Clinical microbiology and antimicrobial resistance

STAPHYLOCOCCI

Staphylococci are gram positive cocci, often aggregated in irregular or grapelike clusters. They are pyogenic and catalase positive (enzyme that cleaves hydrogen peroxide into water and oxygen). They are facultative anaerobes, but prefer growing in the presence of oxygen. They are extremely hardy, so they can survive a variety of environmental stresses. They are also halotolerant, so they may grow in conditions of high salinity (the majority of human pathogens do not have this characteristic). The medium commonly used to isolate staphylococci is the mannitol-salt agar, which contains 7.5% sodium chloride that inhibits the growth of most microorganisms. Mannitol-salt agar is a differential media because we can observe diverse colony morphology depending on the capacity of bacteria to ferment mannitol. When mannitol is fermented, acid is produced and the pH indicator of the medium turns from pink to yellow, while in the presence of non-fermenting staphylococci, the medium remains pink.

Staphylococcus aureus can ferment mannitol. Staphylococcus aureus is the clinically most important species, and it is coagulase positive. Other species are S.epidermidis, S.haemolyticus, S.saprophyticus and S.lugdunensis which are all coagulase negative. Coagulase is an enzyme that causes formation of clots, to perform its test we use rabbit plasma inoculated with 0.5 ml bacterial culture and incubates at 37°. Another method consists in adding a drop of rabbit plasma onto a microscope slide and mix one or few colonies into the drop of plasma. Then they observe the formation of clumps within 30 seconds. First method in the real coagulases test, the second one is capable to detect the presence of clumping factor, a protein located to staphylococcus surface and able to bind fibrinogen.

Staphylococci are common commensals of human and they can also be responsible of important human infections (hospital- acquired infections and community-acquired infections). They express remarkable antibiotic resistance.

they are associated to biofilm formation and infection.

Staphylococcus aureus

Gram positive cocci arranger in grapelike cluster, catalase positive and halotolerant, coagulasespositive and mannitol fermenter. It is a human commensal at 30-50% in nares and other tracts. Itcan cause localized infection and systemic infections. Colonization represent a risk factor fordeveloping infection.

Is able to produce a large number of virulence factors some of them localized in cell wall, inparticular the protein A is important because it is able to bind Fc region of IgG preventingopsonization from phagocytic cells. Variable region of IgG recognizes antigens, but if proteins A bindto constant region of iGg this mechanism cannot occur. At the level of cell wall there are alsoclumping factor and other adhesion factors which help colonization

Other virulence factors are exoenzymes like catalase, coagulase, staphylokinase (dissolves clots),hyaluronidases, proteases, lipases (tissue

Colonization state represents a risk factor for developing infection, so physicians must decide if it can be appropriate to perform a decolonization strategy in high-risk patients.

Staphylococcus aureus is the most relevant species of staphylococci spp. Staphylococcus aureus has some different virulence factors:

• Protein A: binds to the Fc region of IgG preventing opsonization and expressing an antiphagocytic effect.
• Clumping factor or bound coagulase: binds fibrinogen favoring clustering of bacterial cells in plasma.
• Other adhesion factors: bind to host macromolecules.
• Exoenzymes: catalase, coagulase, staphylokinase, hyaluronidase, proteases, lipases, etc.
• Cytolytic exotoxins: different groups of hemolysins, the alpha hemolysin subunits can assemble and create pores at the level of the plasma membrane of erythrocytes. Panton-Valentine Leukocidin is made by two peptides which are able to polymerize and interact with polymorphonucleates.

causing the release of inflammatory mediator and ROS and then citolysis. In low concentration it can damage mitochondrion. We can observe neutropenia in necrotizing pneumonia.

• Exotonxins which act as superantigens: antigens are able to force the interaction between MHC 2 and TCR of T cells and induce a non-specific activation of T cells. In this group we can find enterotoxins, exfoliatin or epidemolysin and toxic shock syndrome toxin.

Principal clinical manifestations (all except for exotoxin related disease are associated with endogenous self inoculation):

• Skin and soft tissue infections: folliculitis is a superficial infection, furuncle is a subcutaneous infection, carbuncle is formed by several furuncle (needs surgey), impetigo is a superficial infection common in child. Impetigo is caused also by streptococcus pyogenes, usually starts with painless blisterns on the face, then blisters fill with clear or yellow
• Bacteremia
• Osteomyelytis
• Pneumonia

Endocarditis

Abscessed

Exotoxin-related diseases: food poisoning is caused by consumption of food contaminated with staphylococcal enterotoxins (intoxications non infection) and do not require ingestion of viable staphylococci. Enterotoxins stimulate vague nerve, the CNS vomiting center and increase peristalsis. Few hours after ingestion of contaminated food.

Staphylococcal scaled skin syndrome is caused by strains encoding exfoliatin (also called epidermolysin), which degrades intercellular desmosomes, which provide tight binding between adjacent epithelial cells. This causes flaccid fluid-filled bullae resulting in lesion that may be very extensive and far from the primary source of infections, but remain superficial. Common in children.

Toxin shock syndrome is caused by strains encoding toxic shock syndrome toxin which enters the circulation and acts as superantigen stimulating lymphocytes and macrophages to produce cytokines that mediate shock syndrome. It is a life-threatening multisystem diseases.

associated to sunburn-type rash with fever, hypotension, confusion, nausea, vomiting and diarrhea. Possible evolution in multiple organs failure. It is associated to the use of superabsorbable tampons (withdrawn from market). Cases are mostly hospital acquired. Staphylococcus colonizes vaginal environment and cause the syndrome.

CNS-coagulase negative staphylococci. They are part of skin microbiota but they are opportunistic pathogens. They are frequent contaminants of blood cultures. They can create biofilm and biofilm-associated infection, also they can cause endocarditis.

Evolution of antibiotic resistance in staphylococcus aureus. In a pre-antibiotic era, staphylococcus aureus was susceptible to Penicillin G, and other beta-lactams but since Penicillin have been used in chemotherapy, staphylococcus acquired plasmid-mediated bet-lactamases which leaded to penicillin resistance. these resistant strains rapidly spread out in all over the world. after that new type of penicillins were introduced.

in therapy, but Staphylococcus acquire mecA genes encoding for PBP2a which are methicillin-resistant. Today all Staphylococcus aureus is able to produce beta-lactamases and methicillin-resistant Staphylococcus are common in hospitals but also in the community life. This type of staphylococci has been treated with glycopeptides, but in 1996 they became vancomycin-intermediated and in 2002 they became vancomycin resistant. The acquisition of the Van operon may occur during the colonization of the human or animal intestine by enterococci. This operon seems to be not very suitable for staphylococci as totally resistant strains emerge with very low frequency. In 2000, linezolid and daptomycin were introduced, but some years later, some resistant strains emerged. Cefoxitin is not used for therapy but it demonstrates to be the best surrogate for mecA-mediated methicillin/oxacillin resistance. Isolates that test as mecA positive should be reported as methicillin resistant, and all other beta-lactams.should be reported as resistance or should not be reported, because associated to clinical failure. Antibiotic resistant S.aureus can be found in hospital, communities, environment, farming etc.. in hospital preoperative screening and decolonization are associated with decreased transmission and surgical site infections, the majority of infecting strains are concordant to colonizing strains. In the community the prevalence of MRSA can vary from 0.2, to 7,4%. Test to identify a colonization are performed in nares, oropharynx and axilla, inguinal region and intestinal region (important because there are specific strains). It is not easy to find strategies to defeat or contain MRSA strains, which can circulate in all situations. When it is possible to do a decolonization, we use mupirocin. The antibacterial reagent mupirocin targets the bacterial IARS synthetic active site by blocking ile-AMP binding, it is use when there is the necessary. 5/11/2020 STREPTOCOCCUS The genus streptococcus comprisesmany different species, the most clinically relevant are S.pneumoniae, S.pyogenes and S.agalactiae, some other species are responsible for many different infections but have a minor role (S.dysgalactiae, S.suis, S.anginosus group ecc..) 21 They are gram positive cocci, aggregated in pairs or in chains (when grow in liquid medium), pyogenic (associated to infection with production of pus). They are negative for catalase test, anaerobes (unable to use oxygen), but aerotolerant so they can grow in the presence of oxygen. Usually increased carbon dioxide concentration stimulates their growth. Blood agar is required for optimal growth, this medium is use because streptococcus can produce exotoxin with hemolytic activity. Some streptococci are unable to produce hemolysis (medium unchanged), other can produce partial hemolysis (the medium becomes green or brown) and are called alfa-hemolysis, finally other can produce total hemolysis and are called beta-hemolysis (clearing of blood from the medium).

under and surrounding colonies). Streptococci are classified by their ability to induce hemolysis. Another important feature commonly used to classify streptococci is the antigen serogroup referred to a carbohydrate group located in the cell wall. This classification is called Lancefield classification. The group antigen is anchored to the plasma membrane and it crosses the peptidoglycans layer, there are different groups (20 indicated by letters). This carbohydrate is called C polysaccharides and depending on this nature, the classification is established.

Streptococci can be part of human or animal resident microbiota, the clinical relevance of such bacteria concerns upper and lower respiratory tract, urinary tract infections, endocarditis exc….

Streptococcus Pyogenes

It belongs to the Lancefield group A (GAS), it is beta-hemolytic and causes pharyngitis, scarlet fever, pyogenic infections, impetigo, rheumatic fever and glomerulonephritis. It is usually arranged in chain, it is gram

be concluded that the bacteria is Gram-negative and catalase-negative.

It can be concluded that the bacteria is Gram-negative and catalase-negative.