Clinical microbiology and antimicrobial resistance

Clinical microbiology and antimicrobial resistance

Textbook references

Medical microbiology 8th edition by Murray, Rosenthal, Pfaller
Textbook of diagnostic microbiology 7th edition by Mahon Lehman

Oral exam dates

• 22 December 10.00
• 15-29 June 10.00
• 22 January 10.00
• 9-23 February 10.00

Basic knowledge of general microbiology

Chapters: 12-14-15-16-17-18-19-20-22-23-24-25-26-27-30-32-33-35

Clinical microbiology overview

Clinical microbiology is the section of microbiology dealing with clinical issues, such as microbial pathogens causing clinical syndromes, laboratory diagnosis of infectious diseases, and antimicrobial susceptibility tests. The role of the medical biotechnologist includes understanding the mechanism of diseases, development of new diagnostic tools, and development of new analytical tools.

Examples of human clinical syndromes

• Central nervous system infections: meningitis, encephalitis
• Respiratory tract infections: URTIs, LRTIs
• Skin and soft tissue infections
• Bone and joint infections: related to bacterial biochemistry
• Gastro-intestinal infections
• Intro-abdominal infections
• Urinary tract infections
• Surgical-site infections: different kinds of pathogens
• Diabetic foot infections: a peculiar syndrome difficult to treat
• Bloodstream infections

Syndromes can be combined (e.g., bloodstream infections following another one). Communicable diseases are those caused by infectious agents.

Global health statistics

The most updated analysis of the top 10 causes of death in upper-middle-income countries in 2016 lists low respiratory tract infections in the 6th position, while cardiac diseases are in the first one. In low-income countries, low respiratory tract diseases are the first cause of death, especially in children, followed by dermatological diseases, malnutrition, HIV, malaria, tuberculosis, and others. In the global top 10, the lower respiratory disease is in the fourth position.

Bacteria

Bacteria are prokaryotic cells (smaller than eukaryotic cells), and their genome consists of a single chromosome not contained by a nuclear membrane, having a circular form and being haploid (with some exceptions). There is a rigid cell wall crucial to withstand internal osmotic pressure, so if the bacterium cannot synthesize its cell wall, it reaches lysis and death. Their size varies from 0.3 μm to 0.5 μm. Prokaryotic ribosomes are 70s (50s+30s), whereas the eukaryotic are 80s (60s+40s). Mitochondria, Golgi apparatus, and endoplasmic reticulum are not present. In prokaryotic cells, the plasma membrane doesn’t contain sterols (in particular cholesterol), except for the mycoplasma, which is the only bacterium without a cell wall. The cell wall is mainly composed of peptidoglycans. The enzymes involved in cell respiration locate in the plasma membrane. The bacteria replicate by binary fission.

Bacteria have a rigid cell wall that contrasts the osmotic pressure of the cytoplasm and gives them shape. The spherical bacteria are called coccus, if more elongated bacillus, and if they have a curved form, they are called spirillum (e.g., Vibrio cholera) or spirochete (e.g., syphilis). Cocci can be arranged in pairs (diplococci) or chains (streptococci if regular or staphylococci if irregular clusters). Staphylococcus aureus has a grape shape. Streptococcus pyogenes has a regular chain shape, and when it produces a particular toxin, it is responsible for scarlet fever. Streptococcus pneumoniae (diplococcus) is responsible for meningitis and pneumonia in childhood.

Generally, we can divide the structure of the bacterial cell into fundamental, crucial to consider the cell viable, or accessory. The fundamental structures include chromosomes, cell wall, cytoplasm, and plasma membrane. Accessory structures are important for virulence and responsible for infection but not necessary to be viable; they include capsules, pili, flagella, and plasmids.

The chromosome contains genetic information, is a single DNA double strands molecule with a circular form, and contains approximately 5 million bp. There is no nuclear membrane. To be contained inside the cell, the chromosome must be supercoiled, so there are some enzymes responsible for the supercoiled and opposite structures.

The cytoplasm is a gelatinous solution containing water, proteins, nutrients, and genetic material. It contains ribosomes, storage bodies for nutrient purposes, and plasmids.

The plasma membrane of bacteria has a classical structure with two layers of phospholipids but does not contain sterols, unlike eukaryotic membranes (except for mycoplasma). Inside it, there are a lot of proteins, which can be transmembrane or anchored in the inner or outer layer. The great majority of metabolic pathways involve proteins anchored to the plasma membrane. These proteins perform functions such as electron and metabolite transport, energy accumulation and production, and enzyme involvement in chromosome duplication and cell wall synthesis. The inside of the membrane is lined with actin-like protein filaments that help determine the shape of the bacteria. The membrane contains hopanoids (bacteria steroid surrogates, serving the same role as sterols in our cell membranes) but lacks sterols. The membrane is characterized by a proton motive force used by enzymes, consisting of the accumulation of protons on the outside part of the membrane, which produces an electrical charge gradient for generating energy/exported molecules (Na-proton antiport).

The bacterial cell wall differs between Gram-positive and Gram-negative bacteria. The Gram-positive wall is made of a thick peptidoglycan layer and contains teichoic and lipoteichoic acids. The Gram-negative wall includes a thin peptidoglycan layer (without acids) and an outer membrane with a hydrophobic layer. The outer layer is present only in this kind of bacteria, making it more difficult to recognize and target them with a drug. The inner leaflet of the outer membrane is made of phospholipids, and the outer contains primarily lipopolysaccharides, which are composed of a lipidic part (outer layer) and a polysaccharide part. In the outer membrane, we find protein channels called pores.

The differing response to the Gram stain in Gram-negative and positive bacteria reflects the composition of the cell wall. The first step involves crystal violet entering both kinds, followed by the Gram iodine, which fixes the violet inside the cell. The third step involves using alcohol to decolorize the bacteria; Gram-positive bacteria cannot be decolorized due to the thick layer of polysaccharides that alcohol cannot destroy. The last step involves using safranin red, which enters the negative cells while the positive remain violet.

Peptidoglycan is a complex sugar important for cell stability and shape. It consists of long glycan chains made by two alternating sugars (N-acetyl muramic acid and N-acetyl-glucosamine). These long chains are bound by peptide cross-links; without them, the peptidoglycan is not stable. The peptide part is linked to the N-acetyl muramic acid, as this sugar has a COOH group that can attach amino acids. Specifically, this acid links a tetrapeptide (L-alanine, D-glutamate, L-lysine, D-alanine), and the cross-link is made by its L-lysine and the D-alanine of another acid. The link can be direct or have an interbridge made by 5 glycines.

Lipopolysaccharide (LPS) in Gram-negative bacteria

Lipopolysaccharide (LPS) constitutes the outer membrane of Gram-negative bacteria. It comprises a lipid part named lipid A, which is the endotoxin complex of these bacteria and composes the outer layer. The lipid A is released when the bacterium is lysed. The inner part consists of O-specific polysaccharide, which varies from species to species, and a constant part that is the same for all bacteria. The LPS is one of the most powerful immune stimuli.

Why is this endotoxin so dangerous? During an infection by Gram-negative bacterium, the endotoxin can be released after bacterium destruction. In our blood, there is a protein that binds specifically to the LPS (LPS binding protein); when the protein links to it, it activates macrophages to produce TNF, interleukins, and PAF. All these molecules cause activation of coagulation, prostaglandin leukotrienes production, and complement activation. This cascade modulates vasodilation, leading to endothelial damage, increased blood pressure, and finally, disseminated intravascular coagulation, which halts blood circulation. As a result, we have shock, necrosis of tissue, and organ damage. The deadliness of meningococcus is related to this kind of mechanism.

Accessory structures and their functions

The capsule is an accessory protective envelope, mainly of polysaccharides. It protects bacteria from phagocytosis, favors adherence and biofilm formation, and is the main virulence factor of some pathogens. Without it, they cannot induce diseases (e.g., meningitis). Fortunately, we have vaccines that protect us from meningitis and contain capsule polysaccharides.

Bacteria can express accessory appendages (fimbriae, pili) constituted by the polymerization of a globular protein called pilin. The fimbriae allow bacteria to adhere to specific surfaces of different tissues, based on the specific recognition of a cell receptor by a receptor located in the fimbria's apical part. Bacteria of the same species can cause different types of infections.

The pilus is used for conjugation, which differs entirely from fimbriae and is encoded by a gene contained in the conjugative plasmid.

The flagella are an accessory motility structure (not present in all bacteria). They are a protein-based structure, consisting of a basal body (differing between Gram-negative and positive), a rigid hook, and a rigid filament. The basal body uses energy usually provided by the cell. The location of flagella causes different types of movement, responding to chemotaxis stimuli. In some cases, flagella are very important virulent factors, as in Vibrio cholerae, where only cells with flagella are virulent. When flagella are located around the entire cell, it is called peritrichous; on the contrary, it is called polar. When peritrichous flagella move clockwise, the bacteria rotate. The polar flagellum can push or pull the cell.

Plasmids are accessory genetic elements containing extra chromosomal DNA double circular strands. They can replicate independently from chromosomes. Their size ranges from a few to hundreds of kD pairs. They are often horizontally transferred and can be important in antibiotic resistance, alternative metabolic pathways, virulence, resistance to heavy metals or other toxic elements, and bacteriocins.

Sporulation in bacteria

Some bacteria can form highly resistant spores, enabling them to survive adverse conditions (chemical and physical). The spores are metabolically inert and cannot replicate. The vegetative cell has an active metabolism and can replicate; under specific environmental conditions, this cell starts the sporulation process. At the end of this process, the spore is released. Clostridium tetani, Clostridium botulinum, Clostridium difficile (diarrhea), and Bacillus anthracis can sporulate. The inner part contains a condensed nucleoid (not needing access to the DNA), then a partially dehydrated cytoplasm (more protected from physical and chemical adverse conditions), followed by the plasma membrane and outside of this, the cortex. The cortex comprises peptidoglycans, but it is not as strong as the bacterial wall because there is no peptidoglycan replacement. The coat, made by specific proteins produced by the sporulation process, forms the outer layer. Sometimes, outside the coat, there is an exosporium made by proteins, polysaccharides, and lipids. The protein coat is the most important envelope.

Membrane vesicles in bacteria

Membrane vesicles are released by all bacterial genera (Gram-positive and negative), ranging between 20-400 nm in diameter, and they have been associated with important functions related to virulence, horizontal gene transfer, export of cellular metabolism, infection, and cell-to-cell communication. They have immunomodulatory activity, making them important for vaccines. There are different types of these vesicles with different functions, and related to these differences, there are many routes for vesicle generation. Many aspects concerning these vesicles are currently being investigated. In Gram-negative bacteria, these membrane vesicles are made by blebbing of the outer layer of the membrane and perform functions such as horizontal gene transfers, immunomodulation, toxin secretion, quorum sensing, and protein secretion.

Membrane vesicles have implications in biomedicine and nanotechnology; some are used in vaccination as natural membrane vesicles or with adjuvants or enriched in antigens or gold nanoparticles. Bioengineered membrane vesicles can express antigens. These vesicles can also be used for drug delivery, directing enzymes to the target.

There are different types of membrane vesicles, those produced by Gram-negative are the outer MV and the outer-inner MV (constituted by the outer and the plasma membrane). The Gram-positive bacteria produce cytoplasmic MV, possibly containing phage tails and heads.

• Blebbing of the outer membrane occurs due to imbalanced cell wall biosynthesis (e.g., after exposure to antibiotics) or intercalation of some hydrophobic molecules. The inner-outer membrane vesicles are also called explosive MV because they originate from explosive cell lysis after endolysin.
• In Gram-positive bacteria, cytoplasm vesicles are released by bubbling cell death as a consequence of endolysin of phage.

Polymyxin targets the LPS of Gram-negative bacteria, leading to outer MV formation; ciprofloxacin is associated with DNA damage and induces the activation of SOS response, causing the formation of outer-inner MV and explosive MV. β-lactams inhibit cell wall biosynthesis, causing cytoplasm MV. During polymyxin therapy, outer MV production can increase within biofilm formation, as these vesicles are part of the matrix, the main component of bacterial biofilm. Additionally, vesicles can protect bacteria from polymyxin. Different triggers induce membrane vesicle formation, with antibiotics in chemotherapy being the most important.

Antibacterial agents and antibiotics

Antibiotics must interact with their specific molecular target inside the cell, leading to alteration or blocking bacterial functions. When an antibiotic reaches its molecular target, it has two effects:

• Bacteriostatic effects: Reversible block of bacterial growth. Most antibiotics have bacteriostatic effects, but we cannot use them in immunodeficient patients to avoid exposing them to higher risks.
• Bactericidal effects: Death of bacteria, resulting in a decrease in the population.

The majority of antibiotics successfully used in treatments have bacteriostatic effects. Our immune system is very efficient, so it can kill bacteria after using bacteriostatic antibiotics. The compounds we use must be the most toxic possible for bacteria but the safest for us.

Antibiotic targets

• Each step of peptidoglycan synthesis: these targets are not present in our cells, but only in bacteria (e.g., Fosfomycin)
• Plasma membrane
• Transcription: different enzymes
• DNA topoisomerases: enzymes responsible for the supercoiled structure
• Protein synthesis: ribosomes differ from ours, so it is easy to create compounds that inhibit protein synthesis only in bacteria
• Folate synthesis pathway: absent in our cells

The majority of antimicrobial agents were developed before the 1960s; between 1960 and 2000, no new antibiotic class was introduced, creating an innovation gap. The new antimicrobial compounds mainly act against Gram-positives, so the biggest problem today is the lack of compounds active against Gram-negatives. Today, drug development is primarily based on private companies, making it clear that if these companies are the main investors, they prefer to invest in drugs that are not a resistance risk. It is more attractive for them to invest in chronic and life-long treatments instead of short-course ones like antibiotics.

Antibiotics inhibiting peptidoglycan synthesis

Peptidoglycan is an essential component of most bacterial cell walls; it composes the entire cell wall in Gram-positive, one layer of Gram-negative walls, and some layers of mycobacterial walls. The mycobacterial wall is peculiar, composed of one peptidoglycan layer covered by long-chain fatty acids. The last group has a peculiar wall that contains long-chain fatty acids.

Peptidoglycan is a macromolecule composed of a glycanic part of glycan chains and a sugar part of N-acetyl muramic acid and N-acetyl glucosamine. Peptidoglycan synthesis involves several steps:

1. Synthesis of N-acetyl muramic acid and N-acetyl glucosamine: The reaction starts with a UTO molecule binding one NAG-P to form UDP-NAG and release one PP. Then a transferase (MurA) adds phosphoenolpyruvate to UDP-NAG. A reductase reduces PEP to lactic acid and forms UDP-NAM. Lactic acid has a COOH group that can be used for peptide bonds. Next, the pentapeptide precursor is assembled in the NAM molecule. Amino acids are added in sequence. Two last D-alanines are added as a dimer by a ligase, and a racemase converts the D-alanine to L-alanine. These steps occur in the cytoplasm.
2. UDP-NAM links to undecaprenyl-P with the release of one UMP. NAG links to NAM by glycan bonds, forming the monomer ready.
3. The transporter must export this monomer to the cell wall.
4. The monomer is incorporated into the cell wall by transglycosylases, incorporating monomers into the growing chain through glycosidic bonds.
5. The last step is cross-linkage of nearby glycan by peptide bonds. Transpeptidases catalyze this reaction.

This reaction requires energy, provided by the cleavage of the peptide bond between the two D-alanines at the pentapeptide end. In the final form, there are 4 peptides. Fosfomycin can inactivate the MurA transferase, which adds PEP to the NAG.