In this continuing series looking at microbial control of water systems, the importance of species characteristics is considered.
Assimilable organic carbon
Generally, the bacteria present in pharmaceutical and healthcare water systems are heterotrophs, which means they need to utilise organic compounds as their carbon and energy source.
As addressed previously 2, in both drinking water and applications like pharmaceutical and healthcare water systems, Total Organic Carbon (TOC) is the general measure beloved by chemists, assessed using a combustion catalytic oxidation/nondispersive infrared analysis method. ‘TOC’ refers to the total amount of carbon found in organic compounds within a substance, serving as a key indicator of water quality and purity. A more focused measure is sometimes used - Dissolved Organic Carbon (DOC), a fraction of which is bioavailable 3. Where TOC refers to the total amount of organic carbon in a water sample, DOC is the fraction of organic carbon that passes through a 0.45-μm filter.
DOC represents the organic carbon dissolved in the water, not associated with larger particles or suspended solids. Yet is DOC a reliable predictor of the potential for bacterial growth in water? Several research strands suggest not. A better predictor is Assimilable Organic Carbon (AOC). AOC specifically refers to the fraction of organic carbon that is readily utilisable by microorganisms (the small molecules that can immediately be used by microbes). AOC includes readily biodegradable molecules like sugars, organic acids and amino acids 4.
Will growth be supported?
The degree to which a water system can potentially support the growth of a microorganism recovered from a water sample can be an important consideration. With any sample taken, there exists the potential for it to not be from the actual water system - hence a source of error.
For example:
- Contamination from sample tubing
- Contamination from water residing in a valve
- Splashback from a sink
- Cross-contamination from the person sampling
- Damaged sampling container
- Testing error
The distribution of microorganisms in water does not conform to normal distribution. Instead, it is invariably a negative binomial distribution 5. In the past, Poisson distribution was considered the typical pattern in water but bacteria in water and the water itself are insufficiently homogeneous.
This means a situation of pass-fail-pass-pass is never sufficient to be confident that the water system is again satisfactory (recording ten ‘clear’ samples over ten days would give a higher degree of confidence). As well as the issues of distribution and sample size, if a biofilm is present the release of contamination through sloughing with be inconsistent, erratic and not confirming to a regular temporal pattern 6. Bacterial distribution is also influenced by:
- Planktonic: bacteria that are free-floating in the water column
- Particle-associated: bacteria attached to suspended particles in the water
- Deposits: bacteria within loose sediments and deposits
Growth potential
Research suggests that where organisms truly originate from water (and given the pharmaceutical context, recoveries are low) the type of species affects the propensity of growth. Hence, researchers sometimes talk of the growth potential. This is a marginal assessment, beginning with whether a given species can survive within a low-nutrient aquatic environment. To assess this, AOC is a useful measure.
However, AOC is not absolute. While levels at or above 100 μg liter−1 are considered to have the greatest growth potentials (7), since AOC contains a heterogenous mix of amino acids, sugars and fatty acids, this does not mean that the proportions are correct to enable growth of one species over another.
Since the composition of AOC is distinct to a given water system, the ‘fingerprint’ of utilisable organic carbon compounds is important (8). This is significant because the spectrum of growth-supporting substrates (carbon compounds) of individual bacterial strains is specific. Therefore, characterising a species and profiling it against the AOC composition can play a significant role in understanding the growth potential of contamination (9).
For example, considering AOC and three organisms - Escherichia coli O157, Vibrio cholerae, and Pseudomonas aeruginosa, researchers from the Swiss Federal Institute of Aquatic Science and Technology considered the concentration of cells produced (as measured by flow cytometry) and the fraction of AOC consumed by each organism. The data suggests that bacterial growth is affected by both the concentration (numbers of cells) and by the composition of AOC.
It should be noted that other factors will affect survival, including osmotic pressure and the effectiveness of disinfection treatment.
Summary
This article has demonstrated that the variance influencing microbial survival are complex and a holistic approach is required. What is of interest is the variability associated with distinct species. Identification, coupled with an assessment of AOC, play supporting roles in the pharmaceutical organisation’s Contamination Control Strategy.
