Importance of Bacterial Identification

What's the point?

A rose by any other name would smell as sweet so it doesn't matter what the bug is called that is causing the catastrophic loss of electrolytes and fluid from your gut, you are just as dead at the end of the day. Surely the only really important piece of information is the name of the antibiotic which will stop it.

However, practically speaking, there's a wealth of information contained within a name which is helpful in treating disease or whatever it is that you are engaged with. The name, for example, can tell us how the bug is likely to be transmitted;

how the disease may develop and give forewarning of any complications.

A name is, in fact, shorthand for everything we know about that particular organism. So while knowledge of antibiotic sensitivity is near the top of the list in things we need to know in order to treat many diseases, names are far more important in the long run.

What name?

Having decided that we need to name our microbe, the next obvious question is which name? Well actually we can give it any old name just so long as everyone who deals with this microbe uses the same name to represent the particular package. After all, an elephant is still an elephant even if it’s called a large, thick skinned, big-eared grey mammal with a long nose (but in Latin).

In fact, many years ago before a Swedish Botanist called Linnaeus tried to impose some order on things, this is precisely what used to happen. The problem with this was that there was no system to it. Names were largely fanciful and there was no requirement or necessity to indicate that the animals described as tigers and lions were, in fact, related, closely or otherwise.

So along comes Linnaeus who dedicates his life to imposing order on disorder and inventing a logical system for naming all forms of life. This was a monumental task and the fact that we are now uncovering a large number of problems and inconsistencies in his life’s work should not be allowed to detract us from recognising his achievement.

Linnaeus recognised that some forms of life are more or less closely related and used this to create a hierarchical structure of groups. At the very top he placed his Kingdoms. Linnaeus recognised just two Kingdoms, the plants and the animals. These days we recognise 5 of them, although with taxonomists being naturally argumentative sorts this is considerably debated. Within each Kingdom are a number of Phyla. Within each Phyla there are a number of Classes and so on through Order, Family and Genus down to the lowest level which is Species. Whereas there are many hundreds of thousands of animal species, there are fewer families and even fewer classes. The whole system adopts the shape of a pyramid with Kingdom at the top. In order to give an idea of how it works it might be useful to look at the classification of some familiar animals including a household cat such as my moggy Stavros.

So Stavros is a Felis domestica. However, within this species there is enormous variation as anyone who knows even a little about cats realises. This, in fact, is true of all species including those belonging to the other Kingdoms. Whereas this is obvious in the case of animals and plants, it is less so with bacteria. Consider two humans (Homo sapiens) who are not identical twins. It is almost invariably true that they can be distinguished by any other human. This is because we are highly conditioned to identify individuals based on a summation of their features. Evolution has made us exquisitely sensitive in this respect and for the same reason our ability to distinguish differences within other species is reduced.

Consider, for example, how much more difficult it is to distinguish between two chimpanzees or two giraffes. It is not impossible to make these distinctions and becomes easier the more familiar we become with them. However, as the number of distinguishing features becomes less obvious to our eyes, identification of individuals gets increasingly difficult. How similar do two houseflies or two earthworms appear? This is not to say that there are not a vast number of features which might be used, and probably are by other houseflies and earthworms, it’s just that we do not readily know what they are.

Taxonomy

Of course Linnaeus’ system proved enormously popular because it attempted to impose order on chaos and everyone knew that order was good. However, it quickly became apparent that the Laws of Thermodynamics applied just as much to theoretical concepts as the physical world and an enormous amount of energy was expended on classifying life. The science of taxonomy was born and provided useful, or useless depending on your point of view, employment for lots of pedants for lots of years.

Taxonomists, as they became called, observed the physical, and sometimes chemical, properties and composition of their subjects, compared them with other more or less similar organisms, and made decisions about their relatedness. What quickly became a problem was that these decisions were of necessity entirely arbitrary. Taxonomists, it appears, were called upon and seemed perfectly happy to, decide on the relative importance of different features on the most specious of grounds. For example, is a thick skin a more or less important trait than the length of the proboscis? Clearly taxonomists think so because both rhinos and elephants belong to the Family called Pachyderma. But who is to say they are correct? At every turn such important decisions had to be made so it’s no wonder that many have been brought into question. Often more than once! Imagine the upheaval.

So What About Bacteria?

What is significant?

Now consider bacteria which appear as small spheres, rods or squiggly things down a microscope. Microbiologists have spent many years searching for these distinguishing features and have come up with a vast array ranging from single chemical reactions through to whole metabolic pathways; whole-cell protein profiles; surface antigens; staining reactions. The list is extensive but the problem is that we have little to no understanding of the taxonomic significance of the features we chose to distinguish not only between species, but even between individuals with a species. Actually, to some extent, this is true of all the Kingdoms. What there does seem to be agreement on is that within a species, including bacteria, there is large variation. We humans, acknowledge this variation by ascribing names to each other so that we can agree on the identity of individuals.

Strains

In essence we are identifying strains. So, for example, there is a Homo sapiens var John Smith. To a large extent, the same is true of microorganisms. The difference being is that often we do not have direct knowledge of the nature of the difference. Because we can not discover the difference does not mean that we can assume no difference. So unless we know for a fact that two isolates are derived from the same cell, ie they are clones, we have to assume they are different. Adopting the same principle we use to recognise different humans, different isolates of a microbial species are also given strain names. These often reflect the name of the person who isolated the microorganism, its source, or something which happens to take the fancy of the microbiologist. Hence Streptococcus sanguis NJH1 was isolated from my daughter Nicola Jane.

Bacterial identification

Linnaeus knew little of microorganisms and they didn’t fit into his original scheme of things. Later a new Kingdom, the Monera, was created especially for the bacteria. Fungi used to be in the plant Kingdom but now they have their own and as for viruses, there is still a healthy debate about whether they are actually alive. Almost as soon as bacteria were discovered attempts were made to classify them but it’s easy to appreciate the special problems posed by trying this on something you can barely see down the microscope. All the early bacterial taxonomists had to go on was the Gram stain and the gross morphology (shape) of the cells. Hence we had genera such as Streptococcus (cocci in chains), Staphylococcus (cocci in bunches) and Bacillus (rods). Clearly there were big differences between bacteria which were all Gram-positive rods, or whatever, but the problem was how to tell them apart.

It's a bit like cookery

What developed were numerous, widely different, sometimes highly creative but essentially pragmatic schemes based on a variety of biochemical, chemical and serological reactions. Just like in cookery, schemes (recipes) were tried and discarded until one was found which worked (tasted good). However, the same problem which dogged the classification of plants and animals was present with bacteria. That is, how much importance do you attach to particular characteristics when you try and understand taxonomic relationships? Numerical taxonomy tries to address this issue.

Phenotypic variation

So, the first problem in arriving at a scheme for identifying bacteria, or a plant or animal for that matter, was which characteristics do you use and how much relative importance do you place on each? Even now this is a topic of much argument. There was, however, another problem which was not immediately apparent and was more practical in nature. In all organisms every cell contains a complete copy of the genome but it has been realised for some time that not all of it is functional all of the time. Indeed in cells which have become specialised large amounts of the information contained in the genome is entirely redundant. This is clearly an efficient means of managing the cell but it goes much further because it shows that the genome is under control and genes can be switched off and on as required. After all, why make a group of proteins whose function is to transport the sugar galactose across a cell membrane when there is no galactose present? Before we stray too far from the point, it was soon realised that bacteria switched genes off and on as well, even though they are single cells. They select a repertoire of genes from their genome which are best suited to their current environment and situation.

Phenotype and identification

So what has all this to do with problems of identification? The answer is that if part of your identification scheme tests for the ability of the bacteria to ferment galactose, for example, then you have to ensure that the conditions in which you are growing the bacteria are conducive to the relevant genes being switched on, or expressed in the current jargon. You will then get the definitive answer to the question but what if the chap who devised the scheme you are using didn’t culture the bacteria in exactly the same way and got a different answer? It isn’t important from the point of view of identification whether or not the bacteria can or can not ferment galactose. What’s important is that you do the test in exactly the same way as the person who devised the scheme.

This problem of gene activation applies right across the board of tests used whether they are aimed at identifying a particular antigen on the cell surface, the ability to ferment any range of sugars you care to name or any one of a thousand other feats. This is because classical bacterial identification and, indeed, most current taxonomy, is based on phenotypic traits. That is to say the particular mix of genes currently active in the organism. This normally requires that the bacterium of interest has to be first isolated and then grown in pure culture, a process which is both time consuming and expensive in materials and manpower.

Genotype

The alternative, and one which is theoretically better in that it avoids making assumptions, is based on the analysis of either the cell’s genome or its mitochondrial or ribosomal nucleic acid. These studies are starting to reveal some quite startling relationships and some long-held notions are having to be thrown out of the window. It is even proving possible, through the use of nucleic acid amplification techniques (PCR) to analyse the nucleic acid of tiny fragments of tissue or even single cells to reveal taxonomic data about extinct species (the Dodo is a close relative of the common pigeon) or bacteria which we are, as yet, unable to cultivate. But these techniques offer even more because they do not need the bacteria to be in pure culture which cuts out the time delay required for isolation and cultivation and with it much of the expense. The gain in speed of identification is quite remarkable and techniques are being developed to package the necessary molecular reactions on the tips of small probes which could be used in the field, ward or surgery to give a result within minutes rather than days.

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