial that is monitor by the fluorescent signal in the instrument. The membrane separates the incubation zone from the reading zone. If E. coli is present, it utilizes MUG (4-Methylumbelliferyl-3-D-Glucuronide) through an enzymatic reaction to create fluorescence. A 1:10 dilution is made in TSB or a similar growth medium and this mixture is incubated for 18-24 hours at 35°C. From the TSB dilution, 0.1 mL is added to an EC vial containing the MUG supplement and then tested in the BioLumix instrument. The EC assay runs for 18 hours in the instrument. An example of E. coli curves is shown in the graph. If detection occurs, a confirmation test is performed. From the time the sample is prepared to the time the confirmation is complete, the EC test takes only 2 days, saving valuable time.

The BioLumix assay is much simpler to perform, requiring less labor and disposables as any to the standard method. It is faster (completed in 30 hours), accurate, automated, and it eliminates any product interference due to the two zone vial.