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o Plectridial type – before the process of sporoforming, the cell dilates on one
edge, and it's shape starts looking like a drum stick (Clostridium tetani). Spores are in the enlarged part of the cell. Clostridial and plectrical types of sporoforming are common for the representatives of Clostridium spp. It happens quite often, that different cells of the same species have clostridial and plectridial forms at the same time.
• Spiral bacteria. Depending on the amount of curves, these bacteria can be divided
into the two groups: vibriones and campylobacter (these bacteria, have curve, that is not exceeding, the quarter of a spiral's turn); spirilla, with 2-3 big curves and spirochaetes – mobile microorganisms, with a cell's shape of a spiral and with plenty of curves.
1.3. Structure of Bacteria
Schematic structure of the bacterial cell is presented in Fig 2
Bacteria are organisms with the prokaryotic type of cell's organization i.e., they
do not have a nucleus. Prokaryotic cell has some differences, concerning both its ultrastructure and chemical composition.
It is logical that we start the discussion of bacteria describing internal structures,
that are common for all the major groups.
1.3.1. Internal structures of the bacterial cell.
There is almost no difference between gram-positive and gram-negative bacteria. In the
cytoplasm of any bacteria there are organelles, synthesizing proteins (ribosomes) and nucleic acids (DNA and RNA).
Deoxyribonucleic acid (DNA) is the basic nucleic acid, that is encoding genetic
information. Bacterial DNA exists in a chromosome and plasmid form.
The bacterial chromosome is a ring-shaped molecule of DNA that is coiled and
condensed. It carries all important genes, that make this microorganism unique. Unlike in eukaryotes, the bacterial chromosome is not surrounded by a nuclear membrane.
Plasmids are extrachromosomal ring-shaped DNA molecules much smaller than the
chromosome. Plasmids are mobile genetic elements. That means, that unlike the chromosome, they can be transferred from one bacterial cell to another. This process is one of the major ways
of the genetic exchange between bacteria. Plasmids are not essential components of the microorganism's cell, however, they can be composed of genes, determining bacterium's resistance against its surrounding (including antibiotics), bacteria's virulence and other features, that are not essential, but give bacteria, an advantage in the process of evolution.
Protein synthesis, taking place on ribosomes is a complex, multistage process and its
detailed discussion is beyond the objective of this article. The following are the main steps of protein synthesis. As a result of the transcription process, there appears a single-chain molecule of informational or messenger RNA (mRNA), which is the exact copy of the DNA, encoding the gene. Protein synthesis takes place on the ribosomes of the bacteria during translation, which is a process where the information encoded in the mRNA is translated. Ribosomes consist of big (50S) and small (30S) subunits, which consist of ribosomal RNA (rRNA) and proteins. Ribosomes connect to the mRNA molecule and move along it. While doing so molecules of the transporting RNA (tRNA) bring all the needed amino acids to the ribosomes. Once amino acid is brought, it joins the growing polypeptide chain.
Ribosomal ribonucleic acids (rRNA), that are taking part in forming ribosomes, are one
of the most conservative elements of the microorganism's cell. Studying of the rRNA is the basis in genotypic classification of bacteria.
Ribosomes are targets for many antibiotics, which are suppressing biosynthesis of
proteins. Macrolides, lincosamides antibiotics and chloramphnicol are blocking the big subunit. Small subunit is affected by aminoglycosides and tetracyclines.
1.3.2. Bacterial surface structures.
The cytoplasmic membrane and the cell wall separate the cytoplasm from the
While cytoplasmic membrane has basically the same structure among different types of
bacteria and even eukaryotes, cell wall is a unique feature of prokaryotes, and its structure varies between gram-negative and gram-positive bacteria.
The structure of the gram-negative and gram-positive bacteria is shown schematically at
Cytoplasmic membrane. Every form of life (including bacteria) has cytoplasm
membrane (CPM), protecting the internal content (cytoplasm) of the cell from the environment. Cytoplasmic membrane is represented by a double layer of phospholipid molecules (phospholipid bilayer). CPM also consists of a large amount of proteins (about 200), performing different functions.
Since among membrane's proteins there are cytochromes, one of the major functions of
the membrane's proteins is an energy generating one. Energy is generated in an ATP molecule form.
The second important role of the membrane proteins is transport of small molecules.
CPM is an osmotic barrier, and is not penetrable by the majority of molecules, some special transporting mechanisms are needed. Some proteins penetrate CPM, while others are localized in the internal, or external surfaces. Amino acids, organic acids, non-organic salts and antibiotics, most need active transport in order to get inside the cell. At the same time some products of the cell's metabolism, toxins and other factors of virulence of protein nature need active export from the cell.
Cell wall. The cell wall is an important and essential structural element of most bacterial
cells. In the bacteria that do not have a slime layer, the cell wall serves as a mechanical barrier protecting the bacterial cell from the environment and gives the bacteria its shape.
Depending on the structure of the cell wall, prokaryotes are being divided into two
categories: gram-positive, and gram-negative.
Historically, both terms appeared due to the difference between bacteria, while being
stained using the method, that was suggested in 1884 by Christian Gram (so called gram-staining). It was discovered, that prokaryotic cell walls form a colored complex, with iodine after the use of crystal violet. If then the cell is treated with alcohol and after that with a red
colour, bacteria either stays violet, or turns red, depending on the structure of the cell wall. Bacteria that keep the violet color are called gram-positive, and the ones, that turn red – gram-negative. Later on, it turned out that the difference of color was correlated to fundamental differences in the structure of the cell wall.
Peptidoglycan (murein). On the external side of the cytoplasm membrane, there is a
peptidoglycan layer. It's structure is shown schematically on Fig .
Fig The structure of peptidoglycan In the gram-positive microorganisms, the cell wall is tightly adjoined to CPM, while in gram-negative ones it is separated by a periplasmatic space. Peptidoglycan is the structure that is responsible for the bacteria's shape, and resistance against intracellular pressure, that can be as high, as a few atmospheres. Peptidoglycan consists of a parallel polysaccharide chains, that are forming the framework of the cell. Its tightness is provided with the perpendicular joints between different polysaccharide chains. Peptidoglycan's polysaccharide chains consist of the alternating molecules on N-acetyl muramic acid and N-acetyl glucosamine, which are linked together by means of beta-1-4-glucoside bounds. Transverse joints are formed through connecting the covalent bounds between short amino acid chains, which are deviating at right angles from the main polysaccharide chains. Special enzymes synthesize and connect the transverse joints. Beta-lactame antibiotic's pharmaceutical effect is achieved through suppressing of these enzymes. These enzymes are called penicillin binding proteins (PBP).
Cell wall of the gram-positive bacteria.
Peptidoglycan's heteropolysaccharide molecules form the complex multilayer
dimensional structure in gram-positive bacteria. In many species murein layer is three-valently connected with teichoic and teichuronic acids, which can be a part of the receptors for several bacteriophages. Some gram-positive bacteria have a little number of polysaccharides, proteins and lipids in the cell wall.
Cell wall of the gram-negative bacteria.
Gram-negative bacteria have a more complex cell wall. It consists of a larger amount of
macromolecules of a different structure. Peptidoglycan forms only the internal layer of the cell wall, and is separated from the CPM by a periplasmatic space. The majority of the gram-negative species have a peptidoglycan layer forming one or two layer structure, that is connected
to heteropolysaccharides by means of transversal chains. Chemically peptidoglycan of the gram-negative bacteria is analogous with the gram-positive one.
On the outside of the peptidoglycan layer there is an additional layer of the cell wall –
external membrane. Unlike the cytoplasmic membrane, external membrane of the gram-negative microorganisms is asymmetric. Its internal layer is composed of the phospholipid molecules, while the structure of the external layer is more complex and includes lipopolysaccharides.
LPS molecule ( .) consists of three main parts: conservative structure – lipid A
(practically the same in all the gram-negative bacteria); relatively conservative oligosaccharides structure (core) and highly variable polysaccharide chains (O-specific chains), that are specific for every sero-type. LPS's lipid part possesses hydrophobic features, while the polysaccharide is hydrophilic. In accordance with this features the lipid fragment is oriented inwards in the external membrane, and the polysaccharide one – outside.
Gram-negative LPS, and mainly it's A lipid, plays the key role in the pathogenesis of the
inflammatory reaction, since it is known to be the most active stimulator of the anti-inflammatory cytokines' production (e.g. interleukin–1, interleukin-2, TNF).
Lipopolysaccharide layer is almost impenetrable for the exogenous hydrophilic
substances, including most of the nutrient substances (e.g. carbohydrates, amino acids) and antibiotics. Transportation of all these substances inside the bacterial cell goes through the funnel-shaped protein structures (porines, porine canals), which are inserted in the lipopolysaccharide layer. Hydrophobic substances (among antibiotics quinolones, macrolides, and tetracycline belong to this group) can diffuse through the lipopolysaccharus layer.
Relatively big hydrophilic antibiotics (MW>1000) such as glycopeptides and natural
penicillin hardly can come through the gram-negative bacteria's porine canals. That explains the natural resistance of these microorganisms to the named antibiotics.
Besides porines there are other proteins that are performing the transporting function in
the gram-negative microorganism's membrane.
1.3.3. Superficial structures of bacteria
The superficial structure of bacteria include flagella, fimbriae, pili and glycocalyx.
Flagella are the organelles, responsible for the mobility of the bacteria. The main function of fimbriae and pili is to provide adhesion of the microorganisms to epithelial surfaces.
While flagella and pili are typical of gram-negative microorganisms, glycocalyx is more
often found in gram-positive ones. There are two main types of glycocalyx: capsule and slime. Both forms of the glycocalyx have similar structure, and are represented by a superficial glycoproteine net. The difference applies to the durability of the connection with the cell. Capsule is a polysaccharide firmly connected to the cell, while slime can be easily removed from the bacterial cell. Glycocalyx functions are associated with surface adhesion, antiphagocytic defence, prevention from drying and preservation of nutrients.
1.4. Bacterial metabolism, and biochemical activity.
Differentiation of the microorganisms inside of the big taxonomic groups, based on their
morphology and coloring using the Gram's method is impossible. For example, when using the light microscopy method, Escherichia coli – the representative of the normal microflora looks almost the same as Salmonella typhi (agent of the typhoid).
Character of the microorganism's metabolism plays the main role in their differentiation.
Clinical doctor is frequently meeting such a terms, as "aerobic", "anaerobic", "fermenting" and "nonfermenting" microorganisms.
In order to solve practical problems (separation and identification) bacteria are divided
into several groups, according to their utilization of oxygen.
• Obligatory (strict) anaerobes – bacteria, that can reproduce only when there is no
oxygen as oxygen has a toxic effect on these microorganisms.
• Aerotolerant anaerobes – microorganisms, less sensitive to the free oxygen. These
bacteria stay alive within concentration of oxygen equal to 2-10%.
• Facultative anaerobes – microorganisms, that can reproduce in both anaerobe
conditions and in the presence of the atmospheric air.
• Microaerophiles – microorganisms, that need oxygen for their life, but can not
reproduce in the presents of the atmospheric air. Optimal oxygen concentration for them is around 5%.
• Obligatory (strict) aerobes – relatively small group of microorganisms, that need
atmospheric air for their reproduction and life.
When there is no free oxygen, carbohydrates (mainly glucose), which are the main source
of energy for bacteria, are utilized, called fermentative metabolism. Accumulation of the energy in the form of adenosine triphosphate (ATP) results from substrate phosphorylation. Resulting products of the fermentative metabolism are low-molecular organic acids (lactic acid, and others), alcohols, short chain fatty acids and carbonic gas (CO2).
As an alternative to the fermentative metabolism there is an oxidative one, aerobic
respiration. ATP accumulating in the oxidative metabolism is a result of oxidative phosphorylation. In this case, electrons are transported along the respiratory chain and their final acceptor is oxygen. Glucose is fully metabolised into water and CO2.Therefore, the effectiveness
of the oxidative metabolism is much higher than the fermentative one.
Facultative aerobes utilize carbohydrates either using fermentative, or oxidative
metabolism, depending on the presence of oxygen. Historically, the group of microorganisms, which utilize glucose using only the oxidative metabolism are called "non fermentative" (unable to ferment). In this group there are such pathogens as P. aeruginosa, Acinetobacter spp. and some others.
2. Clinical significance of the different groups of
In the discussion of the clinical significance of microorganisms, the most frequently used
phenotypic classification will be used. The most significant kinds and species will be discussed
here.
To get a reliable bacteriological diagnosis one must ensure that sampling technique, storage
before transportation and transportation are correctly performed. This is the duty of the
clinician. The laboratory must use validated methods. The clinical significance of the
microorganisms found in the sample depends on the clinical situation and must be
thoroughly evaluated to avoid overuse of antibiotics.
2.1. Aerobic and facultative aerobic microorganisms.
2.1.1. Gram-positive microorganisms.
Gram-positive cocci.
All of the aerobic gram-positive cocci are divided into the two subgroups: catalase-
positive and catalase-negative. This division is based on the definition of the enzyme catalase in the simple test with hydrogen peroxide.
Catalase-positive cocci. Representatives from this group are the components of the
normal human microflora. The following microorganisms fall into this group: • Staphylococcus
• Micrococcus • Stomatococcus
Staphylococcus (Staphylococcus spp.). There are currently 34 species of
staphylococcus described. Since Staphylococcus aureus has the biggest clinical significance, as it is the most virulent one, its identification is a very important practical task. This can be easily achieved e.g. by means of revelation of the microorganism's coagulase activity in the simple test with rabbit plasma.
Since the coagulase activity is seldom seen but in S. aureus the term "coagulase-positive"
staphylococci is used as the synonym of Staphylococcus aureus, while all the other species are united under the term "coagulase-negative" staphylococci. Staphylococci are able to produce many different virulence factors especially S. aureus. The most important virulence factors of S.aureus are:
• Superficial proteins, which help colonization of the epithelium and other tissues by
means of binding together with proteins of the extracellular matrix (laminin, fibronectin and others)
• Superficial factors, protecting microorganisms from phagocytes – polysaccharide
capsule and A protein. Protein A joins antibodies within their Fc-fragment, preventing the normal process of opsonization. It results in the antibody's orientation on the bacteria's surface in the way that makes the process of phagocytosis impossible.
• Factors that assist bacterium's survival in the phagocyte (cartenoids, production of
• Extracellular proteins
o Toxins, destroying eukaryotic cell's membrane and contribute in spreading of
the microorganisms in tissues (α-β- γ- δ- hemolytic toxins and leucocidine).
o Pyrogenic exotoxins: entrotoxins (A to H) and toxic stress syndrome toxin
(TSST). All the named toxins work as the super antigens. They activate T-lymphocyte regardless of the process of antigen recognition and induct massive cytokine liberation that leads to the development of the toxic stress syndrome.
o Exfoliative toxins o Enzymes, stimulating fibrin synthesis and fibrinolysis – coagulase and
o Hydrolytic enzymes – lipase, nuclease, urease and hyaluronidase
In spite of all the named virulence factors, the immunocompetent organism can resist
the development of a staphylococcus infection if he has epithelial cover intact. Further staphylococci, mainly coagulase negative, are the basic and essential components of the human normal skin microflora. The biggest amount of the different species of staphylococci is found in orifice of the hair follicle, sweat glands and sebaceous glands. Some species of staphylococci have a tropism to different areas of the skin.
For example, adults have the biggest amount of S. capitis on their head, S. epidermidis on
their face and S.auricularis - in the area of the external acoustic meatus. There are many staphylococci in the axillary area and in the perineum, since those areas have favourable conditions for bacterial growth. The predominating species there are S. epidermidis and S. hominis. In the perineum area also there are S. aureus and S. saprophyticus. On the skin cover of extremities and trunk there is a small amount of staphylococci, usually S. epidermidis and S. hominis.
For the healthy adults, the most common place to find S. aureus is the internal surface of
the nose wings. It seems that the phenomena of carrying of S. aureus has genetic nature. Data of many researches show, that the total percentage of resident and transitional S. aureus carriers in a human population equals 19 to 68%, some times showing the persistence of the same strain in a human for 10 years.
Due to the big amount of S. aureus carriers in the human population, there is no
appropriate need in total checkup of all the medical workers for the purpose of finding S. aureus carriers.
At the same time it has a big practical significance to detect all the carriers of S. aureus
resistant to methicillin/oxacillin (MRSA – Methicillin Resistant Staphylococcus aureus) among both patients and medical workers. In case the medical workers carry MRSA they should be sanitated with mupirocin. (This is debated and not the current Swedish opinion).
S. aureus. The fact, that S. aureus is found in almost any clinical material proves its
etiologic significance. When evaluating the role of S. aureus in the structure of human pathology, one has to distinguish between infections occurring in the society and hospital infections.
Community acquired Infections. In immune-competent persons, staphylococcal skin
and soft tissue affections are most frequently seen. Their onset is the result of the problem in the skin integrity (microtrauma). In most cases, infections are localized – impetigo, furuncle, carbuncle and others.
The second most important entrance gate for the S. aureus infection is the respiratory
tract. In this case, as in skin diseases, a problem in the superficial protecting structures is needed for the disease to occur. One of the basic factors, favoring the development of the extra-hospital staphylococcal pneumonia is virus infections of the respiratory tract (mainly influenza).
The role of S. aureus in the development of infections of the urinary tracts is
Compared to the relatively large number of minor localised infections with S.aureus in
the society the number of deep or generalised infections is small .Nevertheless, the absolute number of staphylococcal osteomyelitis, arthritis, endocarditis, septicaemia, and septic shock causes serious problems. The only coagulase negative staphylocci of importance outside the hospital is S.saprophyticus which causes urinary tract infections in fertile women.
Among hospital infections associated with S. aureus the most often seen are infections
of the incisional wound. The volume of tissue's lesion can vary in this infections (e.g. from slight infection in the area of a stitch, up to mediastenitis after coronary artery bypass grafting). However, their pathogenesis has no principal difference, compared to extra-hospital infections.
S. aureus is the frequent etiological agent of such common hospital in fections as
ventilator associated pneumonia, catheter-associated infection and infections of implants.
S. epidermidis and other coagulase-negative staphylococci (S. haemolyticus, S. hominis,
S. warneri, S. capitis, S. simulans and some others) have a smaller clinical significance, in comparison with S. aureus, since they lack many factors of virulence. Unless there is a serious failure of resistance in the host, these microorganisms do not have much of significance in the human pathology. However in some cases S. epidermidis and other coagulase-negative staphylococci can be the leading agents of pathology.
Some of a few virulence factors of coagulase-negative staphylococci are capsule and
other adhesives, providing the adhesion of the microorganism on plastic and other surfaces and on proteins of the tissue matrix (fibrinogen, fibronectin and others). The capsular "slime" forms a biofilm on practically all intravascular and other implanted devices (artificial joints, osteosynthesis material, different catheters, shunts, artificial heart valves, pacemakers and others) that provide favourable conditions for the adhesion of staphylococci. Inside the biofilm bacteria are protected from factors of host resistance and antibacterial medication.
Thanks to the adhesion ability coagulase-negative staphylococci are the leading pathogen
agents of vascular catheter-associated infections and infections on implants.
While evaluating the significance of coagulase-negative staphylococci in intrahospital
infections it should be mentioned, that these microorganisms almost never cause infection of the respiratory tract (e.g. pneumonia) and urinary tracts, even in the immunosupressed organism.
It is also important to mention, that coagulase-negative staphylococci are the most
common microorganisms contaminating blood samples. The clinical significance of their presence in a blood sample should therefore be carefully evaluated.
Catalase-negative cocci. This group of microorganisms is quite numerous. It includes:
• Streptococcus spp.
• Enterococcus spp. • Lactococcus spp.
• Leuconostoc spp.
• Pediococcus spp.
Streptococcus spp. This kind of microorganisms is characterized by a big variety of
species and the significant difference in virulence between species. Here are some troubles, connected with the classification of streptococci. In everyday practice such terms, as "streptococcus A, B" which refer to serological groups, and "alpha-" or "beta-haemolytic streptococci" which refer to the appearance on agar containing, blood are used. Such a terminological variety can be explained by the usage of the different ways of differentiation and may cause confusion.
Since phenotypic methods of classification are not sensitive enough, contemporary
taxonomy mainly uses genetic methods.
Presently there are known around 40 species of streptococci. They are united in 6
clusters (groups):
• Pyogenic • Anginosus
• Salivarius • Bovis
Streptococci are representatives of the normal microflora of man, and can be found in the
intact respiratory tract's mucous membrane, gastro-intestinal and urogenital tract of a human and some animals. However, when they get into the initially sterile locuses of the human organism, they can cause infections. That is why any species of streptococci, that are found in blood have to be considered as etiological agents.
Out of streptococci, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus
pneumoniae are proven to be common pathogens for a human.
Streptococcus pyogenes falls into the beta-haemolytic group of streptococci, serologic
group A (Lancefield).
Streptococcus pyogenes has a big variety of virulence factors, including: • M protein, and M-type superficial proteins. These proteins prevent phagocytosis of
the streptococci by leukocytos. In our days, there are around 100 serotypes of M protein known. Antigen variability of the M proteins is used for typing.
• Capsule consists of the hyalyronic acid, which is similar to the mammal's one. It
forms the antigen mimicry, which results in difficulties when the immune respond is formed.
• F protein mediates the process of microorganism adhesion on the fibronectin.
• Pyrogenic exotoxins (A-C) have features of super antigens
• Hydrolytic enzymes: hyaluronidase, nuclease, neuraminidase, C5a peptidase (an
enzyme, destroying C5a component of the complement, which is a powerful chemoattractant).
• Hemolysines (streptolysin O and S.) Their biological role is not yet determined, but
it is believed that streptolysine O is responsible for the development of rheumatism.
• Streptokinase – an enzyme with the powerful fibrinolytic activity. Probably it is
involved in the streprococcus dissemination in tissues.
Most often Streptococcus pyogenes causes tonsillitis, pharyngitis, and skin infections
(pyoderm). Sometimes streptococci cause infections in other parts of the respiratory tract (otitis, sinusitis and pneumonia). Erysipelas and scarlatina are considered to be relatively rare infections, caused by Streptococcus pyogenes. Scarlatina is actually a reaction, caused by the pyogenic exotoxines, which circulate in blood. Some cultures of Streptococcus pyogenes show strict tissue tropism, though its mechanism is not yet studied enough.
A rare but feared complication of a localized infection is it's generalization with the
development of e.g. septicemia, bone affection in joints, endocarditis, meningitis followed by development of toxic stress. Necrotizing fasciitis is considered to be the most severe form of infection. It can be characterized by the fast development of necrosis of subcutaneous fat, bordering fascia and muscles due to streptococcal toxins.
Steptococcus pneumoniae (pneumococci). Pneumococci are considered to be alpha-
hemolytic streptococci without a serological type in the Lancefield system, but can be divided into over 50 serotypes based on variation of their capsular polysaccharide. Although they have a marked virulence (produce pneumolysin), pretty often they are found on the intact mucous membrane of the pharynx. Pneumococci are the leading etiological agents of pneumonia, and it is important to keep in mind, that some pneumococcical pneumonias have a very severe course, and are accompanied by bacteriemia. The finding of S. pneumoniae in a blood sample is a more specific proof of the etiological role of S. pneumoniae than the finding in a sputum sample. Also, pneumococci can be etiological agents of meningitis.
"Viridans" group of Streptococcus spp. This group includes around 20 species,
causing alpha-hemolys when cultured on chocolate agar. In the immunocompetent organisms streptococci of the "viridans" group can cause endocarditis. When streptococci are believed to be the cause of endocarditis, they should be identified up to the name of the species.
Streptococci viridans play a significant role in the pathology of patients with neutropenia
(especially in the affection of mucous membranes in mouth). Those patients risk to have bacteriemia accompanied by sepsis.
Enterococcus spp. In 1984 Enterococci were separated as an independent genus. This
was based on the results of studies on their DNA structure. Before that they were included in the genus Streptococcus. Nevertheless, in older literature (and sometimes even in contemporary) such names as Streptococcus faecalis, Streptococcus faecium are used. There are 17 species of enterococci known. These microorganisms cause no hemolysis. Serologically, some enterococci fall into the D group, but the majority can not be grouped.
Enterococci are the resident components of the intestinal microflora of man. However, in
some cases enterococci cause severe infections, but their virulence is relatively unknown. There are some data accumulated, proving that some features of the enterococci can be their factors of virulence. They include:
• The production of cytolysin, which performs lysis of the eukaryotic cell and gram-
positive microorganisms
• The production of metallic endopeptidase, which hydrolyzes gelatin, collagen,
• The production of aggregation protein, which helps enterococcus to stick to the cells
of the epithelium and microorganisms to aggregate. It is believed, that this protein is needed for the process of genetic exchange while performing conjugation.
It is important to mention, that all these virulence factors are common only for E.
faecalis. Rarely they are found in E. faecium.
Most often enterococci cause infection of the urinary tract, especially if it is a hospital
Enterococci are frequently found within intra-abdominal infections, however, almost
always in association with other microorganisms. Their etiological significance in the development of these infections is not apparent. In the case of wound infection enterococci colonize in the nidus of infection. There they can be found among other species who usually represent the original or true pathogen.
A third process, in which enterococci take part is nosocomial bacteriemia. It is usually
connected with central venous catheters.
It is very rare for enterococci to cause infection of any other localization (e.g. respiratory
tract, central nervous system)
Out of the infections, related to the surgeon's practice, and caused by enterococci it is
also logical to mention endocarditis.
Out of many catalase-negative gram-positive cocci that cause generalized infections
Leuconostoc and Pediococcus should be mention for their resistance to vancomycin in immune-suppressed patients. When they are misidentified, it can be reported that a vancomycin resistant enterococcus has been found.
1. Gram-positive rod-like bacteria
Gram-positive aerobic bacteria include representatives of the different taxonomic groups:
corynebacterii and coryne-shape bacteria, listeria, bacilli, aerobic actinomycetes and some other. The role in the human's pathology varies between microorganisms that are included in this group.
Corynebacterium spp. Among different species of the genus Corynebacterium there are
both obligatory pathogens such as the agent of diphtheria (C. diphtheriae), and some species that are typical representatives of opportunistic pathogens.
Clinically diphtheria (specific tonsillitis, myocarditis, kidney affection) depends on the
ability of some strains to produce toxins. Some strains of C. ulcerans can produce toxins and cause a disease that is clinically identical to the classical diphtheria.
Those cultures of C. diphtheriae that do not produce toxins can colonize skin and mucous
membranes of the pharynx without causing any clinical manifestation. Nevertheless, sometimes these cultures can cause skin affection (some epidemic episodes are known), endocarditis, arthritis and osteomyelitis. At present there is an idea that non-toxigenic cultures of C. diphtheriae can possess other virulence factors. • Many corynebacterii are normally present in the human microflora. It is noticed, that some
species have tropism to specific sites of the human body: skin (C. striatum), mucous membranes of the respiratory tract (C. durum), men's urogenital apparatus (C. glucuronoliticum, C. genitalium).
Such Corynebacteri or "diphteroids" are low virulent and are a rare cause of wound
infections, infections of the urogenital apparatus and respiratory tract, catheter associated infections. C. jeikeium (endocarditis, bacteriemia, infections of a foreign body) should be mentioned. Even less important types are C. amycolatum (bacteriemia, wound infections, infections of he urogenital apparatus and respiratory tract), C. glucuronoliticum, C. striatum, C. minutissimum, C. macginleyi, C. pseudodiphtericum and C. ulcerans. The natural antibiotic sensitivity of corynebacteri is not sufficiently studied due to the fact that it varies between different species and methods of evaluation of antibiotic sensitivity are not standardized. For example, C. diphtheriae is highly sensitive to the majority of antibiotics, while C jeikeium is resistant to most of them.
Listeria monocytogenes. Of other gram-positive rod-like microorganisms that are rarely
seen by the surgeon representatives of the genus Listeria should be mentioned, the most important one being L. monocytogenes. Listeria are widespread in nature: they are found in soil, water, milk and meat products. Healthy human and animals can be carriers of Listeria particularily in faeces without clinical symptoms. Clinically expressed listeriosis (meningitis, encephalitis, sepsis) is seen either among patients of the extreme age groups (e.g. newborn, elderly), or under conditions that are associated with troubles in the cell immune system.
Pregnant women can have listeriosis clinically looking like influenza, accompanied by bacteriemia. In case if not treated it can cause in amnionitis and fetus infection.
Diagnostics of listeriosis is based on classical microbiological methods. No express
methods are available. The importance of fast diagnostics of listeriosis if based on microorganism's natural resistance to cephalosporins and moderate resistance to fluoroquinolones. That means that empiric usage of these antibiotics will give no results.
Bacillus bacteria are gram-positive spore-forming rods with occasional clinical
significance in human pathology. The majority of these microorganisms are saprophytes. Their natural habitat is soil, and thanks to the endospore-forming ability they are extremely stable. The only, but extremely important obligatory pathogen of the genus Bacillus is the pathologic agent of anthrax – Bacillus anthracis. Thanks to the spore-forming ability this microorganism can stay alive for an unlimited period of time in the cattle mortuary, if this cattle died of anthrax. This can be a great danger for a very long time.
Human is relatively resistant to anthrax. The mortality is less then 20% in the skin forms,
even if without treatment. The intestinal form associated with usage of infected meat and primary-pulmonary forms caused by inhaling spores are much more severe and are accompanied with high mortality. Man to man transmission is very rare.
The role of Bacillus cereus in the human pathology is mainly determined by the
production of two toxins: thermolabile and thermostable, causing food poisoning. B. cereus is number two after S. aureus as the most important etiological agent of endophthalmitis. We are not sure this is the case in Sweden.
B. cereus can cause severe infections of different localizations (pneumonia, meningitis,
osteomyelitis, abscesses) in the immunosuppressed patients but these are rare. It is possible, that the etiological role of B. cereus is underestimated, since it is widespread in nature it is often thought that these microorganisms are only contaminates of the clinical samples. Rarely B. licheniformis, B. subtilis, B. circulans and some others can cause diseases in immuno-suppressed patients.
In general bacteria of the genus Bacillus should be considered to be opportunistic, and
therefore it is needed to pay more attention to their correct identification and evaluation of their clinical significance.
Aerobic actinomycetes are very varied taxonomical group of microorganisms. Among
them there are about 20 genera, and the number of species in some of these are up to the few hundreds. Their natural habitat is soil and water. Aerobic actinomycetes are opportunistic pathogens in the immune-suppressed patients, against the background of HIV infection. Out of all the actinomycetes Nocardia asteroids, N. brasiliensis, Rhodococcus equi, Actinomadura madure are causing human diseases. The mose severe clinical forms are invasive pulmonary infections with fast development of necrotic affections. Sometimes pulmonary affections can be developed slowly. The second most frequent clinical manifestations are skin and soft tissue infections of different severity (from slight chronical ulcerative affection to severe forms with generalization of the process). In the tropical areas these infections can occur in immuno-competent persons (mycetoma).
ii. Gram-negative microorganisms.
1. Gram-negative cocci
The group of gram-negative cocci incluses microorganisms of the genera Neisseria and
Branhamella (Taxonomical position of B. catarralis is not exactly clear). Taxonomically all the named microorganisms are close to microorganisms that have cocco-bacillary and rod-like shape – Kingella and Moraxella.
Neisseria includes: the obligatory pathogen N. gonorrhoeae, pathogenic microorganism
N. meningitides, and also a large number of nonpathogenic neisseria that colonize mucous membranes. In all the cases that discharge contains N. gonorrhoeae it means an infectious process. It is important to remember, that ghonorrhoea can proceed not only in local infections
(urethral, otopharyngeal and anorectal), but also in the form of the disseminated process with the affection on skin, joints, development of bacteriemia, meningitis and endocarditis.
In spite of that N. meningitides is highly pathogenic, causing e.g. meningitis, the
nasopharyngeal carriage rate in healthy humans may be high.
B. catarrhalis has the main clinical significance as the pathogenic agent of infections of
respiratory tract, though some cases of endocarditis and meningitis are known.
2. Gram-negative rod-like bacteria
Family of Enterobacteriaceae
Representatives of the Enterobacteriaceae family play a great role in the infectious
pathology of a human. It includes more then thirty genera and several hundred species of microorganisms such as Escherichia, Klebsiella, Enterobacter, Citrobacter, Serratia, Proteus, Providencia, Morganella. The role of many enterobacteria in the etiology of the abdominal and enteric infection is proven. These microorganisms have a number of virulence factors, helping bacteria in adhesion on epithelium, destroying eukaryotic cells and inducting anti-inflammatory cytokine production.
• In spite of all the research proving the importance of the microorganisms named, their
clinical significance should be evaluated every time when they are found in previously non-sterile locuses.
In the community enterobacteria most frequently cause infections of the urinary tract.
Their role in respiratory tract, skin and soft tissues diseases is relatively small. In hospital infections they are ranked as number 1-2 along with staphylococci due to their ability to cause severe infections of respiratory, urinary tracts, wound and intra-abdominal infections. No matter where the infection is localised there is always also a risk of a generalised process.
Elimination of the gram-positive flora from the upper respiratory tract and skin with
future colonization of those surfaces with gram-negative microflora is most common during the corse of hospital stay. There are several reasons for this e.g. physiological changes in the host, treatment with antibiotics.
Other gram-negative rod-like bacteria
Haemophilus spp. Bacteria of the genus Haemophilus are representatives of a human's
residential microflora in the upper air passages.
The most significant microorganism is H. influenzae, causing not only localized
infections of the upper air passages (otitis, sinusitis, exacerbation of chronical bronchitis) but sometimes also pneumonia and other severe invasive infections. It is typical for the strains causing severe infections to have polysaccharide capsule, which is considered to be the main virulence factor. Capsulated strains can be serologically typed into 6 serotypes. The most important one is serotype b. Most often H. influenzae of b serotype causes invasive infections in children up to 5 years including meningitis and epiglottitis, septic arthritis, osteomyelitis and pericarditis.
Non-encapsulated, and therefore not serotypable H. influenzae usually only cause
infections in the upper respiratory tract.
Out of other microorganisms of the genus Haemophilus H. parainfluenzae and H.
aphrophilus have some clinical significance due to their ability to sometimes cause the same type of infections as H. influenzae.
H. ducreyi causes chancroid.
Legionella spp. The genus Legionella includes more then 40 species. Approximately
half of them can cause human infection. The most widespread species are L. pneumophila and L. micdadei. Legionellae are free living microorganisms, and often can be found in soil and water, but aslo as as intracellular parasites of protozoa, living in water (e.g. amoeba). Inhalation of
small droplets of water contaminated with legionella may cause infection in humans. Outbreaks of legionellosis, connected with the massive colonization of cooling towers and the following contamination of the air conditioning systems are well known. Outbreaks due to contaminated water in large buildings such as hotels and hospitals have also been described. The risk is connected with taking a shower. There are many other situations when water with legionellae may be sprayed on people, e.g. city fountains. Microaspiration of water with legionella is also possible but is mainly regarded as a risk for the severely immunocompromised host.
The clinical picture in legionellosis can vary from light flu-type of disease up to severe
pneumonia. Extrapulmonary processes of the legionella etiology – pericarditis, myocarditis and endocarditis, pyelonephritis, peritonitis, abscesses of the gastro-intestinal tract are also known. Important for the pathogenesis is the ability of the bacteria to parasitize in human phagocytes. This complicates antibacterial treatment of legionellosis. Those antibiotics, that hardly penetrate in phagocytes (e.g. beta-lactames and aminoglycosides), show no clinical effect, drugs of choice are e.g. macrolides.
When dealing with severe therapy resistant pneumonia it is important to keep in mind the
possibility of legionella etiology.
Vibrio spp. Representatives of the genus Vibrio are free living microorganisms, whose
natural habitat is water of tropical seas and not so often freshwater. Microorganisms can be found in different aquatic life forms, mainly mollusks.
Traditionally V. cholerae, the pathologic agent of cholera, is considered to be the most
significant. The clinical symptoms of cholera depends on the production of the cholera toxins, which binds to ganglioside receptors of the intestine epithelium. Then it activates enzyme adenylate cyclase which leads to hypersecretion of liquid and electrolytes inside the intestine lumen.
It is important to mention that some vibrios (V. alginolyticus, V. parahaemoliticus, V.
damsela and others) can not only affect the gastro-intestinal tract, but also cause wound infections, including severe ones, which may be accompanied by bacteriemia. The usual way of getting the infection is swimming in the sea.
V. vulnificus may cause severe bacteriemia, accompanied by secondary suppurative
focuses, which need urgent treatment. This infection may occur after eating uncooked oysters. The infection strikes more often people that have accompanying liver diseases.
Aeromonas spp and Plesiomonas spp. As vibrios Aeromonas are free living water
microorganisms. However they usually live in fresh water. Aeromonas can cause both affection of gastro-intestinal tract, and extra-intestinal infections (skin and soft tissues, bones and joints, intra-abdominal, septicemia). Clinically the most significant species are A. hydrophila and A. veronii.
Plesiomonas shigelloides are also widespread in nature: they can be found in soil,
freshwater and sea-water. Also they can often be found in mollusks, sea and freshwater fish. Infection occurs after eating fresh or not enough cooked sea-products. The main clinical symptome of the infection is gastroenteritis. Also extra-intestinal affects (arthritis, meningitis, bacteriemia, cholecystitis, abscess of pancreas) can be found, usually among patients with immuno-suppression.
Basics of clinical microbiology for surgeons
Continued
Nonfermenting gram-negative bacteria
Pseudomonas spp. and allied microorganisms. In this group of bacteria Pseudomonas
aeruginosa is of the largest clinical significance.
Pseudomonas spp. are free living microorganisms, widespread in nature (soil and water).
Pseudomonas spp.often contaminate food products (fruits and vegetables). They are rarely found in healthy persons but can often cause hospital infections. Thanks to their ability to live in a humid environment, pseudomonas usually contaminate different solutions (including
disinfectants), equipment (in places where water is standing still) and wet surfaces. As a result of widespread contamination with pseudomonas in the environment, mucous membranes and the skin of vulnerable patients (e.g. immunocompromised or on antibiotics) are fast contaminated.
The clinical significance of pseudomonas is due to its ability to resist antibiotics, survive
and reproduce in the hospital environment, and the fact that these microorganisms (mainly P. aeruginosa) have many virulence factors (exotoxin A, exotoxin S, thermostable and thermolabile hemolysines, different proteases, collagenase and some others). Those virulence factors have the ability to bind GM-1 ganglionic receptors of the epithelium. The enzyme neuraminodase, is secreted by the microorganism and helps the specific adhesion by means of liberating sialic acids.
The lipopolysaccharide of P. aeruginosa is a powerful stimulator of the inflammatory
reaction. Some cultures of P. aeruginosa can produce capsule polysaccharide alginate. Cultures, producing alginate are usually found inpatients with chronic diseases such as mucoviscidosis (Cystisc fibrosis). Alginate helps bacteria to form the film on the cell's top (mucous surrounding the cell, biofilm). This film protects microorganisms from the resistance factors of the macroorganism and antibiotics. P. aeruginosa is described to have system of proteins's secretion (so called III type), which helps exotoxins excrection out of the bacterial cell, and their translocation into the eukaryotic cells, straight to the sensitive targets.
Since P. aeruginosa has virulence factors, infections caused by this microorganism is
potentially more dangerous, than those caused by potentially or opportunistic pathogenic microorganisms. They can occur in patients with burns, acute leucosis, mucoviscidosis and those on artificial ventilation. Infection is preceeded by antibiotic prophylaxes or therapy and is usually found in places where there is an accumulation of fluid. in tracheostomies, lower parts of the lungs, permanent catheter in urinary bladder, moist wounds and others. The problem of colonization of vascular catheters with P. aeruginosa is very important. The second most important members of the group are Burkholderia cepacia and Stenotrophomonas maltophilia. In general, these microorganisms are less virulent, compared to P. aeruginosa, and do not cause such severe infections. When these microorganisms are found, the question, whether it is a true infection or colonization has to be solved. The participation of B. cepacia in affections of the lung in patients with mucoviscidosis is its characteristic feature. S. maltophilia is characterized by its natural resistance to a large number of beta-lactam and other antibiotics, including carbapenems, though they are sensitive to co-trimoxazole. All the named microorganisms are most likely to be found when superinfection occurs during antibiotic therapy.
Burkholderia species includes B. mallei – pathologic agent of glanders and B.
pseudomallei – pathologic agent of myelosis.
Acinetobacter spp. Of the non-fermenting microorganisms bacteria belonging to the
Acinetobacter spp. are the second most important after P. aeruginosa. Acinetobacter is widespread in nature and in clinics, especially surgical ones, resuscitation departments and intensive care units. Microorganisms stay alive in many solutions, on dry and wet surfaces of the equipment and are able to colonize skin and mucous membranes of vulnerable patients very fast.
Acinetobacter is almost non-pathogenic for the healthy adults, while they can cause
severe infections in resuscitation departments and intensive care units. Most often these microorganisms cause ventilator-associated pneumonia, though their role in development of infections with different localizations (infections of the urinary tract, meningitis, endocarditis, osteomyelitis, peritonitis and others) is also proven. There are some cases of hospital epidemics, caused by these bacteria, described.
• When actinetobacter is found, the evaluation of its clinical significance and the
differentiation of infection from colonization should be performed.
Acinetobacterspp. strains causing hospital outbreaks are usually multiresistant and
selected by antibiotics.
Anaerobic microorganisms
Anaerobic microorganisms include a large number of taxonomically different microorganisms. This group includes all the morphological forms of gram-positive and gram-negative microorganisms. Many of them are able to form spores. Sometimes, Gram stain does not correspond with the structure of cell wall in anaerobic microorganisms. That is why special staining methods, developed especially for anaerobes (e.g. Kopeloff's method) should be used. Anaerobes are widespread in nature – they are found in soil, water, uliginous sediments, in food products and the majority of them do not play any role in the human pathology. At the same time some anaerobes are the main components of the human normal microflora. They colonize skin, vagina and gastro-intestinal tract from the mouth and down to the lower parts of the intestine. In some cases this fact can cause trouble in evaluating the clinical significance of anaerobes, when they are found in clinical material.
If a dedicated search for anaerobic microorganisms is performed, they can be found in
practically any clinical material. However, their clinical significance is questionable. In practice, clinical symptoms have the biggest significance in the diagnostics of the majority of anaerobic infections. This applies to infections, caused by spore-forming anaerobes (clostridia) that have a distinct clinical picture (gas-gangrene, tetanus, botulism), as well as to infections caused by non spore-forming anaerobic ("non-clostridia") bacteria that do not have such specific clinical symptoms. In the case of non-clostridia infections, color and smell of the discharge of the wound and a microscopic examination of smear, is more important for a rapid diagnosis, than a culture. Species' identification is associated with difficulties, and may require chromatographic analysis.
For practical purposes the following classification of anaerobes is appropriate:
• Spore-forming rod-like bacteria – pathogenesis and clinical symptoms of infections,
caused by these microorganisms are mainly due to the production of exotoxins. Spores may not be found when the primary material is examined.
• Non spore-forming rod-like bacteria and cocci - grouping rod-like bacteria and cocci
into one group is done because of the polymorphism they show on direct microscopy of clinical material.
• Rod-like bacteria
2.2.1. Spore-forming gram-positive rod-like bacteria
From clinical point of view spore-forming gram-positive rods belonging to the genera
Clostridium are the most significant ones among anaerobes. Their natural habitat is soil and the intestines of man and animal. Some Clostridium species are air-tolerant, though they do not form spores in the presence of oxygen. Man gets into contact with a lot of clostridia daily, while eating. As the result, the concentration of C. perfringens in the intestinal content is about 103-108 CFU per gram of faeces. Infection can be endogenous or exogenous. Clinical significance of different species of clostridia is determined by the variety of the exotoxins produced.
Gas-gangrene and similar diseases. In addition to C. perfringens, C. novyi, C.
septicum, C. histoliticum, C. bifermentans and some other species cause this clinical picture. The most severe clinical form is gas-gangrene (myonecrosis). Microorganisms produce a number of toxins: alpha, beta, epsilon and iota. Alpha-toxin is the main one. It has phospholipase, lecithinase and hemolytic activity. Also it has direct lethal and necrotizing
effects. Clostridia are typical opportunistic pathogens, since they cannot reproduce and produce toxins in intact tissues. The main factor, promoting their multiplication is the decrease in tissue oxygenation, i.e. necrosis associated with trauma, operative intervention, and blood supply disturbance. When the named conditions are met clostridia infection can develop in almost any area of the human body.
Gas-gangrene (clostridial myonecrosis) is the direct toxic destruction of muscular tissue,
accompanied by growth and spread of the microorganism, gas accumulation (hydrogen and nitrogen), expressed toxicoemia, and often bacteriemia. Most common localization for gas-gangrene is striated muscular tissue of the lower extremities. However, smooth muscular tissues, such as myometrium, can also be affected, for example after criminal abortion, or complicated parturition.
In addition to clostridia other bacteria that initiate production of host cytokines can cause
necrotic processes accompanied by gas accumulation in soft tissues but without muscle affection (crepitant, anaerobic cellulites). In some cases these diseases can be as severe as gas-gangrene. It is important to keep in mind that such effects accompanied by soft tissues crepitation (gas accumulation) can be caused by other microorganisms (anaerobic cocci, Bacteroides spp., Fusobacterium spp.).
For the diagnosis of gas-gangrene, and its differentiation from similar conditions, direct
microscopy of smears from lesions are most informative. In the case of gas-gangrene direct microscopy gives a typical picture with large relatively short and wide gram-positive rods. Spores and capsule may be absent. Microorganisms with other morphology are usually not found, or are present in small numbers. As a rule, inflammatory signs (leucocytes) are also absent. The finding of a polymicrobial flora and leukocytes in the smear is typical for anaerobic cellulites.
The leading role of clostridia in the development of necrosis in ischemic tissues is
obvious, but their significance in polymicrobial infections, especially intraabdominal ones is to be evaluated both clinically and microbilogically every time they are found.
When blood analysis is performed, clostridia (C. perfringens, C. ramnosum, C.
clostridioforme, C. bifermentans, C. sordelii, C. tertium and others) are often found in patients, which have had surgical intervention in the abdominal cavity, oncological processes of different localizations, pancreatic diseases, diabetes, chronic alcoholism and other conditions. If the general condition of the patient does not cause alarm, then such a finding should be interpreted as a transient bacteriemia, which does not require therapeutic treatment. Some peculiarities are connected with finding C. septicum in blood. There is a strongly marked correlation between bacteriemia, caused by C. septicum, and oncological affects in the intestines (lymphoma, carcinoma). Its mechanism is not studied yet.
Antibiotic-associated diarrhea and pseudomembranous colitis.
The extremely important microorganism is C. difficile. It causes an affection in the
gastro-intestinal tract varying from slight diarrhea to the severe pseudomembranous colitis. The pathogenesis being attributed to the production of toxins (A and B), which have a direct cytotoxic activity, and to the "substance, slowing the intestines' motion activity". The ability to produce toxins is not present in all cultures.
C. difficile is considered to be the main pathologic agent of diarrhea and
pseudomembranous colitis related to antibacterial therapy. However, it is important to keep in mind that this microorganism can be found in about 2-15% of healthy persons. The frequency of C. difficile in faeces rapidly increases in hospital patients, especially in those in surgical departments, and in intensive care units (this is considered to be the result of reduction of the part of the normal flora that is sensitive to antibiotics giving room for other bacteria). However, not all patients get diarrhea or pseudomembranous colitis. It should be accepted, that only the finding of C. difficile in faeces is not sufficient to make the etiological diagnosis of diarrhea. Only when toxin-generating ability of the culture or toxin in faeces is detected, it can be referred to as etiologically significant.
In spite of the obvious significance of C. difficile in the etiology of antibiotic-associated
diarrheas, the possible role of S. aureus and C. perfringens should not be forgotten.
Clostridia, producing neurotoxins.
Botulism. Botulinus toxin is the most powerful of the known toxic substances, produced
by microorganisms. Its mechanism of action lies in the blockade of neuromuscular transmission at the synapse level, due to the inhibition of acetylcholine discharge. The main microorganism, producing botulinic toxin is C. botulinum, but some cultures of C. butyricum and C. baratii can also do it. The possibility of some cultures of C. argentinense to produce botulinic toxins is questionable.
Classical botulism, which is associated with eating food, containing botulinus toxins, and
botulism, caused by the production of toxin in the wound should be distinguished clinically.
Tetanus. Toxic affection also includes tetanus or lockjaw – the pathology is caused by
the tetanus toxin (tetanospasmin), produced by C. tetani. Unlike botulinus toxin, tetanospasmin acts at central nervous system level (it blocks exocytosis of gamma-aminobutyric acid – negative regulator of the motor neuron's activity). The absence of negative regulation in motor neuron explains tetanus' clinical symptoms – simultaneous spastic tone disturbance of flexor and extensor muscle groups.
C. tetani is widespread in nature, including in intestines of mammals, but as a rule, the
development of the disease is connected with exogenous infection of small, but deep wounds (e.g. stab, gunshot wounds). In these cases the anaerobic conditions for growth and toxin production as well as connection with neural tissue are met.
Since the clinical picture is very typical, there is usually no need for a microbiological
2.2.2. Non spore-forming gram-positive rod-like bacteria
This group of microorganisms includes Actinomyces spp. (anaerobic actinomycetes),
Propionibacterium spp., Bifidobacterium spp., Lactobacillus spp., Eubacterium spp., Mobiluncus spp. and some others. Anaerobic gram-positive cocci can also obtain the cocco-bacillary form. This complicates their identification. The majority of microorganisms in this group are present in the normal human microflora, and can be characterized by the low pathogenicity.
Mucous membranes of mammals are the natural habitat for the majority of anaerobic
actinomycetes. Bifidobacterium spp., Lactobacillus spp., Eubacterium spp., Mobiluncus spp. are important components of the normal microflora in gastro-intestinal tract and vagina. Propionibacterium spp. is usually found on face, other skin areas and mucous membranes of the oral cavity.
Anaerobic actinomycetes, which cause chronic granulomatous inflammation with abscess
and fistula forming (usually on head and neck areas; less frequently – in the thoracic and abdominal cavities) have clinical significance. A. israelii is most common. Less often the disease is caused by A. naeslundii, A. odontoliticus, A. meyeri, A. pyogenes and A. viscosus. Usually, actinomycosis has a low-grade clinical course.
Propionibacterium spp. are often found in the mixed microflora during skin and soft
tissue's diseases. The role of Propionibacterium acnes in the development of acne is universally recognized. Propionibacterium spp. belongs to microorganisms, which often contaminate blood samples. However, some cases of clinically significant bacteriemia caused by these microorganisms are known.
Lactobacillus spp., very rarely can cause endocarditis, meningitis among newly born,
choriamnionitis and bacteriemia among immuno-suppressed patients.
2.2.3. Gram-positive cocci
Anaerobic gram-positive cocci are represented by two kinds – Peptococcus (consists of
the only species P. niger) and Peptostreptococcus (P. asacchrolyticus, P. magnus, P. anaerobius, P. micros, P. tetradius, P. prevotii and many others, with a total of 20 species). Occasionally
species of strictly anaerobic streptococci, which do not have an exact systematic position, can be found.
Anaerobic gram-positive cocci belong to the normal microflora of skin, upper airways,
gastro-intestinal tract and urinary tracts. The rate of these microorganisms in the vagina of pregnant women rises rapidly, and can be up to 90%. Gram-positive cocci have a low pathogenicity, due to the lack of virulence factors, and are considered opportunistic microorganisms in all the cases.
Traditionally, gram-positive anaerobic cocci are considered to be most likely in
infectious processes, localized above diaphragm. However, this is correct only in some cases. Indeed, gram-positive cocci are more often seen in infections of head and neck, chronic infections of the upper respiratory tract (otitis, sinusitis), periodontitis, in brain and lung abscesses and paratonsillar abscesses, rather than in other localizations. At the same time, the clinical significance of Peptostreptococcus spp. and P. niger is proven in the development of postpartum septic complications and in inflammatory diseases in the female pelvis minor. In case of infection these microorganisms are usually found in a mixed microflora. All the above can also be said about intra-abdominal infections.
2.2.4. Gram-negative rod-like bacteria
Out of the group of gram-negative anaerobic rod-like bacteria Bacteroides spp.,
Prevotella spp., Porphyromonas spp., Fusobacterium spp. have clinical significance. Their morphology can remind of rod-like, or cocco-bacillary forms. Taxonomy of anaerobic gram-neganive rod-like bacteria changed in the past years. Chromogenic microorganisms were singled out of Bacteroides spp and placed in the Prevotella and Porphyromonas group.
Gram-negative anaerobes are components of the normal microflora. Some of them are
mainly met in the distant parts of the digestive tract (Bacteroides spp.), others in the mouth cavity and upper air passages (the majority of Prevotella spp., Porphyromonas spp.).
In spite of the fact that microorganisms of Bacteroides spp usually are components of a
normal microflora in large intestines, they have some pathogenicity (they produce metalloproteases and enterotoxin). In a large number of cases (when the biomaterial is sampled correctly) can they be identified as the real pathologic agents of the infectious process. In small quantities bacteroides can also be found in the genital tract of women. Sometimes they are met in the mouth cavity and in upper air passages.
Within the Bacteroides spp, the group of so-called "Bacteroides fragilis" is singled out.
Out of this group B. fragilis and B. thetaiotaomicron are the most common pathologic agents of intra-abdominal infections, plus they can be met in infections of any other localization. In abscesses and other intraabdominal infectious Bacteroides are usually found as part of a synergistic infection together with aerobes (E. coli, eneterococci).They are often found in blood, and in most cases such findings can be evaluated as clinically significant. B. fragilis group also includes such a rare bacteria as B. diatasonis, B. ovatus and some other. Bacteroides, which are not included in the Bacteroides fragilis group are non-homogeneous taxonomically and have smaller clinical significance.
The majority species in the genera Prevotella, Porphyromonas and Fusobacterium cause
infectious processes, localized above diaphragm (mouth cavity, head and neck, upper and lower air passages). However there are some exclusions known. For example, Prevotella bivia and Prevotella disiens are most often found in the inflammatory diseases of women's pelvis minor.
2.2.5. Gram-negative cocci
Of the gram-negative cocci only microorganisms of Veillonella kind, which are included
in the normal microflora of gastro-intestinal tract have some clinical significance. Single species are present mostly in faeces' flora (V.parvula), others – in the mouth cavity and upper air passages. All the named microorganisms can be found in infections in different localizations. However, their clinical significance is always questionable.
C Yeast-like pathogenic fungi
At present there is a marked increase in infections caused by potentially pathogenic
yeasts belonging to Candida spp. The main factors, favouring the development of candidal affections are:
• Pancreatitis • Diabetes
• Immune status disorder (immunodeficiency)
• The usage of immunodepressing and cytostatic medications • The usage of corticosteroids
• The usage of antibacterial medications, especially broad spectrum antibiotics Candida spp and many other yeasts most often cause the affection of mucous membranes in
the mouth (thrush); vulvovaginitis, as the result of disturbance of the vaginal microflora; skin affections mainly in areas with high humidity (axilla, gluteal sulcus, inguinal region and some other). Candida infections can affect lungs, kidney, complicate the clinical course of wound infection. Some cases of candidal endocarditis were described among patients with different immunodeficiencies. These fungi can cause fungiemia in patients of the resuscitation department and intensive therapy units.
Yeast-like fungi of the Candida kind are included in Fungi imperfecti and form a
separate genus, including more then 80 species. However, not more than 10 species have clinical significance: Candida albicans, C.tropicalis, C.pseudotropicalis, C.krusei, C.guilliermondii, C.parapsilosis, C.glabrata and some other rarely met species. The most widespread among ill people is C. albicans.
In our days Cryptococcus neoformans gained big clinical significance, especially among
patients with immunodeficienses. This microorganism is very widespread in nature, particularly it is found in the pigeon's dried droppings. Human diseases, caused by cryptococcus usually are secondary. Most often patients with immunodeficiensis (e.g. aids), or ones that are taking immunodepressing therapy are affected. Infection can be accompanied by non-specific pulmonary symptoms. While the pulmonary infection gets disseminated, the affection of central nervousystem and other organs is very possible. CNS affection usually leads to chronic meningitis with frequent remissions and exacerbations. Cryptococcic meningitis has no typical symptomatology, and often there is a need to perform differentiatial diagnostics with tumour, brain abscesses and meningitis, caused by mycobacteria. Cryptococcic meningitis is characterized with long clinical course and high lethality, if no treatment is provided. This disease cannot be transferred from one human to another.
3. Microbiological diagnostics of infections
Methods and clinical interpretation of the results
The aim of microbiological diagnostics is to find the relevant microorganisms in the
clinical material and to perform antibiotic susceptibility testing.
When microorganisms are found in the clinical material, bacteriologist faces the problem
of interpreting the results. In the case when the microorganism is an obligatory pathogen, then finding even few microorganisms from any source, will have clinical significance.
In the case potentially pathogenic or opportunistic microorganism are found they should
be placed in one of the following categories: • Microorganism - true agent of the disease. The microorganism should be considered a true
agent of the disease when it is found in a prior-sterile locus of the human body, or when it is found in large numbers in a place where it is normally not found. For example, finding of E. coli in phlegm in a concentration of >105-6 /ml colony-forming units allows us to consider
this microorganism as the agent of pneumonia provided there are no epithelial cells in the smear.
• Microorganism – colonizing the locus. This category includes lightly pathogenic
microorganisms, when they are found in the unusual for them places in small quantities. For example, as the rule, Enterococcus spp., will not cause pathologic process in the wound, if the patient's immune system is competent.
• Microorganism – component of this locus' normal microflora. For example, this category
includes S. epidermidis, when it is found on skin and streptococci of the "viridance" group, when found on the mucous membranes of pharynx.
• Microorganisms, contaminating the clinical material. This category includes solitary
colonies of slightly pathogenic microorganisms (representatives of skin and air microflora). Contamination is possible not only while the material is gathered, but also in the process of examination (bacterial inoculation, filling of the Petri dish with nutrient medium).
The described method of interpreting the results is of great practical significance, since only
the real pathogenic microorganism should be the object of therapy. Knowledge about the contents of the normal microflora in non-sterile locuses of the human organism and pathogenic features of the microorganisms, described in previous chapters is the fundamental basis of the judging clinical significance of microorganisms.
Basics of microbiological investigations of particular biomaterials and
interpreting the results.
Blood examination.
Blood examination is the most important method to diagnose generalized infections in the
clinical practice. Direct bacterial inoculation of blood samples on dense nutrient medium is not used (except for some special situations), due to its lack of sensitivity. Traditionally, many laboratories use a vial with different nutrients in order to examine the blood. According to the existing normative documents, vials sowed with blood are incubated in thermostat at the temperature of 37° C. Vials are to be examined daily during the first 8 days (smears are prepared and Gram-stained). If bacterium is found, then culture on the 5% chocolate agar is made in order to separate the pure culture and identify it.
The usage of commercial vials with pre-made nutrient medias and arobic or anaerobic
atmosphere is a definite break through. When the volume of blood to investigate is relatively small, their use can be valid. Its disadvantage is that signs of bacterial growth (appearance of turbidity) can appear rather late. In order to reveal growth earlier, the periodical cultures on solid nutrient medium should be done.
The combination of automatic analyzers with commercial nutrient medium in vials is the
most progressive method of blood culturing. Dynamical registration of the growth of bacteria with special sensors is based on measurement of the physicochemical indexes (pH, oxidation-reduction potential, concentration of CO2) of the medium. In most cases the use of
hemoculture's analyzers permits registration of growth in the first 20 hours of incubation and excludes the need for making control cultures when the incubation period ends. High sensitivity of the nutrient medium in commercial vials (exposure of bacteria even if there are only few microorganisms) exacerbates the problem of false-positive answers, caused by contamination with skin microflora. There is also a clear risk of false negative blood cultures when the bacterial counts are very low, so that none are present in the limited volume of blood that can be cultured.
In order to reveal the false-positive answer, the following schedule for blood analysis was
suggested. After the puncture of an intact peripheral vein (the blood uptake by means of central catheter is performed only in special cases, e.g. for diagnostics of catheter-associated infections) two vials are filled (one for aerobic and second for anaerobic bacteria). After 20-30 minutes the
second intact vein is punctured and two more vials are filled (one for aerobic and second for anaerobic bacteria). The result is considered positive (real bacteriemia) if the same microorganism grows in 2 or more vials. The only disadvantage of this method is the high price of such an examination.
The final evaluation of the etiological significance of the microorganism found in blood,
is performed by the clinician with the respect to the following factors:
• The severity of patient's condition
• Presence of the manifest nidus of infection • Antibacterial therapy performed, and it's character
• Data of other laboratorial analyses
Bacteriological investigation of wound infection
Both surgeon and microbiologist have to consider the character of the wound and
the stage of the wound process, while planning microbiological examination of a wound. In general meaning a wound is any breach of the skin permitting microorganisms to penetrate into the deep, normally sterile tissues. From pathogenetic point of view, all wounds can be divided into acute and chronic [Bowler P.G., Duerden B.I., Armstrong D.G., 2001].
Environment (air and objects, that can cause mechanical damage), skin and mucous
membranes, bordering the wound are sources of microorganisms getting into the wound. In accordance with the source, either infection by free-living microorganisms from the environment or from the endogenous flora of skin and mucous membranes of upper air passages, gastrointestinal tract or urogenital area occurs.
The penetration of microorganisms into the wound can result either in its colonization, or
in development of clinically significant disease (wound infection). Infection process in wound leads to the additional destruction of tissues, and substantially slows the process of reparation. Besides, the risk of generalization of the infection appears. Subcutaneous and profound tissues of the human organism are quite favourable media for the growth of many microorganisms. If tissues lose viability, or their anti-infectious resistance is reduced it becomes an optimal situation for the bacteria. The main factor, determining the result of wound infection is the condition of tissue perfusion. It is well known, that well vascularized anal area wound infections are relatively rare, in spite of the massive microbe colonization.
The aim of microbiological analysis of wounds (same as for any microbiological sample)
is to discard or confirm the assumption of presence of a nidus in the wound, and in case it is found identify the main pathogen and choose adequate antibacterial therapy.
The most important criteria of an infectious nidus in the wound is a clear clinical
presentation. Finding of microorganisms together with oedema, hyperemia, pain in the area of the acute wound permits the conclusion that it is an infectious process. The situation is more complicated if it is a chronic wound. If microorganisms (usually a mixed flora) are found in the wound and the clinical presentation is not clearly indicating an infection, surgeon or microbiologist faces the question weather these pathogens affect the speed of wound repair.
There are two ways to solve this problem.
The role of high-virulence microorganisms in the pathogenesis of wound infections.
In accordance with the first point of view, the main role is played by high-virulence microorganisms, which cause an additional destruction of tissues and slowing the reparation of a wound. S. aureus, P. aeruginosa, S. pyogenes and enterobacteria are considered to be such microorganisms.
In chronic wounds the sign of infection and the indication for antibacterial therapy is
finding S. pyogenes and P. aeruginosa [Consensus meeting of the European Tissue Repair Society and the European Wound Management Association, 1998].
The hard task is to evaluate the role of anaerobic microorganisms in the pathogenesis of
wound infections. For instance, the American microbiological society's recommendations state,
that it is pointless to examine the open wound for anaerobic microorganisms. It is supposed, that such wounds are always contaminated with skin anaerobes and it is impossible to evaluate their clinical significance when found. The material's analysis with the purpose to find anaerobes is considered valid if dealing with closed cavities (abscesses, empyema and others), or intra-operative samples and biopsy material.
At the same time, it is known that many anaerobes (Prevotella and particularly,
Porphyromonas spp., Bacteroides, Fusobacterium spp., Peptostreptococcus spp., Clostridium spp. such as C perfringens) have determinants of virulence and can cause tissue destruction. Here it is appropriate to mention one more time, that in order to diagnose anaerobic infections such simple signs as wound appearance, smell and results of microscopic examination of Gram-stained smears are highly informative.
The role of a mixed flora in the pathogenesis of wound infections.
The second point of view is based on profound microbiological examinations of wound materials. In accordance with this point of view, in most cases wound microflora represents a complicated microbe association, and it is impossible to single out the leading pathogen. It is postulated, that additional destruction of tissues is appearing as the result of synergistic action of several pathogens. There are numerous examples of the increase in the growth of one microorganism when accompanied by other species. For example, staphylococci stimulate the growth and induct the production of virulence factors in anaerobes. Klebsiellae and other enterobacteria may perhaps stimulate the production of virulence factors in anaerobes. Numerous research reports reveal that wound repair slows down when the total microbe count is above 104-106 CFU per gram of tissue.
Based on the general ideas about the pathogenesis of wound infections it is possible to
make an assumption that most important clinically microorganisms will be found in samples,
which were gathered within vitalized tissues (in the area of granulation). In, pus discharge and
on superficial parts of the wound flora can be different and of little or no significance. However,
when the control is made, it is not always possible to prove the advantages of biopsy
examination
Cooperation of doctors with different specialities . The important conclusion of what has
been said above is the need for close cooperation between the surgeon and the microbiologist.
This is further illustrated with the following common practical example. A swab of cotton wool
in transport medium was received in the laboratory with the note "wound discharge". In this
situation the qualified microbiologist faces the question about the extent of examinations needed,
whether to culture on a limited number of media in order to search only for the most pathogenic
microorganisms or to examine also for anaerobes. It is obvious that in order to plan and perform
the analysis some additional information about the diagnosis, character of wound, its localization
and sampling and transport method is needed.
The same information is necessary for interpreting the results and as a base for
therapeutic recommendations. For example, moderate growth of S. aureus (even methicillin-resistant) in discharge from the surface of a wound without signs of inflammation is not a sign of infection and is not an indication for antibacterial therapy.
Cooperation between surgeons and microbiologists is also necessary in order to decrease
the number of examinations that give little information. The use of microbiological analysis is helpful when there are clinical signs of inflammatory process, especially at their beginning. If the wound process goes normally (wound cleansing, appearing of granulations), repeated examinations usually give no significant additional information. Slow healing of a wound, with the absence of clear clinical signs of wound infection is the second indication for microbiological analysis.
Hence, the volume of microbiological analysis totally depends on the surgeon's
knowledge of the process of wound healing.
Practical recommendations. In spite of the fact, that wound examination is one of the
most complicated question in clinical microbiology and many theoretical problems are still not solved, some practical recommendations can be given.
Getting the material – biopsy of tissues. The gain of samples with vitalized tissues out
of the deep parts of a wound, after it's cleansing and the removal of detritus is considered to be the most adequate method for both finding possible leading pathogens, and quantitative assessment of microbe semination.
It is necessary to follow rules of asepsis, while getting the biopsy material. Preliminary
management of wound is an important issue, since topical antiseptics, used for treatment of the wound, can cause distortion of the results if in the clinical material. Systemic antibacterial treatment also affects the results of analysis. It decreases the chance of finding the pathogen. Considering this fact, it is better to gather material, especially out of deep wound areas, right before the start of antibiotic therapy. It is also important to keep in mind that in case of vast wound, the use of several samples, gathered from different areas is advisable. Preference should be given to viable tissues.
Material gathering – wound liquids. Aspiration of the wound discharge with the help
of syringe is the most adequate method. Its use is advisable in order to get the material out of closed abscesses and deep parts of a wound (recess).
The most widespread, simple and cheap method of gathering the material is imprinting of
the wound discharge with swab of cotton wool. The noninvasiveness of this method is also very important advantage of it. It was suggested to use velveteen tissue, paper discs and similar devices for gathering the wound discharge, but no significant advantages were noticed, while using these methods.
Transportation of the material. The best results of microbiological examination are
seen when material is received in laboratory and the analysis started immediately. The amount of time between gathering of the material and the beginning of analysis should not exceed 2 hours. Transportation should be held within the room temperature, since when it is higher, microorganisms might reproduce, and that will affect magnitude relation of microorganisms in the results. If the temperature is below the suggested one, oxygen diffusion increases, and that will negatively affect viability of anaerobes.
Examining and evaluating results. The extent of microbiological examinations of the
wound material has to be evaluated separately in every single case. It can vary from quantitative evaluation of the main aerobic pathogens, up to quantitative contents of all the components of microflora, including anaerobes. Microscopy of a Gram-stained smear is the essential component of the analysis. At direct microscopy of a sample not only the morphology and quantitative ratio of different groups of microorganisms, but also the presence of leucocytes should be noticed. In most cases, the results of microscopy are really guiding the choice of antibacterial therapy (in case of the obvious predominance of gram-positive or gram-negative microorganisms).
The evaluation of the results of the analysis has to be composite, taking into account the
data of clinical presentation, microscopy of the smear and results of other examinations.
Significant problems are faced in interpreting the results, if there is no growth in clinical
material. There could be several reasons, leading to that:
• Presence of high concentrations of local or systemic antibacterial medicines in the
• Disturbance of taking, keeping and transporting the sample. • Anaerobic microorganisms represent the leading microflora.
• Methodological mistakes in laboratory
• Effective control of the infectious process with antibacterial drugs (especially while
examining biopsy materials of viable tissues).
If all the possible reasons of mistake are excluded, and the positive dynamics in wound is
seen, the last reason is very possible.
In the microbiological concluding information, characterizing the significance of the
pathogens found, that can help surgeon in the final evaluation of their role in infectious process has to be written.
Examining of the material, taken from the upper air ways.
The etiological diagnosis of pneumonia is based on examination of the blood (see above)
and material, collected from the lower air ways. Such a material can be airway secretions, sputum or discharge, gathered with help of invasive methods such as endotracheal aspirate, bronchoalveolar lavage, biomaterial, gathered with protected brushes or telescoping catheters. The examination of sputum, gathered while expectorating, is the least sensitive method of diagnostics. However, since in some cases sputum is the only accessible material, there is a need to be especially careful while gathering it.
Rules of sputum gathering: • Sputum has to be taken in the morning before having meal
• Mouth cleaning has to be done before sputum gathering (oral rinsing with boiled
• Patients have to be instructed about the need to get the contents of the lower parts of
air passages, not of the oral cavity or nasopharynx.
• Sputum has to be gathered in sterile disposable containers
The storage period of sputum gathered should not exceed 2 hours at room temperature. In ventilated patients, the use of invasive methods of gathering the material is indicated.
It is important to keep in mind, that material, gathered through tracheostoma has low diagnostic value. Usually material is gathered while doing bronchoscopy. In order to get lavage out of the affected lung area broncoscope is moved until "jams" in the appropriate segmental (to get bronchial scourage), or subsegmental (to get broncho-alveolar lavage) bronchus. Bronchial and broncho-alveolar lavages are gathered before getting the sample with cytological brush, or before biopsy in order to prevent blood from getting into tracheobronchical secret (blood can change the concentration of both cellular and non-cellular components of secret). The catheter is driven in the canal of bronchoscope, through which 5-15 ml of sterile 0.85% solution of sodium chloride is injected. After a few minutes the solution, is aspirated into the sterile container or sterile test-tube. Lavages gathered are quickly (during 15-20 minutes) transported into bacteriological laboratory. While transporting, it is important to keep samples from cooling!
When organizing the diagnostic process of pneumonia it is important to gather material
before the beginning of antibiotic therapy.
Before examining any material, gathered from lower air passages, Gram stained smear
should be prepared and microscoped. It is important to evaluate both: the character and quantity of microorganisms, and the amount of epithelial cells and leucocytes.
In conclusion it should be stated, that negative results of microbiological examination of
lower air ways (especially if repeated) can indicate a noninfectious nature of the process.
Urine analysis.
Urinary tract infections (UTI) can cause the hospitalization into the resuscitation
department or intensive care department, or also be the manifestation of different complications and intra-hospital infections. Microbiological diagnostics of UTI infections has to include both: urine and blood analysis. It should be considered a rule, in reanimation patient, to collect urine for analysis by special catheterization of urinary bladder by means of new sterile catheter.
The evaluation of the significance of microorganism, extracted from urine can be quite a
hard problem. The main guiding line should be quantitative data about the level of microorganism's semination in urine. Traditionally, the concentration of 105 CFU/ml and above in voided urine is considered significant. Nevertheless, in some cases even much lower
concentrations (104-103 CFU/ml or less) should be considered significant. In complicated diagnostic cases, in order to get urine without the contamination by microflora of the distant parts of UT, suprapubic puncture of urine bladder is needed and any level of bacteria is then indicating UTI.
The examination of faeces.
The practice of faeces examination is a point of contradiction in our medicine. The
analysis "dysbacteriosis check", focused on revelation of the quantitative and typical disorders in the contents of intestine's microflora, is widespread in Russian hospitals but not used in Western countries. Not touching the value and significance of such an analysis in other areas of medicine, it should be mentioned, that patients who after an operation stay in the intensive care unit or resuscitation department with multiorganic disorder and receive total or partly parenteral nutrition and massive antibacterial therapy, often have achange of the intestinal microflora with accompanying stool disorders. However, in most cases, after the diet normalizes and antibiotic therapy stop, these changes disappear without any additional medical therapy.
In patients of surgical and resuscitation departments the faeces analysis should be focused
only on the revealing the pathogenic microflora. Performing "dysbacteriosis check", due to it's high cost and low self-descriptiveness should be excluded. When a patient with signs of intestinal infection is received in surgical department, the analysis should be pointed on revealing such pathogens as Salmonella spp., Shigella spp., Vibrio cholerae, Campylobacter spp,Yersinia enterocolitica, Escherichia coli (enteropathogenic and enterotoxic cultuses, e.g. O157:H7). The presence of liquid stool in such a patient (especially if the patient is aged, or was receiving a long course of antibiotics) has to arouse suspicion of the development of antibiotic-associated diarrhea, caused by Clostridium difficile. The severe enterocolitis with blood and pus impurities in faeces indicates the development of pseudomembranous colitis. In this case, microbiological analysis has to be focused on finding C. difficile or rather on detection of its toxins.
Source: http://www.epicourse.narod.ru/SSI/English/Refs/Clinmicsr.pdf
INTRODUZIONE (Mt. 13,1-3a) Le Parabole del Regno (Mt. 13) I 5 "discorsi" del vangelo di Matteo Le 7 Parabole del Regno Discorso della montagna (Mt. 5-7) Parabola del seminatore Discorso Missionario (Mt. 10) Parabola della zizzania Discorso delle parabole (Mt. 13) Parabola del chicco di senape
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