The significance and detection of VBNC microorganisms

The purpose of this article is to assess the potential significance of Viable but Non Culturable (VBNC) microorganisms in the pharmaceutical industry; consideration is given to the definition of the VBNC state, current methods for the detection of such organisms are outlined and potentially significant new methods, which may impact the industry, are considered. The article will also discuss the future significance of VBNC organisms within pharmaceutical microbiology and its ongoing development.

Microorganisms are very tenacious life forms capable of surviving in some very extreme environments.They normally exist as vegetative cells but some species have the ability to produce highly resistant structures known as ‘spores’. Spores are a well known method of helping microorganisms survive adverse conditions as they are able to protect a dormant organism until conditions are right for growth to occur. Not all microorganisms are able to sporulate; however, there is an alternative utilised by some microorganisms which falls somewhere between the vegetative state and spores; known as ‘Viable but Non Culturable’ (VBNC). The VBNC state is useful to organisms unable to sporolate because it offers greater protection from environmental threats than the vegetative state; helping to increase chances of survival. The existence of bacteria which survive in the VBNC state is now well established and documented (Calwell et al 1985, McDougald et al 1999). The state of VBNC gained its name because the organisms in question are still viable but lose the ability to be grown on media. This presents a problem to microbiologists because many methods used today for enumeration or identification rely on growing the organism in question. Pharmaceutical microbiology is no exception; finished product testing such as Microbial Limit Testing of non-sterile products and Sterility testing of sterile formulations rely on growth-based methods. Water evaluation, raw material testing, environmental monitoring of air, surfaces and personnel all rely heavily on growth based methods. Much of the investigative work has centred on organisms from the natural environment, particularly aquatic organisms. Little information has been produced on the potential impact of VBNC organisms from industrial environments or processes; this applies in particular to the potential impact of VBNC organisms in the pharmaceutical industry. It is therefore important to consider the potential significance of VBNC organisms within pharmaceutical manufacture and production processes.

The significance of VBNC in the pharmaceutical sector

Many current Microbiological methods in the pharmaceutical sector include growth-dependant techniques such as agar plating, membrane filtration and broth enrichments. These methods represent the main tools used for the microbiological analysis of pharmaceutical input raw materials, water and finished products. Unfortunately these types of methods are wholly unsuitable for the recovery of organisms in the VBNC state; when using these techniques it is impossible to assess how representative VBNC organisms are in the pharmaceutical manufacturing environment and what sort of challenge they represent to the microbial quality of input materials, including water, and manufacturing processes. The significance, therefore, of VBNC organisms in the industry is subject to some debate. What is apparent, however, is that microbiological analytical methods will need to change if a true assessment of the significance of such organisms is to be made, and there is evidence that this is happening.

Areas of interest in pharmaceutical analysis include:

• Raw material testing
• Water analysis
• In-process bioburden testing
• Environmental monitoring
• Finished product testing

Maintaining the patient safety of pharmaceutical products is of paramount importance; microorganisms are a major source of risk to patient safety; impacting significantly upon it in two main ways;

1. Infection – occuring when an organism is present in sufficiently high numbers to become pathogenic to patients. The level at which disease is triggered depends on the type of microorganism and is usually high. However, for some organisms, such as Salmonella , the infection level is low; this is because the organism is capable of replicating to pathogenic levels inside a person once ingested.
2. Intoxication – the production of toxins by a microorganism which has the potential to cause illness. The toxin can survive even the elimination of the organism which produced it.

VBNC organisms include pathogenic organisms which retain pathogenicity during the VBNC state (Pommepuy et al 1996). (Refer to table 1 for some species known to be capable of becoming VBNC). VBNC organisms include both infectious and intoxicating species; does this indicate that there is a potential risk to pharmaceutical products and ultimately patients? The answer is probably not; infectious organisms will be reduced or eliminated by processes such as heat, filtration and use of antimicrobials. Typical bioburden levels present in pharmaceutical ingredients are low. The ability of VBNC organisms to replicate is limited by the VBNC state. Therefore, organisms in the VBNC state will have great difficulty in multiplying from such low numbers to pathogenic levels because of their limited ability to replicate. In addition, microbial toxins will be denatured due to the aggressive nature of many manufacture processes; thereby reducing the risk to patients of disease from intoxication. Overall, the risk in terms of patient safety from organisms in the VBNC state is low.

Many future advances in pharmaceutical manufacturing will come from greater characterisation of processes. This understanding will be the result of superior data, using more sensitive analytical techniques capable of giving results in real time, or near real time. Inevitably this superior process understanding will lead to better process control. Process control and characterisation of critical parameters is an area where the detection of VBNC organisms may have a significant impact. With the implementation of more appropriate microorganism detection methods it will be possible to understand the types and actual numbers of organisms present in starting materials. It will also be possible to determine more representative in-process controls for bioburden levels; setting appropriate action or alert levels will allow closer control. Microbiological control of pharmaceutical processes relies heavily on an understanding of process parameters; a greater understanding of the type of microbiological challenges presented to a process may help towards a greater understanding of the whole process. The ability to assess VBNC organisms may also have a contribution to the development of improved understanding of current and future manufacturing processes. This understanding is obtained through process simulation trials, equipment validation, revalidation control and maintenance. Due to time demands and the limitations of methods already outlined, process control cannot currently rely on microbiological monitoring. In the future, real time microbiological data may actively contribute toward true process control. Obtaining a more accurate picture of the total numbers of microorganisms present is a process which will require more sensitive and accurate information. Better process understanding will lead to improvements in process control, ultimately leading to greater process assurance.

Water testing

Water is a major input raw material of many pharmaceutical processes; VBNC organisms are widely found in the aquatic environment. This water is treated in various ways to reduce bioburden by filtration, distillation, heating, and use of hypochlorite etc. all of which can induce the VBNC state in environmental microorganisms, some of which are pathogenic. However, it is important that a true picture of the actual bioburden is obtained. Current methods may fail to give an accurate picture of total bioburden levels either because they lack sufficient sensitivity or worse, because they are wholly unsuitable for the job. Failure to detect VBNC bacteria due to inappropriate methodologies could therefore have potentially serious effects on pharmaceutical product quality.

Environmental monitoring

Environmental monitoring is an important role performed by pharmaceutical microbiologists; data is generated illustrating the microbiological quality of air, gases, manufacturing surfaces, facilities and operators. This information gives a snapshot of the microbiological control of facilities both at rest and during manufacture. It is important that accurate information is generated in a timely manner. Failure to detect a significant portion of environmental microorganisms because of their morphological status must be considered in this context. Better understanding of the underlying microbiological situation through detection of VBNC organisms could have potentially far reaching consequences. Use of new technologies with increased sensitivity, accuracy and speed may profoundly change current alert and action limits, environmental methods and how ultimately microbiological data are used. Microbiological monitoring can then contribute to real time process control. In the future, superior characterised processes could even reduce the need for the sterility test in all sterile manufactured products for both terminally sterilised and aseptic processes. Currently this approach can only be considered for terminally sterilised products due to greater process characterisation.

Advantages of VBNC

VBNC is a survival strategy employed by a wide range of gram-negative heterotrophic bacteria and also by many nonsporulating gram-positive and gram-variable bacteria (see Table 1). It appears to be a genetically inducible state (Oliver 2000). VBNC is an advantage to organisms living in changing environments such as the aquatic environment where sudden fluctuations in a range of conditions could potentially threaten survival. The ability to rapidly respond to changing conditions offers obvious evolutionary benefits. Environmental conditions seen to trigger the VBNC state include; fluctuating temperature, nutrient levels, salinity, age, oxygen levels and light levels Which are in fact very similar to many conditions found during pharmaceutical manufacture. The VBNC response to changes in environmental conditions is now being considered as a possible explanation as to why different species dominate the same geographical location at different times of the year (Oliver 2000).

VBNC associated morphological and physiological changes

Generally microorganisms showing enhanced capacity for survival in nutrient limiting environments show reduction in size and lower metabolic rates. In Vibrio spp this reduction in size has been described as ‘rounding up’. Reduction in size from 2.27 to 2.14 µm for the enteric bacterium Escherichia coli has been described. In nutrient depleted aquatic environments microbial isolates have been shown to be smaller than in nutrient rich environments. Bacteria from seawater have been shown to be able of passing through 0.45mm filters (Openheim 1952). Bacteria capable of passing through 0.2µm filters have been termed ‘ultramicrobacteria’ (Tabor et al 1981). Responses such as these have considerable implications for pharmaceutical processes. Filters comprising of nominal pore sizes of 0.4 or 0.2µm are routinely used as sterilising grade filters. Aquatic ultramicrobacteria in the VBNC state with the capacity to pass through such filters could pose a threat to some pharmaceutical processes and could ultimately compromise the microbiological integrity of some product types if no further antimicrobial treatments are used .

Hypochlorite has been shown to induce the VBNC state in Legionella pneumophila ( Bej et al 1991), and Escherichia coli (Singh et al 1986). This observation has some significance to the pharmaceutical sector; hypochlorite is widely used by the industry for its sporicidal properties; it is used to decontaminate plant and water systems. Legionella can be difficult to eradicate completely once a water system has been contaminated; induction of a more resistant physiological state by an agent used to sanitise water systems may help partly to resolve this colonisation.

Methods of detection

Conventional microbiological culture methods using non-selective, selective, differential agars or broth enrichment are not suitable for the recovery of microorganisms in the VBNC state. Methods which are either currently used or which show potential include:

• Direct counting methods
• Dye reduction testes
• Immunological -based methods
• Genetic methods
• ATP bioluminescence
• Fluorescent labelling methods

Direct counting methods

A previously common method for the detection of VBNC organisms was the acridine orange direct count (AODC) (Daley & Hobbie 1975) ; in this test acridine orange (AO) was used to differentiate between viable and non-viable organisms. Monomers of AO produce green fluorescence , whilst dimers of AO produce red/orange fluorescence. This differential fluorescence was used to differentiate between viable and non-viable forms of microorganisms. It was assumed monomers were formed with double stranded DNA and dimers were formed with RNA. Unfortunately this differential reaction was not always a reliable indicator of viability.

A commonly used direct counting method is the direct viable count (DVC) developed by Kogure (Kogure et al 1979). In this method, yeast extract is added to microbial populations along with nalidixic acid. The nalidixic acid prevents DNA replication or cell division, the yeast extract promotes protein synthesis. VBNC organisms respond to the yeast extract but are unable to replicate; these cells become elongated, whilst non-viable organisms remain unchanged. The VBNC cells can be detected by direct microscopic observation. Unfortunately, Gram positive bacteria are generally resistant to nalidixic acid; In addition, the method takes 6 hours or more to produce a result.

Dye reduction tests

The DVC test has been adapted by the addition of soluble p-iodonitrotetrazolium violet (INT). Reduction of the soluble INT by dehydrogenase activity leads to the deposition of insoluble INT-formazan (Zimmerman et al 1978). This method is suitable for detecting viability in all microorganisms possessing an active electron transport system. The method is also significantly faster than the DVC method taking approximately 1 hour to produce a result.

5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is a newer dye reduction method (Rodriquez et al 1992). Reduction of colourless CTC by electron transport activity produces CTC-formazan which fluoresces red and accumulates intracellularly. It is not yet known if this method is suitable for all types of bacteria.

Immunological methods

Monoclonal antibodies combined with DVC may be a promising method for the investigation of known bacterial species in the VBNC state. Monoclonal antibodies must be raised to the species under investigation; the antibodies are then tagged with a fluorescent marker (Xu et al 1982, Grimes & Colwell 1986). The technique has some limitations when looking at mixed microbial populations which is the norm in most environments.

Genetic-based methods

The polimerase chain reaction (PCR) has been used to detect organisms in the VBNC state including Vibrio cholerae and Shigella dysenteriae ( Koch et al 1993, Islam et al 1993). This technique amplifies small amounts of DNA from a target bacterium to detectable levels without the need for cell replication. The use of this technique is increasing and it will develop into a useful way of detecting organisms in the VBNC state. Although used successfully in other industrial sectors, routine use of this technique is not yet widespread in the pharmaceutical sector. It would appear to be a very suitable method for the detection of VBNC organisms. However, a review by Oliver has identified potential difficulties in applying PCR to organisms in the VBNC state (Oliver 1993). Extraction of 400 times more DNA was required from cells of V.vulnificus in the VBNC state for successful application of the PCR technique than from organisms in the culturable state. Some changes to the genetic material of organisms entering the VBNC state are possible. A number of studies have reported such modifications to DNA structure (Linder & Oliver 1989 , Brauns et al 1991). These studies indicate condensation of the nuclear region. Changes such as these could interfere with the effectiveness of the PCR technique.

ATP bioluminescence

All living cells possess adenosine triphosphate (ATP) as the main chemical energy store. This includes organisms in the VBNC state. ATP levels in VBNC Escherichia coli have been shown to initially drop then rise when exposed to 0.8M NaCl (Roth et al 1988). In fact, within 4 hours ATP levels were higher than before entering the VBNC state. VBNC organisms do appear to contain ATP.

ATP bioluminescence-based systems are currently being developed for a number of industrial sectors for the rapid detection of microbial biomass including the pharmaceutical sector for the rapid analysis of water, and non-sterile product testing. As VBNC organisms and culturable organisms both contain ATP, bioluminescent-based methods may be a useful way to rapidly detect total biomass.

Cytometry

Another area of significance to the detection of VBNC bacteria is Laser Scanning Cytometry (LSC).
LSC, also known as solid phase laser cytometry, couples membrane filtration, direct viability staining (epifluorescence) and laser scanning. Chemunex S.A. (Paris, France) have developed the scan RDI®.which utilises LSC in a semi-automated technology. One draw back to the technique is that it is only suitable for filterable materials. However, a distinct advantage of the technique is the sensitivity of 1 CFU per sample with a result in less than 2 hours. The sample is filtered, labelled and then laser scanned. Labelling involves the passive diffusion of a non-fluorescent substrate derived from fluorescein (Flurassure®). Work has shown that in some grades of water scan RDI® detects higher numbers of microorganisms than conventional microbiological methods (Wallner et al 1997).

Flow cytometery is another technique useful for detecting VBNC organisms which uses digital electronics to detect and enumerate viable microorganisms. After labelling with a viability stain, cells pass individually through a laser excitation beam where a sensitive photomultiplier is used for detection. Discrimination parameters are used to differentiate between labelled, viable organisms and autofluorescing particles. The current sensitivity of the systems is in the region of 50 CFU/ml. Results are expressed as direct counts per volume and can be stored on a PC for traceability. Flow cytometry based systems are currently being used to test a range of complex sample matrices such as toothpaste and ointments. Again, like the scan RDI®, microbial detection using flow cytometry does not require culturing of cells and so may be suitable for rapid, possibly real time enumeration of contaminating microorganisms including VBNC organisms.

Conclusion

Microbiologists have long recognised that there are significant numbers of microorganisms in the environment that cannot be cultured successfully with conventional methods. A wide range of nonsporulating Gram negative and Gram positive bacteria can exist in the Viable but Non Culturable state. The VBNC state can be regarded as a survival strategywhich employs enhanced resistance to combat adverse conditions. Pathogenicity is maintained by some species during the VBNC state.; this could be an indication that such organisms pose a potential threat to human health.

The true significance of VBNC organisms to the pharmaceutical industry is only just beginning to emerge. The real risk to patients from undetected VBNC organisms in pharmaceutical products is limited. This is due to the range of antimicrobial processes involved in pharmaceutical manufacturing. However, the real significance of VBNC is likely to be in helping us to understand more about current and future industrial processes. If we can get more information about true levels of viable organism in raw materials, water and the manufacturing environment we are a great deal closer to gaining a greater understanding of manufacturing processes. Emerging alternative technologies may help play a role in significantly improving the quality of pharmaceutical products in the future by increasing our process knowledge. New methods of enumeration and identification with the potential for detecting VBNC organisms may bring about a radical reappraisal of bioburden limits, environmental monitoring techniques and limits. These methods could even drastically reduce the duration of the sterility test and could even remove the need for such a test due to improved process characterisation.

Introduction of new detection methods not requiring cell culture has raised the level of interest in VBNC organisms. The significance of such organisms is beginning to be considered. The full impact of VBNC bacteria on industrial processes has not been given consideration due in part to the widespread use of conventional culture methods incapable of detecting such organisms. There is an emergence of new methods and technologies in pharmaceutical microbiology and other sectors, and these new methods have the potential to detect VBNC organisms. Interest in the significance of such organisms is growing and will continue to do so; a greater understanding of VBNC organisms and their significance in pharmaceutical processes could therefore have profound effect on the way in which pharmaceutical microbiology is conducted in the future.

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