|Adherence [under revision]|
|Aseptic - Antiseptic|
|Colony Forming Unit (CFU)|
|Flagella and fimbriae|
|Lancefield grouping of streptococci|
|Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS)|
|Nomenclature of bacteria|
|Simple stain techniques|
|VBNC (Viable but nonculturable)|
|Virulence and virulence factors|
Adherence [under revision]
IntroductionAdhesion in bacteriological contexts refers to the ability of bacteria to stick to surfaces (e.g. tissues). This ability can be more or less specific. For specific adhesion to take place, there must be a receptor on the surface and a ligand on the bacterium, which can bind to the surface. Bacteria use the so-called adhesins as ligands to attach to different receptors on cells in the host animal tissue. Adhesins are proteins or polysaccharides and their receptors can also be made of these components. Adhesins are important virulence factors because they contribute to the ability of bacteria to colonize different tissues.
Adhesin-receptor systems in bacteriaFimbriae (= common pili) often function as adhesins and sometimes a particular polypeptide, which is localized at the tip of the fimbria is the actual ligand. Membrane proteins may also act as adhesins.
IntroductionAnaerobic bacteria are bacteria that cannnot use oxygen in their metabolism, but are poisoned and killed by this molecule. These bacteria are also said to be strictly anaerobic (= obligate anaerobic) to distinguish them from the oxygen tolerant bacteria and the facultatively anaerobic (= facultatively aerobic) bacteria. Oxygen tolerant bacteria does not use oxygen in their metabolism. However, they are not poisoned by oxygen, but can live and multiply in the presence of oxygen, at least for a certain time. Facultatively anaerobic bacteria are not poisoned by oxygen and can switch their metabolism, so that in the presence of oxygen they utilize oxygen in metabolism, but in the absence of oxygen, they can extract energy in other ways (e.g. by fermentation or anaerobic respiration).
How can oxygen be toxic for cells?
The oxygen molecule consists of two atoms of oxygen, making the molecule stable because the two atoms can then share a pair of electrons in the outer electron shell (see Fig. A). In the presence of oxygen (O2) in an aqueous solution (e.g. in a cell) small amounts of hydrogen peroxide (H2O2) and superoxide radicals are always formed by an equilibrium reaction. The superoxide radical (see Fig. B) is usually designated O2·-, to show that it consists of an oxygen molecule, which has taken up an extra electron (·), making it negatively charged. Hydrogen peroxide and the superoxide radical in particular are highly toxic to cells because they are very reactive and can affect a variety of substances, e.g. oxidize unsaturated fatty acids, leading to the so-called oxidative stress. In order to survive in an oxygen-containing environment, the cells, therefore, have enzymes that can metabolize (detoxify) hydrogen peroxide and the superoxide radical.
Detoxification of hydrogen peroxide and superoxide radicals
Strictly anaerobic bacteria lacks all enzymes which metabolize
hydrogen peroxide and superoxide radicals. Other bacteria have
superoxide dismutase (SODM), which converts superoxide radicals
into hydrogen peroxide and oxygen (see Fig. C). Aerobic and most
facultatively anaerobic bacteria have catalase, which converts
hydrogen peroxide to water and oxygen (see Fig. C). Many
oxygen-tolerant anaerobic bacteria have a peroxidase, which
converts hydrogen peroxide to water by utilizing NADH2
(see Fig. C; click on it to enlarge it).
IntroductionSome pathogenic bacteria are anaeribic and have to be cultivated in an oxygen-free atmosphere. The method of choice is dependent upon how anaerobic the bacterium to be cultivated is. There are strictly anaerobic bacteria, which cannot withstand oxygen at all, and there are aerololerant anaerobic bacteria, which will survive if the exposure to oxygen is not too long.
Strictly anaerobic bacteriaIf you work with strict anaerobes, or if you have large amounts of samples to cultivate from, you may prefer a so-called anaerobic chamber where you work with rubber gloves sealed to the chamber. The anaerobic chamber maintains a constant temperature and the atmosphere may e.g. consist of nitrogen (N2) and carbon dioxide (CO2).
Aerotolerant bacteriaIf you work with aerotolerant anaerobes maybe you prefer a so-called anaerobic jar where chemicals are used to absorb oxygen (O2) and generates CO2. There are commercial systems for this purpose. The anaerobic jar can be placed in thermostatic cabinet during the incubation.
An antibiotic (pl. antibiotics) is a substance that selectively kills (bactericidal) or inhibits (bacteriostatic) growth of bacteria. Below is a compilation of various antibiotics, which are used in veterinary medicine. However, not all antibiotics mentioned below are necessaily registered for use on animals in Sweden or other countries. As for treatments of specific diseases in different species we refer to The Guidelines of the Swedish Veterinary Association (SVF). There you can find all the general guidelines for antibiotic treatment of animals and specific guidelines for small animals, horses and farm animals.
Bacteria may be resistant to some antibiotics and the resistance may be natural or acquired. Natural resistance means that a bacterium is resistant to an antibiotic due to its natural capacity. Mycoplasmas are e.g. naturally resistant to antibiotics which inhibit cell wall synthesis, because they have no cell wall. Acquired resistance means that a bacterium that was initially sensitive to a certain antibiotic, have developed resistance (by selection) because it has been exposed to this antibiotic. Antibiotic resistance is a very large and, unfortunately, growing problem in all health care because prescription of antibiotics has been too liberal in the past, and this is still the case in some countries, even within the EU.
From the National Veterinary Institut's (SVA's) website you can download the so-called SVARM- and SVARM/SWEDRES-reports, which are published annually by the Public Health Agency and SVA. The SVARM and SVARM/SWEDRES-reports provide a summary of antibiotic consumption and the resistance situation in Sweden in both veterinary and human medicine. The ability of bacteria to survive in the presence of an antibiotic can be tested by so-called susceptibility testing. The result of a susceptibility test is expressed as a MIC (Minimum Inhibitory Concentration) value, which has the unit mg/l (= µg/ml). In order to facilitate the interpretation of a MIC value, bacterial isolates are usually classified as sensitive (S), intermediate (I) or resistant (R) depending on the MIC value in relation to the "normal" MIC distribution of the corresponding bacterial species (see below).
Antibiotics are usually divided into different groups depending on their modes of action and chemical structure. Antibiotics, which are mentioned in the SVFs policy document appear in bold when they are first mentioned in the text below.
I. Inhibitors of cell wall synthesis.
A. Beta-lactams, where the so-called beta-lactam ring is included in the structure.
B. Other inhibitors of cell wall synthesis.
Examples of resistance mechanisms:
A transpeptidase is involved in the synthesis of the cell wall of bacteria. This transpeptidase binds to the dipeptide -ala-ala- and links peptides to be included in the cell wall, into a network. The beta-lactam ring is a structural analogue of -ala-ala- and can, therefore, compete with ala-ala which means that a stable cell wall cannot be formed. Mutations in the transpeptidase or other so-called penicillin binding proteins (PBP) may lead to penicillin no longer binding and, therefore, the bacterium becomes resistant.
Some bacteria are naturally possessing a chromosomal or plasmid encoded penicillinase, i.e. an enzyme that hydrolyzes the beta-lactam ring. In the presence of beta-lactams, these bacteria may be stimulated to increase production of beta-lactamase and if the beta-lactamase gene is plasmid-encoded, it can be transferred to other bacteria.
Clavulanic acid, which is a structural analogue of penicillins, can inhibit penicillinases by blocking their active centers. Clavulanic acid is particularly active against plasmid born beta-lactamases and is used therapeutically. Clavulanic acid is not an antibiotic in itself, since it only works in combination with certain beta-lactams.
II. Inhibitors of nucleic acid synthesis.
A. The fluoroquinolones (such as ciprofloxacin, difloxacin, enrofloxacin, ibafloxacin, marbofloxacin and orbifloxacin) inhibit the enzymes DNA gyrase and topoisomerase, which are needed for DNA replication. Fluoroquinolones must be used very restrictive for the treatment of animals because they are also used for the treatment of life-threatening diseases in humans. Use of the third and the fourth generation of fluoroquinolones are regulated in the Constitution SJVFS 2013: 42, Saknr D9.
B. Rifampicin inhibits DNA-dependent RNA polymerase, which in turn blocks the protein synthesis since the synthesis of the mRNA is inhibited. Rifampicin must only be used for the treatment of Rhodoccus-infectioner (Constitutional SJVFS 2013: 42, Saknr D9, Annex).
C. Nitroimidazoles (e.g. dimetridazole, metronidazole and ronidazole) reacts with nucleoproteins under anaerobic conditions, which in turn results in breakage of the DNA helix. Metronidazole is one of the few antibiotics that are effective against Clostridium difficile-induced diarrhea in humans and should, therefore, only be used for animals in exceptional cases. Nitroimidazoles are currently not approved for use on animals in Sweden.
Examples of mechanisms of resistance:
Resistance to fluoroquinolones arise by mutation(s) in the genes for the DNA gyrase and/or topoisomerase, which makes the fluoroquinolones unable to block these enzymes.
III. Inhibitors of protein synthesis.
A. Aminoglycosides (e.g., gentamicin, neomycin,
streptomycin and dihydrostreptomycin) bind to the
30S subunit of the bacterial ribosomes and cause misreading of the
B. Tetracyclines (chlortetracycline, oxytetracycline and doxycycline) interfere with the aminoacylated tRNA chains and prevents binding of amino acids to the growing polypeptide chain. This effect leads in turn to inhibition of protein synthesis.
C. Chloramphenicol (chloromycetin) inhibits the enzyme peptidyl transferase, linking amino acids to the growing polypeptide chain. This inhibition results in blocking of protein synthesis. Chloramphenicol can cause severe side effects and should absolutely not be used in animals that will go to food production.
D. Macrolides (e.g. tylosin and erythromycin) and lincosamides (e.g. lincomycin and clindamycin) binds to the 23S rRNA of the bacterial ribosome and prevents translocation along the mRNA chain. Ketolides belong to the group of macrolides, but they have a broader bacterial spectrum. Azalides (e.g. azithromycin) and streptogramins also belong to the group of macrolides.
E. Pleuromutilins (e.g. valnemulin and tiamulin) bind to the peptidyl transferase of the 50S subunit, thereby preventing amino acids from connecting with the growing polypeptide chain.F. Fusidic acid is a steroid derivative, which interferes with the release of elongation factor EF-G when amino acids are coupled to the growing polypeptide chain.
G. Oxazolidinones bind to the 23S rRNA of the bacterial ribosome preventing translocation along the mRNA chain. Oxazolidinones are today prohibited to be used for the treatment of animals, as they are also used for the treatment of life-threatening diseases in humans (constitution SJVFS 2013: 42, Saknr D9, Appendix).
Examples of mechanisms of resistance:
Resistance to aminoglycoside may arise by mutations in the ribosomal proteins, so that the macrolide can no longer bind to the ribosome. Some bacteria carry plasmids encoding aminoglycoside modifying enzymes. Such enzymes inactivate aminoglycosides and can easily spread in a bacterial population.
Lincosamide and macrolide resistance can occur by bacterial methylation of a specific nucleotide in the 23S rRNA or by mutations in the 23S rRNA gene, which result in that the macrolide will no longer bind to the ribosome.
IV. Inhibitors of functions in the cell membrane.A. Polypeptide antibiotics (such as polymyxin B and colistin) affect cell membrane permeability by binding to LPS, and interfering with phospholipids. This allows the essential metabolites to leak out from the bacterial cell.
B. Cyclic lipopeptides (such as daptomycin) bind to the bacterial cell membrane, which is then depolarized. This leads to inhibition of DNA, RNA and protein synthesis.
V. Inhibitors of different steps in the metabolism.
A. Folic acid antagonists
B. Furantoins (such as nitrofurantoin and furadantin) are transformed by bacterial flavoproteins to reactive intermediates, which inhibit bacterial energy metabolism as well as the synthesis of DNA, RNA, proteins and the cell wall. Furantoins affect so many different steps in of bacterial metabolism and, therefore, resistance does not easily arise. Consequently, furantoins are used against urinary tract infections in primarily dogs to reduce the use of aminopenicillins, against which resistance is more easily incurred. Resistance against furantoins occurs, but is uncommon.
Examples of mechanisms of resistance:
Point mutations in the genes for some of the enzymes involved in the synthesis of folic acid may lead to resistance because these modified enzymes are no longer blocked. Enterococci are naturally resistant to sulfonamides because they absorb folic acid from the environment.
VI. General mechanisms of resistance:
By decreased permeability or increased efflux, bacteria can develop resistance mechanisms, which are effective against different types of antibiotics. Decreased uptake may be due to mutations in porins and increased efflux may be due to mutations in the so-called efflux pumps. Efflux pumps are protein complexes which can pump the drugs out of the cell by active transport. Most often, the natural substrates of efflux pumps are bile salts, adhesins, toxins and other proteins.
ESBL, means "Extended-Spectrum Beta-Lactamases", which implies that the bacterium has a beta-lactamase with extended spectrum (can hydrolyse different kinds of beta-lactams). ESBL is sometimes used, a bit sloppy, for a group of multi-resistant bacteria, but is actually a name given to a group of enzymes, which are present in some multi-resistant bacteria. ESBL confer resistance to penicillins and many cephalosporins. ESBL are often plasmid encoded and may be transmitted between gram negative bacteria.
ESBL-CARBA (or ESBLCARBA), is the term for a mechanism of resistance in enteric bacteria, which causes resistance to most penicillins, cephalosporins and carbapenems. In Sweden, findings of ESBLCARBA in animals are notifiable to the County Administrative Board and the Board of Agriculture.
MRS means Methicillin Resistant Staphylococci. MRS is a designation for staphylococci in general, which is resistant to all penicillins (such as methicillin) and cephalosporins. In Sweden, findings of MRS in animals are notifiable to the County Administrative Board and the Board of Agriculture.
MRSA means Methicillin Resistant Staphylococcus Aureus. MRSA is the designation of Staphylococcus aureus strains, which are resistant to all penicillins (such as methicillin) and cephalosporins. In Sweden, findings of MRSA in animals are notifiable to the County Administrative Board and the Board of Agriculture.
MRSP means Methicillin Resistant Staphylococcus Pseudintermedius. MRSP is the designation of Staphylococcus pseudintermedius strains, which are resistant to all penicillins (such as methicillin) and cephalosporins. In Sweden, findings of MRSP in animals are notifiable to the County Administrative Board and the Board of Agriculture.
VRE means Vancomycin Resistant Enterococci.
VIII. More information
More information about the antibiotic resistance situation and antibiotic consumption in various European countries (including Sweden) can be obtained on the website of the European Centre for Disease Prevention and Control when clicking on "Health Topics" and then select "Health Topics A-Z" and then "Antimicrobial resistance". There you can select e.g., "Antimicrobial resistance interactive database (EARS-Net)" under Interactive Databases. Another important source of information on antibiotic susceptibility in the form of MIC values is the website EUCAST (European Committee on Antimicrobial Susceptibility Testing), where you can search a database for a specific antibiotic or a particular bacterium.
Aseptic - Antiseptic
To work aseptically means that you work in a way that will prevent disease-causing microorganisms to contaminate the material you are working with, without the use of chemical disinfectants. Chemical disinfectants (= germicides) are substances used to inhibit the growth of or to kill microorganisms on either an object or a body surface.
An antisepticum is a substance that is used to inhibit the growth of or to kill microorganisms on a body surface.
A bactericide is a substance that kills bacteria. A bacteriostatic agent (= bacteriostat) is a substance that inhibits the growth of bacteria.
A germicide is a substance that kills not only bacteria but also other microorganisms.
BacteriophagesIntroduction Viruses are organisms which parasitize the host cell's protein synthesis machinery. Bacteriophages (phages or bacterial viruses) are viruses that infect and replicate in bacterial cells. Phages may have dsDNA, ssDNA, dsRNA or ssRNA as chromosome and the chromosome can be circular or linear. Phages have either a lytic or a lysogenic cell cycle.
Lytic phagesLytic phages injects their chromosome into the bacterial host. To be able to do this, the phage has to adhere to specific receptors on the surface of the host bacterium. Then, the chromosome is replicated and phage proteins are synthesized by means of the host bacterial ribosome etc. When new phage progeny has formed, the bacterial host cell will lyse and the phage particles will find new bacterial host cells.
Lysogenic phagesLysogenic (= temperate) phages does not lyse the host bacterium immediately, but its chromosome can instead be integrated into the bacterial genome and there it exist as a so-called prophage (endogenous phage). The prophage will then replicate during the bacterial cell division and is passed on to successive generations. The phage will exist as a prophage until the environment for the bacteria deteriorate. Then the prophage become active, form new phage particles and finaly lyse the host cell.
Lysogenic conversionSome bacteria (e.g. Corynebacterium diphtheria, Clostridium botulinum, Shigella dysenteriae, Escherichia coli type VTEC and Streptococcus pyogenes) are pathogenic only if they carry a prophage. They are then said to have undergone lysogenic conversion and it is thus the genes of the prophage that encode important virulence factors.
Phages as diagnostic toolsPhages are not only species-specific, but in many cases also strain-specific and therefore, they can be used for subtyping of bacteria. Phagetyping is used for epidemiological studies of Salmonella enterica subsp. enterica. Phagetyping is perfomed by testing as a set of phage types and examine which of these can lyse the current salmonella strain. To perform the testing, a drop of bacteria is spread onto an agar plate, which is then allowed to dry on the surface. Then, small drops of phages with defined specificities are added. After incubation, the agar plate is inspected for plaques, which are formed where phages for which the bacterium is sensitive, have been applied. Exemples of other bacterial genera for which phage typing is used: Bacillus, Campylobacter, Clostridium and Staphylococcus.
Phage therapySince the problem of multi-drug resistant strains of bacteria increases worldwide, the interest in treating bacterial infectious diseases by phage therapy has increased. For this purpose, lytic phages are best suited. The method is still in the experimental stage, but many researchers believe that it has great potential in both human and veterinary medicine.
BiofilmBacteria that grow freely in liquid media are said to be planktonic, but some bacteria can also grow on solid substrates, forming a so-called biofilm. Biofilms can exist on live or dead surfaces and occur in nature, on materials in industrial processes and in different health care situations. Biofilms consist of population(s) of bacteria, which adhere to a surface and to each other and are enclosed in a network (matrix) of biopolymers. The formation of a biofilm starts with bacteria that adhere to a surface by means of e.g. fimbriae (pili) where they bind irreversibly and initially grow as a monolayer. Then they form several layers and start to produce some kind of a biopolymer (extracellular matrix) that often consists of the same material as the capsule, but in a looser structure. The biopolymer is made up of polysaccharides and is called glycocalyx (capsule). Dextran is one example of such a polysaccharide. Biofilms consist of one or more bacterial populations (species), glycocalyx, DNA and proteins.
A bacterial species that can not itself adhere to a surface, can often become attached to pre-existing bacterial glycocalyx and grow as a biofilm. Bacteria in biofilms are more resistant to antibiotics, detergents and phagocytosis than planktonic bacteria. Bacteria (Streptococcus spp.) can grow as biofilms on teeth (= plaques), on implants (e.g. heart valves), in plastic tubes, which transport nutrients etc. In patients with cystic fibrosis Pseudomonas aeruginosa may grow as a biofilm in the lungs. In biofilms, bacteria can communicate with each other by means of chemical signal substances (quorum sensing) to control gene expression in the whole population.
Link to a video montage on YouTube which is recommended: "What Are Bacterial Biofilms?"
Coliform bacteriaColiform bacteria (coliforms) are Gram-negative, non-spore forming and rod-shaped bacteria, which are also facultatively anaerobic, lactose-positive (during formation of gas) and oxidase-negative when grown for 24-48 h at 37°C. Heat tolerant coliforms are bacteria that also produce gas at a temperature of 44.0°C during cultivation for 24 h.
Coliform bacteria are used in food production as indicator bacteria for general hygiene. For microbiological analysis of water, coliforms are always used as indicator bacteria.
Examples of coliforms: Escherichia spp. (including E. coli), Citrobacter spp. Enterobacter spp., Hafnia spp. and Klebsiella spp.
Colony Forming Unit (CFU)The number of bacteria in a liquid sample is often expressed as colony forming units per ml (CFU/ml). This value is determined by first making a ten-fold dilution series of the sample to be investigated. Then you take a known volume (e.g. by using a calibrated 1 µl plastic loop) from each tube and streak onto appropriate plates. After incubation, select a plate on which you recognize about 100 colonies, and by counting the colonies, you will get a good estimation of the number of CFU/ml in the sample. Do not forget to multiply with the correct conversion factor. The reason for using the term CFU is that it is not certain that each colony originates from a single bacterial cell, because some bacterial species easily form aggregates in suspension cultures. Only living (or rather culturable) bacteria will give rise to colonies.
The number of bacteria (dead and alive) in a sample can be determined by counting them under a microscope in a calibrated chamber (a so-called Bürkner chamber).
For urinary tract infection (UTI), the term CFU is used and the amount of bacteria in the urine is used to be classified in the following way:
0 cfu/ml: no growth <25 000 cfu/ml: sparse growth 25 000 - 100 000 cfu/ml: moderate growth >100 000 cfu/ml: strong growth
Bacteria can be counted in different ways, and here are three fundamentally different methods:
Direct smearDirect smear is perfermed by making a suspension of a clinical sample and smear it out directly onto a microscope slide without first making a bacterial cultivation. Then the sample is fixed and stained by simple stain techniques or differential stain techniques, to provide an indication of whether the sample contains pathogenic bacteria.
Flagella and fimbriae
Bacteria can have different types of protrusions from the surface, known as flagella and fimbriae. Fimbriae are also called pili.
IntroductionThe term genome refers to the complete set of genetic material in a cell. Genome size is usually expressed in Mb or Mbp (= mega base pairs). Thus, 1 Mbp = 1 000 000 bp. The size of the bacterial genome varies between 0.5 and 10 Mbp. Bacterial genomes comprise the chromosome(s) and possibly also plasmid(s). Bacteria are haploid, unlike higher organisms, which are diploid. Gametes of higher organisms are, however, also haploid. The complete genome sequences have been determined for about 4000 bacterial strains and there are approximately 14 000 ongoing genome projects for bacteria.
ChromosomeThe chromosome represents the primary genetic material, which is essential for the bacterium. Bacteria in general have a circular chromosome, but there are exceptions (see below).
PlasmidPlasmids (see also Genome above) are secondary genetic material, not always essential for the bacterium. Bacteria which carry plasmids generally have one or more circular plasmids, but there are also exceptions. Plasmids generally constitute up to 10% of the genome and they are replicated independently of the chromosome.
Gram staining is a so called differential staining techniques, since one can distinguish two major groups of bacteria by this method. These two groups are gram positive and gram negative bacteria, which are stained purple and pink to red, respectively.
Gram-positive bacteria have a thick cell wall (peptidoglycan), which consists of several layers and can be likened to a network. Gram negative bacteria have a much thinner cell wall and also an outer membrane. Crystal violet (CV+), which is the primary dye binds to the negatively charged groups on the bacteria and stain them purple. Then iodine (I-) will be used to form a large complex (CV-I) with CV and thereby bind the stain to the bacterium. When Gram-positive bacteria are treated with the decolourizing solution (ethanol-acetone), the bacteria will be dehydrated and the colour retained. When Gram-negative bacteria are treated with the decolourizing solution the outer membrane will be dissolved and the thin peptidoglycan exposed, so that the CV-I complex is washed out. Then a counterstaining with safranin or basic fuchsin is performed to stain gram negative bacteria pink or red.
Members of the phyla Firmicutes and Actinobacteria (exception: genus Mycobacterium).
Members of the phyla Proteobacteria (exception: some members of the order Rickettsiales), Cyanobacteria and Spirochaetes
Bacteria multiply by binary fission (two identical daughter cells are formed upon the cell division). If the availability of nutrient is constant, and the physico-chemical properties of the culture medium does not change, they divide by the same speed all the time. This means that growth is exponential. The exponential growth phase (log phase) can not continue indefinitely, because the culture medium will become depleted of nutrients and the pH of the medium usually changes. When a bacterial culture is initiated in a liquid medium, it takes some time before the bacteria will start to grow, especially from clinical specimens or from hypothermic cultures and this phase of cultivation is known as the lag phase.
The time required for the number of bacteria to double during cultivation is called generation time or doubling time and it can vary greatly for different bacteria. Escherichia coli, which is cultivated under optimum conditions, has a generation time of 20 min, whereas Mycobacterium avium subsp. paratuberculosis has a generation time of about 24 hours. This means in practice that if you spread these two bacteria on appropriate culture plates, you can easily see colonies of E. coli after one day, while colonies of M. avium subsp. paratuberculosis cannot be observed until after at least 3 months!
Bacterial growth can be described by a so-called growth curve with four different phases: lag phase, log phase, stationary phase and death phase (= decline phase) (labelled A, B, C and D, respectively, in the figure). Note that the scale of the y axis is logaritmic. During the stationary phase bacteria die at about the same rate as they are formed during cell divisions, and in the death phase, bacteria die faster than they regenerate.
Construction of a growth curve
When a growth curve is constructed, you most often want to be able to follow the growth of bacteria in real time to know when to make e.g. addition of a substance, or when cultivation should be interrupted. The fastest and easiest method is to regularly collect samples from the culture and determine light scattering by spectrophotometry. With a spectrophotometer you normally determine absorbance at different wavelengths, but in a suspension (e.g. of bacteria) light scattering (OD = optical density) can also be measured. Light scattering is proportional to the number of bacteria per ml, and by means of a standard curve, one can determine the number of bacteria in absolute terms.
Hemolysis (Brittish spelling: haemolysis) means that the red blood cells (erythrocytes) burst apart (hemolyse) and release the cell contents (hemoglobin). Some bacteria produce so-called hemolysins, which give them hemolytic capacity. Most hemolysins are proteins (enzymes or porins), but there are also other types of hemolysins like rhamnolipids and biological detergents (biosurfactants).
Hemolysins are membrane distupting exotoxins which can be divided into two groups: toxins with enzymatic activity, and channel-forming toxiner (= porins).
Enzymatically active hemolysins are often lipases such as α-toxin of Clostridium perfringens, which is a phospholipase. When lipase cleaves lipids in plasma membranes of the host animal cells, the membrane will become fragmented and the cell contents leak out.
Porins are composed of subunits, but are secreted by the bacterium in monomeric form. In the cell membranes of the host animal, the monomers aggregate to channel-forming polymers (heptamers), which makes it impossible for the ion gradient across the plasma membrane of the host cell to be maintained and the osmotic pressure in the cell will increase until it lyses.
A function of hemolysins is that the bacteria can utilize hemolysis to release and utilize nutrients from the host animal cells. Iron e.g., is essential to many pathogenic bacteria, but is only present in very low concentrations outside the cells. If the bacteria have access to free hemoglobin, it can utilize the iron, which is bound to the heme groups of hemoglobin. Hemolysins do not act only on erythrocytes, but can also lyse other types of cells.
Identification of bacteria based on haemolysis
By cultivation on blood agar, bacteria can be differentiated based on their capacity to secrete hemolysins. The hemolysis will cause a clearing zone of the blood agar around the colonies. Bacteria can cause different types of hemolysis:
Note that the α-hemolysin of staphylococci causes complete hemolysis, whereas their β-hemolysin causes incomplete hemolysis.
The capacity to produce hemolysins may vary between different strains of a particular bacterial species.
Figs. 1 and 2, show colonies of some bacterial species that exhibit different hemolys patterns. The colonies have been lit from above (Fig. 1) and from below (Fig. 2) during photography. The easiest way to observe haemolysis is with the illumination from below and by looking at the plate in the "right" angle. The following bacteria have been used to illustrate hemolysis:
A. Streptococcus uberis, causing no hemolysis. This is sometimes is called γ-hemolysis, which is a bit unfortunate.
B. Streptococcus agalactiae, which gives a clear (complete) β-hemolysis.
C. Streptococcus dysgalactiae (subspecies not defined), that gives incomplete greenish α-hemolysis.
D. Staphylococcus pseudintermedius, giving double hemolysis.
Note that in Fig. 1A and 2A hemolysis cannot be observerd. In Fig. 1B, one can discern a thin hemolysis zone and in Fig. 2B, the clear β-hemolysis is evident around all colonies. In Fig. 1C it is possible to discern hemolysis around some colonies, and in Fig. 2C, one can clearly see the green α-hemolysis around some colonies (white arrows). In Fig. 1D one can see the outer hemolysis zone (white arrow) and in Fig. 2D, it is possible to see both the clear inner β-hemolysis zone and outer turbid α-hemolysis zone (white arrows).
Note also that all the colonies of Streptococcus dysgalactiae do not give rise to α-hemolysis, although the strain used is pure with respect to species. However, it may be that different strains (or clones) of the same species exhibit different hemolysis patterns.
In order to cultivate bacteria from certain types of samples (including food and feed) it is important to disperse the sample in the dilution broth without damaging the bacteria. A Stomacher® is an apparatus used for homogenisation of samples in food microbiology. A large amount of viable microorganisms can be released from different kinds of food samples by this method. The apparatus does not have to be sterilized because the samples are contained in sterile plastic bags to which sterile medium is added.
A. The homogeniser, which is loaded with a plastic bag containing the sample and culture fluid
Click on the image to enlarge it.
IntroductionRobert Koch was a German physician (1843-1910), who was interested in the relationship between microorganisms and disease. Koch formulated four criteria (Koch's postulates), which must be met to prove that a particular microorganism has caused a certain disease (viruses however, require other criteria).
In the original version of the postulates must was used, but with today's knowledge, one must use should as there are many exceptions. The first criterion had to be abandoned when it was discovered that there are asymptomatic carriers of certain microorganisms (subclinical infections). The second criterion must sometimes be abandoned because there are microorganisms that cannot be cultivated. The third criterion is not always valid because external factors can affect the results of an experimental infection.
ConclusionIf all criteria are met, a relation between microorganisms and disease has been proven, but if not all criteria are met, then it may still be a connection.
A number of new criteria to prove a relationship between microorganisms and disease was published in 1996 (Fredricks and Relman, Clin. Microbiol. Rev. 9:18-33). These criteria are based on detection of specific DNA sequences associated with disease.
Lancefield grouping of streptococciStreptococci are sometimes classified in so-called Lancefield groups after the American microbiologist Rebecca Lancefield (1895 1981), who developed a system for serological classification based on the carbohydrate composition of the cell wall. The different groups are called Lancefield groups A to V (except I and J). There is also a group NG (= non-groupable).
IntroductionLipopolysaccharides (LPS), which are also known as lipoglycans, are only present in the outer membrane of gram negative bacteria. LPS is a so-called endotoxin (se även toxin nedan) and the toxicity is associated to lipid A (see below).
StructureLPS is an amphipathic molecule, i.e. it has both hydrophilic (water loving) and hydrophobic (water repellent = lipid loving) regions. The hydrophobic portion (hydrocarbon chains) anchors LPS in the outer lipid layer of the bacterial outer membrane and the hydrophilic region (charged groups) points outward against the bacterial environment. Chemically, LPS consists of a lipid moiety and a polysaccharide moiety. The lipid moiety comprises lipid A, which is a phosphorylated glucosamine disaccharide with several (about 6) linked hydrocarbon chains, which constitute the hydrophobic portion of the molecule. The carbohydrate moiety consists of a so-called core oligosackaride (core antigen or R antigen), which is directly bound to lipid A. The polysaccharide (O-polysaccharide, O-antigen or somatic antigen) is then bonded to the R antigen.
SpecificityThanks to the diversity, found in LPS from various gram-negative bacteria, its antigenic properties can be used for typing and subtyping of bacteria. The antigenic properties will also vary in different parts of LPS:
Effects on the host animalAnimals (including humans) are constantly exposed to small amounts of LPS in the blood circulation because of the turnover of intestinal gram negative bacteria and thus the innate immune system is contiuously stimulated. Exposure to large amounts of LPS, as during for instance sepsis, will cause release of cytokines, leading to fever and inflammation. At toxic concentrations of LPS, blood clots form in the capillary system, which in turn can lead to life-threatening conditions.
Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS)
The instrument in the image belongs to the Department of Biomedical Sciences and Veterinary Public Health at the Swedish University of Agricultural Science (SLU). Lise-Lotte Fernström (+46 18 672389) and Lars Frykberg are responsible for the equipment and it is possible to get samples analyzed.
Mass spectrometry based on the MALDI-TOF means that the sample to be analyzed, is adsorbed to some type of carrier material (matrix). The sample is then irradiated with laser UV light, so that the molecules in the sample are broken into charged fragments (ionization), which are thrown towards a detector. The time it takes for the fragment to reach the detector (time of flight) is measured. The time is dependent on fragment size and charge. Also very large molecules (proteins and nucleic acids) can be fragmented and ionized in this way. Large molecules give rise to many fragments and a characteristic mass spectrum, which can be used for identification.
More recently MALDI-TOF MS has been used for the identification of bacteria. One can perform these analyzes directly on bacterial colonies and you will get an analytical response within a minute. The resulting mass spectrum is then compared with stored mass spectra of known bacteria and the method is considered to be very reliable. The more mass spectra of known bacteria you have to compare with, the safer the method will be.
MALDI-TOF MS is already used in some laboratories for veterinäry bacteriology and many researchers believe that this technique will be tomorrow's routine method for identification of bacteria. The instrument is still very expensive, but material costs are low.
IntroductionMany bacteria are motile and they can migrate by utilizing mechanisms based on different principles. Motility mechanisms have been developed in bacteria in order for them to be to be attracted or repelled by certain stimuli. Motility is a feature, which is used for characterization and identification of bacteria and methods have, therefore, been developed to detect motility. Bacterial locomotion must be distinguished from so-called Brownian molecular motion, which is random and depends on the thermal motion of water molecules, which can push the bacteria so that it is perceived as if the bacteria move under the microscope.
Swimming movement by means of flagellaBacteria can have flagella, which may be one or several in number, and which act as propellers and allow the bacteria to move in a predetermined direction. Very simplified, you can say that the flagellum consists of a moving filament, which is linked to a molecular engine (basal body) in the bacterial envelope via a hook. The flagellum consists of many subunits of the protein flagellin, which form a hollow and flexible cylinder. The molecular engine, which is built up by protein subunits, is driven by the proton gradient (or a Na+ ion gradient) over the cell membrane and can get the filaments to rotate clockwise or counterclockwise. This rotation causes the bacteria to swim in a certain direction and to tumble, respectively. The direction is dependent on outer stimuli and during tumbling, the bacteria can change direction of the movement.
Bacteria within the phylum Spirochaetes have so-called periplasmic flagella (endoflagella or axial filaments), which are localized in the periplasmic space between the cell membrane and the outer membrane. This arrangement result in a screw like motility (or flat-wave motility), which makes it possible for these bacteria to move in highly viscous material (like mucus).
Twitching motility by means of type IV pili (fimbria)Bacteria which have so-called type IV pili can move by using the external ends of the pili, which have hooks, and can adhere to a solid substrates like the surface which the bacteria colonize or to other bacteria. When the pilus contracts, the bacteria are pulled forward. Movement produced by type IV pili is typically jerky, and thus it is simply called twitching motility. Pseudomonas aeruginosa has this twitching motility.
Gliding motility on surfacesSome bacteria can glide on wet surfaces, but the molecular mechanisms are incompletely understood. Examples of bacteria, which have this ability are: members of the phylum Cyanobacteria and the genera Flavobacteria and Mycoplasma.
Motility by utilizing the cytoskeleton of the host cellSome pathogenic bacteria can move inside the host cell by using its cytoskeleton. The cytoskeleton is normally used to move organelles inside the cell. By stimulating actin polymerization at one pole of the host cell, these bacteria can form a kind of tail, which pushes them through the cytoplasm of the host cell. Examples of bacteria that can use this mechanism are Listeria spp. and Shigella spp.
Motility assayMotility tests can be performed in test tubes containing a semi-solid medium, i.e a medium with a low concentration of agar. The medium is inoculated with a plastic loop, which is inserted into the agar and pulled up again. If the bacterium is motile, the medium in the whole tube will turn turbid, but if it is non-motile turbidity will only be obtained where the plastic loop was inserted. The picture shows a negative control (A), which consists of Staphylococcus aureus subsp. aureus (non-motile) and Escherichia coli (B), which is motile.
Nomenclature of bacteria
Nomenclature of bacteria refers to naming and bacteria and other organisms are named according to the binomial system, which was introduced by Carl Linnaeus (1674-1748). This means that a bacterium has a species name, which is composed of a genus name that tells you to which genus it belongs and a species epithet which, together with the genus name, is unique to the bacterium. An example of this is Moraxella bovis, where the genus name indicates that the bacterium belongs to the genus Moraxella and the species name indicates that the bacterium has been isolated from cattle. The genus name and the species epithet form together the scientific name of the species, which is always written in italics. Bacterial names are international and Latin or latinized Greek are used to form the name. If misunderstandings cannot occur, you can abbreviate the genus name after it has been written for the first time in a text, e.g. M. bovis. However, note that there are also bacteria called Mycoplasma bovis and Mycobacterium bovis.
There are strict international rules for how bacteria should be named and these rules are published in a book named: "International Code of Nomemclature of Bacteria". In order to get a proposed name accepted, a scientific paper on the proposed species must be published and approved by an international taxonomy committee.
Trivial names are often used as a simplified way of naming a bacterial genus. A trivial name should neiter be written with capital first letter nor in italic. Examples of trivial names are: lactobacilli, mycobacteria, salmonella, staphylococci and streptococci. The scientific names for these groups are: genus Lactobacillus (or Lactobacillus spp.), genus Mycobacterium (or Mycobacterium spp.), genus Salmonella (or Salmonella spp.), genus Staphylococcus (or Staphylococcus spp.), genus Streptococcus (or Streptococcus spp.), respectively.
If you refer to a specific bacterial species, a trivial name refering to a complete genus should never be used.
Subspecies, biovars and serovars
Sometimes there is a need to divide bacterial species into subspecies, because they are too closely related to be regarded as different species, but too distantly related to be regarded as the same species. In this case a subspecies is introduced by adding a subspecies epithet and write subspecies (subsp. or ssp.) in front of it. An example of this is Streptococcus equi subsp. equi. When you divide a species into several subspecies, the original species always gets the same subspecies epithet as the species epithet.
There is often a need to divide species and subspecies in different biovars (biological variants) or different strains, but this is not strictly regulated, which means that researchers themselves can name their strains or biovars. One type of biovar is serovar (serological variant), by which various surface antigens can be identified with specific antibodies. Contact tracing and epidemiology is based on identification of different variants of the same bacterial species.
Serovar vs. serotype
Serovar and serotype are synonyms and thus, interchangeable terms, but according to the Rules of the Bacteriological Code (1990 Revision), serovar is the preferred term. Serogroup is a group of bacteria containing a common antigen. A serogroup may contain several serotypes. Serogroup is not an official designation, but has been used to classify bacteria belonging to the genera Leptospira, Salmonella, Shigella and Streptococcus.
A bacterial subspecies that occurs in several thousand different serovars is Salmonella enterica subsp. enterica. A common serovar is Dublin and if you you want to write the complete and correct name of the bacterium, it becomes Salmonella enterica subsp. enterica serovar Dublin. Please note that the name of the serovar is capitalized, but not italicized. If the name appears in several places in the text, you can write S. enterica subsp. enterica serovar Dublin. However, because even this abbreviated writing is rather lengthy, it has been agreed that it is acceptable to simply write Salmonella Dublin, except on the first occurrance in a text, where the name must be given in full.
ParameterParameter is a term, which is usually used to identify the variable and measurable characteristics, which define a system. The system may e.g. consist of a bacterial culture and examples of parameters are: number of added bacteria, growth temperature, incubation time, salt concentration, glucose concentration, CO2 concentration, etc.
Testing parameter in food microbiology refers to standardized routine methods for the determination of e.g.: number of slow-growing bacteria, intestinal enterococci, culturable microorganisms etc.
IntroductionThe ability of a microorganism to cause any kind of damage (i.e. disease) to the host animal is called pathogenicity and microorganisms that have this ability are called pathogenes (or pathogenic organisms). Pathogenic is an "all-or-none-characteristic", i.e. a microorganism is either pathogenic or non-pathogenic in a given host animal. Virulence is not synonymous with pathogenicity, but describes the degree of damage that the pathogen has caused. A highly virulent bacterium is very contagious and/or gives severe symptoms.
Obligate or opportunistic pathogensA microorganism may be an obligate pathogen or an opportunistic pathogen. An obligate pathogen can be found in the host animal only in connection with disease. Microorganisms, which are found in healthy host animals, but which may cause a disease in certain circumstances are known as opportunistic pathogens. Such circumstances may be an immunocompromised host, other infection, tissue damage, etc.
Characteristics that make a bacterium pathogenic
PlasmidPlasmids constitute secondary genetic material in bacteria, which can be used if the need arises (see also Genome above). Plasmids can be present in one or several copies in a cell, as they replicate independently of the chromosome. A bacterium may carry multiple different plasmids.
FunctionPlasmids may contain genetic information, which is necessary for:
Practical usePlasmids have been of great use in molecular biology including cloning of genes. Genetically modified plasmids, which contain only a few genes and a suitable cloning site, are then used.
IntroductionA clinical sample seldom contains bacteria in pure culture, but they are usually found in a mixed flora. Thus, the pathogenic bacterial species is often isolated in combination with the normal bacterial flora during a bacterial infectious disease. To identify key bacteria in clinical samples, you have to have them in pure culture. A pure culture thus contains only one species of bacteria, and this can be accomplished by streaking (see below) onto agar plates.
PyogenicA pyogenic (= suppurative or purulent) bacterium is a pus-forming bacterium. If mucus is also generated, the bacterium is called mucopurulent. Examples of pyogenic bacteria are Burkholderia mallei, Klebsiella pneumoniae, Staphylococcus aureus, Sta. epidermidis, Streptococcus pyogenes och Str. pneumoniae.
Pyogenic bacteria may cause pyemia (see sepsis below).
IntroductionQuorum sensing is a system for signaling and response in a population of organisms (e.g. bacteria), regulated by the population size. In other words, the gene expression (= protein synthesis) of certain proteins can be regulated in response to changes in population density. Quorum comes from the Latin and means "of them", which refers to the minimum number present, as required for taking a decision.
Why do bacteria communicate with each other?If very few bacteria are present in a particular area, it is a waste of energy to produce and secrete e.g. certain enzymes and, therefore, gene expression is regulated by quorum sensing. Formation of biofilm (see above) is also regulated by quorum sensing. Synthesis of the constituents of a biofilm is initiated when the bacterial population density has reached a certain level.
How do bacteria communicate with each other?Bacteria use quorum sensing if necessary, to regulate gene expression in relation to the population size. The bacterium releases chemical messengers in the form of complex organic molecules (polypeptides in gram positive and N-acyl homoserine lactones in gram-negative bacteria) to achieve this. The concentration of signaling molecules is not high enough for the bacteria to communicate with each other in a population of planktonic bacteria (see Biofilm above). However, in a biofilm it is, and therefore they can communicate.
SepsisSepsis (blood poisoning) is a potentially deadly medical condition with a whole-body inflammatory state [systemic inflammatory response syndrome (SIRS)], which is usually caused by a bacterial infection.
Septicemia (septicaemia) is a related medical term referring to the presence of pathogenic organisms that multiply in the bloodstream, leading to sepsis.
Bacteremia (bacteraemia) is transiently presence of bacteria in the blood and this term is not equivalent to sepsis.
Pyemia (pyaemia) is a form of septicemia, which results in widespread abscesses of a metastatic nature. Pyemia is usually caused by the presence of pyogenic (pus-forming) bacteria in the blood.
IntroduktionThe word siderophore originates from Greek and means iron carrier. Siderophores are low molecular weight substances that have very high affinity for iron (Fe3+) and they bind iron by so-called chelation. Iron is essential for almost all forms of life. Many bacteria can secrete siderophores, which allows them to absorb iron even in an environment where the concentration of free iron is very low. In mammals, iron is tightly bound to various proteins (e.g. ferritin, lactoferrin, hemoglobin and transferrin) and therefore, pathogenic bacteria bind iron in the form of soluble complexes with siderophores. Siderophores are considered as virulence factors.
Examples of siderophores