5D Health Protection Group Ltd

Global Microbiology Testing Services

5D Health Protection offer microbiology and antibiofilm testing services. We work closely with our customers to fully understand their requirements, offering both standardised and customised methods to suit all their testing needs. We generate the scientific data that is necessary for marketing support, innovations and regulatory submissions of products for CE mark, 510(k), pre-IND and de novo applications.

Our certified contract microbiology testing services are being utilised by many start-ups, SME and global multinational customers in many different industries when they need to ensure that the microbiological quality of their products, processes and services are being maintained to the highest standard to protect public health.

Fundamental Microbiology Testing Services

Many industries require certified microbiology testing services in order to protect products, processes as well as human health. The colonisation and proliferation of microbes on and in materials and on surfaces represents a major concern to public health. These problematic microbes include bacteria, fungi, yeasts and viruses. There are many microbes which are well known to cause contamination of products during and after production and, in healthcare, represent a high risk to human health. Our leading independent world-class certified microbiology testing services are being utilised by many different industries including, as examples, woundcare, medical devices, cosmetics, drinking water and healthcare.

Our microbiology testing services are employed routinely to evaluate the efficacy of antimicrobial based liquids, gels, wipes, wound dressings, medical devices and catheter coatings.

Microbiological Testing and Methods

Microbial Bioburden Testing

Microbial bioburden testing is an important component for monitoring raw materials and control of manufacturing processes. Also, it is important to evaluate the microbial bioburden of both non-sterile and sterile medical devices. The microbial bioburden is used to describe the numbers of microorganisms that may be present in a product. There are many ways that microorganisms may get into a product or a technology particularly during development, from the environment, contaminating raw materials and during manufacturing and packaging products. By monitoring the microbial load or burden in a product this will help to determine if there are any issues with a manufacturers process and help to determine it sterilisation techniques are effective. A sterile medical device has to be free of any viable microorganisms.

5D Bioburden Testing

At 5D we perform many different types of microbial bioburden tests. In particular we perform testing in accordance with BS EN ISO 11737-1:2018 a standard for the sterilisation of health care products which determines the population of microorganisms within a product.

At 5D we have a large amount of experience in evaluating the bioburden of many different types of devices. In the US for bioburden testing procedures such as USP <61> and USP <62> are undertaken. USP <61> is an enumeration test that quantifies microbes in raw materials or nonsterile products and USP <62> is a procedure undertaken to determine the presence or absence of specific microbes in a product. These may include Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella or Clostridium species.

For bioburden testing 5D apply a number of techniques that may include membrane filtration, standard plate counting, spread or pour plating, most probable number, direct enrichment or membrane filtration enrichment.

Log Reduction Assays

We carry out log reduction assays routinely at 5D on many different technologies and products. Log reduction assays are used to assess the antimicrobial effectiveness of wound dressings and antimicrobial impregnated medical devices.

We routinely evaluate the performance of antimicrobials such silver, iodine, PHMB, benzalkonium chloride, hypochlorous acid, acetic acid, peracetic acid, chlorine, hypochlorite and chlorhexidine based products. We have extensive experience testing and evaluating all wound dressings ranging from hydrogels to fibrous materials.

Total Viable Count (TVC)

The total number of microbes (aerobic or anaerobic bacteria, fungi, yeasts) with a sample is referred to as the total viable count (TVC). It is measured as the Cfu/ml. We use a wide range of different nonselective and selective media to isolate different microbes from a product or sample.

Zone of Inhibition (ZOI)

The zone of inhibition or Kirby-Bauer Test is used to evaluate the antibiotic sensitivity of bacteria. It is the circular area around an antibiotic disk in which bacteria do not grow. This test measures the susceptibility of the bacteria to the antibiotic i.e. susceptible, intermediate or resistant. This method can also be performed to evaluate the efficacy of other antimicrobials not just antibiotics.

Corrected Zone of Inhibition (cZOI)

The corrected zone of inhibition is often applied to evaluate the performance of antimicrobial based wound dressings. This method is similar to the ZOI but takes into account wound dressings of different shapes and sizes and takes into account the fact that some wound dressings shrink once they become hydrated.

The CZOI corrects any variances in the size of different materials or wound dressings.

Minimum Inhibitory Concentration (MIC)

The MIC is the lowest concentration of an antimicrobial which prevents or inhibits the visible growth of a bacteria. This is used to indicate if the antimicrobial is bacteriostatic (prevents the growth of bacteria).

MIC must be determined for different microorganisms as they will all have different values.

Minimum Bactericidal Concentration (MBC)

MBC is the minimum concentration of an antimicrobial that results in the death of bacteria. This assay helps to indicate the potency of an antimicrobial i.e. bactericidal ability. MBC is often referred to the minimum concentration of an antimicrobial to achieve a 3-log decrease (99.9%) in the microbial population.

MBC must be determined for different microorganisms as they will all have different values.

Minimum Biofilm Eradication Concentration (MBEC)

The MBEC of antimicrobial agents is a measure of in vitro antibiotic or antimicrobial susceptibility of biofilm producing microorganisms. The method is dependent on a number of variables including the surface, medium and the contact time to an antimicrobial agent.

At present there are no standardised measurement parameters for the MBEC and so these are presently research laboratory values that lack clinical validation.

Etest

Epsilometer testing (Etest) is an alternative to the MIC. Rather than using circular disks which have been impregnated with antibiotics there are commercially available plastic strips that contain a gradient of an antimicrobial. As the bacteria grows, the antibiotic from the plastic strips diffuse into an agar plate.

Microbial Limits Test (MLT)

This MLT is often referred to as the bioburden test. MLT is used to measure the total number of viable microorganisms on a medical device before final sterilisation or before being implanted.

Antimicrobial Challenge Testing/ Preservation Efficacy Test (PET)

A PET or Antimicrobial Effectiveness Test (AET) is required to assess the antimicrobial preservation of a product. We model the preservative challenge test in line with ISO 11930. This is a standard used to evaluate the antimicrobial protection of a cosmetic product.

The test involves firstly screening a product for microbial contamination. This test is also referred to as a total viable count (TVC), a total Yeast and Mould count (TYAMC) or a test that determines what the specific microbes are within the product.

Speed of Kill or Time-Kill Assays (Antimicrobial Efficacy Testing)

The Time-Kill assay is used to study the efficacy of an antimicrobial agent over a specified time period. The test can be used to determine whether an antimicrobial is bactericidal or bacteriostatic. Often an antimicrobial with bactericidal activity must generate a 3 log₁₀ decrease in colony forming units (cfu) which indicates a 99.9% kill. For speed of kill assays we can also employ the use of flow cytometry to provide a more accurate number of microorganisms that have been killed.

Antimicrobial Effectiveness Test

The antimicrobial preservative effectiveness test (United States Pharmacopeia-US 51P) is used to determine the antimicrobial effectiveness of products. In particular it is used to determine the effectiveness of an antimicrobial preservative. The test is undertaken over 28 days.

Microbial Identification

At 5D we utilise rapid methods for the identification of microorganisms which may be encountered in environmental, pharmaceutical or medical devices industries using different techniques. We are able to identify an vast array of different microorganisms using a combination of different methods, both traditional and molecular.

At present we utilise API systems, traditional microbiological identification techniques, matrix-assisted laser desorption/ionisation-Time OF Flight (MALDI-TOF) mass spectrometry (MS) and quantitative polymerase chain reaction (qPCR).

The use of MALDI-ToF enables us to identify microorganisms within minutes due to microorganisms producing a unique protein fingerprint. This fingerprint profile is correlated to a database of already identified microbes. As MALDI-TOF sometimes has limited reproducibility, which can therefore increase variability between laboratories, we routinely employ the use of PCR for the identification of microbes.

Antibiotic Resistance Testing (Frequency of Resistance Assay)

For antibiotic resistance studies bacteria can be evaluated by the use of either single or multi-step resistance studies. Both studies will help to predict the frequency of the formation of resistant mutants over a period of time. Such studies assist in determining the types of resistance strains that may develop in the clinical setting.

A spontaneous mutation frequency assay is used to determine the frequency at which mutants occur following the exposure to inhibitory concentrations of an antibiotic or an antimicrobial. This assay is used to develop resistance in bacteria by continuously passaging bacteria that are grown in sub-inhibitory concentrations of an antimicrobial agent.

The mutant prevention concentration (MPC) assay is used to determine the concentration of an antimicrobial that prevents bacteria from forming resistant colonies.

Bacterial Endotoxin (BET)/LAL Testing

The Bacterial Endotoxin (European Pharmacopoeia 8.8 (2016), 2.6.14. Bacterial Endotoxins) or Limulus Amoebocyte Lysate (LAL) test is an established pharmacopeia method for the screening of irrigation fluid, dialysis solutions, water, materials, products etc. It is often used to assess medical device safety which are make contact directly and/or indirectly to body fluids and surfaces. If there is evidence of endotoxins this indicates that there may have been microbial contamination of a material or medical device. If endotoxins are present in a sample this will potentially give rise to a febrile response. There are standard levels set down by regulatory bodies of acceptable levels of endotoxins in materials and products.

There are three standard methods that can be employed for testing for the evidence of endotoxins produced by bacteria. These include the gel clot method, the kinetic chromogenic method and the kinetic turbidimetric method. In addition to this test we also employ other test methods including the monocyte activation test.

Customised Microbiological Testing

In addition to the examples of microbiological and antibiofilm models, methods and assays mentioned above we also undertake other microbiological testing services which are customised for our customers in different industries. For example, these could include validating microbiology tests undertaken in scientific publications or comparing different products in bespoke models or validating different biocidal treatment methods. We are also often asked to reproduce assays and tests that have been published on scientific journals. Competitor products and evaluations of medical device prototypes has been an on-going requirement by all our customers. 5D represents an independent service to help validate a companies internal testing results.

To discuss more customised microbiology and scientific testing please contact us on info@5Dhpg.com or call on +44 (0) 151 794 9279.