MINISTRY OF HEALTH CARE OF UKRAINE Kharkiv National Medical University D.P. Grynyov department of microbiology, virology and immunology STANDARD PROTOCOLS TO LABORATORY CLASSES IN SPECIAL MICROBIOLOGY for the II and III year English media students PART 3 student year group Surname Teacher Kharkiv 2018 Standard protocols to laboratory classes in special microbiology for the II and III year English media students of medical and dentistry faculties / M.M. Mishyna, Yu.A. Mozgova, N.I. Kovalenko, T.M. Zamaziy, O.O. Vovk. – Kharkiv: KNMU, 2018. – 152 p. Special microbiology Protocol № 33 Theme: Grampositive cocci (staphylococci, streptococci). I. Observe the smears below. Using appropriately colored pencils draw the following cells. Staphylococcus aureus (Gram stain) Streptococcus pyogenes (Gram stain) Streptococcus pneumoniae (Methylene blue stain) II. Study scanning electron micrographs: Staphylococcus Streptococcus pyogenes Streptococcus pneumoniae III.Study nutrient media for cultivation of staphylococci and streptococci: а) MPB and MPA, blood agar, egg yolk salt agar for staphylococci; б) serum and sugar broth for streptococci; в) ascetic broth for pneumococci; г) Hiss media. IV.Study culture and biochemical properties of staphylococci: а) growth on MPB: ___________________________________________________________; b) growth on MPA: ______________________________________________________; c) growth on blood agar: ______________________________________________________; d) growth on egg yolk salt agar: ________________________________________________; g) media with mannitol (only S. aureus can ferment mannitol) and optochin (S. pneumoniae is sensitive while S. mitis (normal flora) is resistant). е) sugar fermentation and protein hydrolysis by staphylococci on Hiss media and MPB: Species Lactose Glucose Mannitol Maltose Sucrose MPB H2S Indol S.aureus acid acid acid acid acid + - S.epidermidis acid acid - acid acid + - S.saprophyticus acid acid acid acid acid + - http://student.ccbcmd.edu/courses/bio141/labmanua/lab14/dkspyog.html S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ V. Spesific therapy of staphylococcal infections - immunoglobulin. Spesific prophilaxis of Streptococcus pneumoniae infections - groups A, C, AC, and ACYW135 capsular polysaccharide vaccines are available. VI.Therapy of staphylococcal infections: a first- and second-generations cephalosporin: cefasolin, cefuroxim, cefaclor, cefoxitin; a third-generation cephalosporin: cefotaxime or ceftriaxone; penicillins: oxacillin, ampicillin, amoxicilin+clavulanic acid (Amoxiclav), ampicillin+oxacillin (Ampiox); fluroquinolones: lomefloxacin, moxifloxacin, levofloxacin; vancomycin, clindamycin, erythromycin, tetracycline, etc. VII. Study antibiotics sensitivity testing of Staphylococci – disk diffusion test. VIII. Study the schemes of laboratory diagnosis of staphylococcal and streptococcal infections. Phage typing after Fisher (to detect origin of the source of infection): • It is the identification of bacterial species and strains by determining their susceptibility to various phages. – The testing strain is infected by bacteriophages. – After incubation, a lytic phage infection will result in plaques = no bacterial growth. 2 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ ADDING THEORETICAL MATERIAL Bacteria in the genus Staphylococcus are pathogens of man and other mammals. Traditionally they were divided into two groups on the basis of their ability to clot blood plasma (the coagulase reaction). The coagulase-positive staphylococci constitute the most pathogenic species S. aureus. The coagulase- negative staphylococci (CNS) are now known to comprise over 30 other species. The CNS are common commensals of skin, although some species can cause infections. It is now obvious that the division of staphylococci into coagulase positive and negative is artificial and indeed, misleading in some cases. Coagulase is a marker for S. aureus but there is no direct evidence that it is a virulence factor. Also, some natural isolates of S.aureus are defective in coagulase. Nevertheless, the term is still in widespread use among clinical microbiologists. Staphylococci can cause many forms of infection. (1) S. aureus causes superficial skin lesions (boils, styes) and localized abscesses in other sites. (2) S. aureus causes deep-seated infections, such as osteomyelitis and endocarditis and more serious skin infections (furunculosis). (3) S. aureus is a major cause of hospital acquired (nosocomial) infection of surgical wounds and, with S. epidermidis, causes infections associated with indwelling medical devices. (4) S. aureus causes food poisoning by releasing enterotoxins into food. (5) S. aureus causes toxic shock syndrome by release of superantigens into the blood stream. (6) S.saprophiticus causes urinary tract infections, especially in girls. (7) Other species of staphylococci (S. lugdunensis, S. haemolyticus, S. warneri, S. schleiferi, S. intermedius) are infrequent pathogens. Structure. Staphylococci are Gram-positive cocci 1μm in diameter. They form clumps. Classification. S. aureus and S. intermedius are coagulase positive. All other staphylococci are coagulase negative. They are salt tolerant and often hemolytic. Identification requires biotype analysis. S aureus colonizes the nasal passage and axillae. S. epidermidis is a common human skin commensal. Other species of staphylococci are infrequent human commensals. Some are commensals of other animals. Pathogenesis. S. aureus expresses many potential virulence factors. (1) Surface proteins that promote colonization of host tissues. (2) Factors that probably inhibit phagocytosis (capsule, immunoglobulin binding protein A). (3) Toxins that damage host tissues and cause disease symptoms. Coagulase-negative staphylococci are normally less virulent and express fewer virulence factors. S.epidermidis readily colonizes implanted devices. Antibiotic Resistance. Multiple antibiotic resistance is increasingly common in S. aureus and S. epidermidis. Methicillin resistance is indicative of multiple resistance. Methicillin-resistant S.aureus (MRSA) causes outbreaks in hospitals and can be epidemic. Diagnosis is based on performing tests with colonies. Tests for clumping factor, coagulase, hemolysins and thermostable deoxyribonuclease are routinely 3 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ used to identify S.aureus. Commercial latex agglutination tests are available. Identification of S epidermidis is confirmed by commercial biotyping kits. Identification of Staphylococci in the Clinical laboratory Staphylococci are Gram-positive cocci about 0.5 – 1.0 μm in diameter. They grow in clusters, pairs and occasionally in short chains. The clusters arise because staphylococci divide in two planes. The configuration of the cocci helps to distinguish micrococci and staphylococci from streptococci, which usually grow in chains. Observations must be made on cultures grown in broth, because streptococci grown on solid medium may appear as clumps. Several fields should be examined before deciding whether clumps or chains are present. Catalase Test The catalase test is important in distinguishing streptococci (catalase- negative) staphylococci which are catalase positive. The test is performed by flooding an agar slant or broth culture with several drops of 3% hydrogen peroxide. Catalase-positive cultures bubble at once. The test should not be done on blood agar because blood itself will produce bubbles. Isolation and Identification The presence of staphylococci in a lesion might first be suspected after examination of a direct Gram stain. However, small numbers of bacteria in blood preclude microscopic examination and require culturing first. The organism is isolated by streaking material from the clinical specimen (or from a blood culture) onto solid media such as blood agar, tryptic soy agar or heart infusion agar. Specimens likely to be contaminated with other microorganisms can be plated on yolk salt agar, milk salt agar or mannitol salt agar containing 7.5% sodium chloride, which allows the halo-tolerant staphylococci to grow. Ideally a Gram stain of the colony should be performed and tests made for catalase and coagulase production, allowing the coagulase-positive S. aureus to be identified quickly. Another very useful test for S. aureus is the production of thermostable deoxyribonuclease. S. aureus can be confirmed by testing colonies for agglutination with latex particles coated with immunoglobulin G and fibrinogen which bind protein A and the clumping factor, respectively, on the bacterial cell surface. These are available from commercial suppliers (e.g., Staphaurex). The most recent latex test (Pastaurex) incorporates monoclonal antibodies to serotype 5 and 8 capsular polysaccharides in order to reduce the number of false negatives. (Some recent clinical isolates of S. aureus lack production of coagulase and/or clumping factor, which can make identification difficult.) Because S. aureus is a major cause of nosocomial and community-acquired infections, it is necessary to determine the relatedness of isolates collected during the investigation of an outbreak. Typing systems must be reproducible, discriminatory, and easy to interpret and to use. The traditional method for typing S. aureus is phage-typing. This method is based on a phenotypic marker with poor reproducibility. Pathogenesis of S. aureus Infections. S. aureus expresses many cell surface-associated and extracellular proteins that are potential virulence factors. For the majority of diseases caused by this 4 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ organism, pathogenesis is multifactorial. Thus it is difficult to determine precisely the role of any given factor. This also reflects the inadequacies of many animal models for staphylococcal diseases. However, there are correlations between strains isolated from particular diseases and expression of particular factors, which suggests their importance in pathogenesis. With some toxins, symptoms of a human disease can be reproduced in animals with pure proteins. The application of molecular biology has led to recent advances in the understanding of pathogenesis of staphylococcal diseases. Genes encoding potential virulence factors have been cloned and sequenced and proteins purified. This has facilitated studies at the molecular level on their modes of action, both in in vitro and in model systems. In addition, genes encoding putative virulence factors have been inactivated, and the virulence of the mutants compared to the wild-type strain in animal models. Any diminution in virulence implicates the missing factor. If virulence is restored when the gene is returned to the mutant then “Molecular Koch's Postulates” have been fulfilled. Several virulence factors of S. aureus have been confirmed by this approach. S. aureus cells express on their surface proteins that promote attachment to host proteins such as laminin and fibronectin that form part of the extracellular matrix. Fibronectin is present on epithelial and endothelial surfaces as well as being a component of blood clots. In addition, most strains express a fibrinogen/fibrin binding protein (the clumping factor) which promotes attachment to blood clots and traumatized tissue. Most strains of S. aureus express fibronectin and fibrinogen-binding proteins. The receptor which promotes attachment to collagen is particularly associated with strains that cause osteomyelitis and septic arthritis. Interaction with collagen may also be important in promoting bacterial attachment to damaged tissue where the underlying layers have been exposed. Evidence that these staphylococcal matrix-binding proteins are virulence factors has come from studying defective mutants in vitro adherence assays and in experimental infections. Mutants defective in binding to fibronectin and to fibrinogen have reduced virulence in a rat model for endocarditis, suggesting that bacterial attachment to the sterile vegetations caused by damaging the endothelial surface of the heart valve is promoted by fibronectin and fibrinogen. Similarly, mutants lacking the collagen-binding protein have reduced virulence in a mouse model for septic arthritis. Furthermore, the soluble ligand-binding domain of the fibrinogen, fibronectin and collagen-binding proteins expressed by recombinant methods strongly blocks interactions of bacterial cells with the corresponding host protein. Capsular Polysaccharide The majority of clinical isolates of S. aureus express a surface polysaccharide of either serotype 5 or 8. This has been called a microcapsule because it can be visualized only by electron microscopy after antibody labeling, unlike the copious capsules of other bacteria which are visualized by light microscopy. S. aureus isolated from infections expresses high levels of polysaccharide but rapidly loses it upon laboratory subculture. The function of the 5 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ capsule is not clear. It may impede phagocytosis, but in in vitro tests this was only demonstrated in the absence of complement. Conversely, comparing wild-type and a capsule defective mutant strain in an endocarditis model suggested that polysaccharide expression actually impeded colonization of damaged heart valves, perhaps by masking adhesins. Protein A Protein A is a surface protein of S. aureus which binds immunoglobulin G molecules by the Fc region. In serum, bacteria will bind IgG molecules the wrong way round by this non-immune mechanism. In principle this will disrupt opsonization and phagocytosis. Indeed mutants of S. aureus lacking protein A are more efficiently phagocytozed in vitro, and studies with mutants in infection models suggest that protein A enhances virulence. Leukocidin S. aureus can express a toxin that specifically acts on polymorphonuclear leukocytes. Phagocytosis is an important defense against staphylococcal infection so leukocidin should be a virulence factor. This toxin is discussed in more detail in the next section. S. aureus can express several different types of protein toxins which are probably responsible for symptoms during infections. Some damage the membranes of erythrocytes, causing hemolysis; but it is unlikely that hemolysis is relevant in vivo. The leukocidin causes membrane damage to leukocytes and is not hemolytic. Systemic release of α-toxin causes septic shock, while enterotoxins and TSST-1 cause toxic shock. Membrane Damaging Toxins (A) The best characterized and most potent membrane-damaging toxin of S. aureus is α-toxin. It is expressed as a monomer that binds to the membrane of susceptible cells. Subunits then oligomerize to form hexameric rings with a central pore through which cellular contents leak. Susceptible cells have a specific receptor for α-toxin which allows low concentrations of toxin to bind, causing small pores through which monovalent cations can pass. At higher concentrations, the toxin reacts non-specifically with membrane lipids, causing larger pores through which divalent cations and small molecules can pass. However, it is doubtful if this is relevant under normal physiological conditions. In humans, platelets and monocytes are particularly sensitive to α-toxin. They carry high affinity sites which allow toxin to bind at concentrations that are physiologically relevant. A complex series of secondary reactions ensue, causing release of eicosanoids and cytokines which trigger production of inflammatory mediators. These events cause the symptoms of septic shock that occur during severe infections caused by S. aureus. The notion that α-toxin is a major virulence factor of S. aureus is supported by studies with the purified toxin in animals and in organ culture. Also, mutants lacking α-toxin are less virulent in a variety of animal infection models. (B) β-toxin is a sphingomyelinase which damages membranes rich in this lipid. The classical test for β-toxin is lysis of sheep erythrocytes. The majority of 6 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ human isolates of S.aureus do not express β-toxin. A lysogenic bacteriophage is inserted into the gene that encodes the toxin. This phenomenon is called negative phage conversion. Some of the phages that inactivate the β-toxin gene carry the determinant for an enterotoxin and staphylokinase. In contrast the majority of isolates from bovine mastitis express β-toxin, suggesting that the toxin is important in the pathogenesis of mastitis. This is supported by the fact that β-toxin-deficient mutants have reduced virulence in a mouse model for mastitis. (C) The δ-toxin is a very small peptide toxin produced by most strains of S. aureus. It is also produced by S. epidermidis and S. lugdunensis. The role of δ- toxin in disease is unknown. (D) The γ-toxin and the leukocidins are two-component protein toxins that damage membranes of susceptible cells. The proteins are expressed separately but act together to damage membranes. There is no evidence that they form multimers prior to insertion into membranes. The γ-toxin locus expresses three proteins. The B and C components form a leukotoxin with poor hemolytic activity, whereas the A and B components are hemolytic and weakly leukotoxic. The classical Panton and Valentine (PV) leukocidin is distinct from the leukotoxin expressed by the γ-toxin locus. It has potent leukotoxicity and, in contrast to γ-toxin, is non-hemolytic. Only a small fraction of S. aureus isolates (2% in one survey) express the PV leukocidin, whereas 90% of those isolated from severe dermonecrotic lesions express this toxin. This suggests that PV leukocidin is an important factor in necrotizing skin infections. PV-leukocidin causes dermonecrosis when injected subcutaneously in rabbits. Furthermore, at a concentration below that causing membrane damage, the toxin releases inflammatory mediators from human neutrophils, leading to degranulation. This could account for the histology of dermonecrotic infections (vasodilation, infiltration and central necrosis). S U P E R A N TI G EN S : E NT ER O T OX I NS A ND T O XI C S H OC K S Y N DR OM E T OX I N S. aureus can express two different types of toxin with superantigen activity, enterotoxins, of which there are six serotypes (A, B, C, D, E and G) and toxic shock syndrome toxin (TSST-1). Enterotoxins cause diarrhea and vomiting when ingested and are responsible for staphylococcal food poisoning. When expressed systemically, enterotoxins can cause toxic shock syndrome (TSS) - indeed enterotoxins B and C cause 50% of non-menstrual TSS. TSST-1 is very weakly related to enterotoxins and does not have emetic activity. TSST-1 is responsible for 75% of TSS, including all menstrual cases. TSS can occur as a sequel to any staphylococcal infection if an enterotoxin or TSST-1 is released systemically and the host lacks appropriate neutralizing antibodies. Tampon-associated TSS is not a true infection, being caused by growth of S aureus in a tampon and absorption of the toxin into the blood stream. TSS came to prominence with the introduction of super-absorbent tampons; and although the number of such cases has decreased dramatically, they still occur despite withdrawal of certain types of tampons from the market. 7 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ Superantigens stimulate T cells non-specifically without normal antigenic recognition. Up to one in five T cells may be activated, whereas only 1 in 10,000 are stimulated during antigen presentation. Cytokines are released in large amounts, causing the symptoms of TSS. Superantigens bind directly to class II major histocompatibility complexes of antigen-presenting cells outside the conventional antigen-binding grove. This complex recognizes only the Vβ element of the T cell receptor. Thus any T cell with the appropriate Vβ element can be stimulated, whereas normally antigen specificity is also required in binding. E P I D ER M O L Y TI C ( E XF O L I A TI VE ) TO XI N ( E T) This toxin causes the scalded skin syndrome in neonates, with widespread blistering and loss of the epidermis. There are two antigenically distinct forms of the toxin, ETA and ETB. There is evidence that these toxins have protease activity. Both toxins have a sequence similarity with the S. aureus serine protease, and the three most important amino acids in the active site of the protease are conserved. Furthermore, changing the active site of serine to a glycine completely eliminated toxin activity. However, ETs do not have discernible proteolytic activity but they do have esterase activity. It is not clear how the latter causes epidermal splitting. It is possible that the toxins target a very specific protein which is involved in maintaining the integrity of the epidermis. OTHER EXTRACELLULAR PROTEINS Coagulase is not an enzyme. It is an extracellular protein which binds to prothrombin in the host to form a complex called staphylothrombin. The protease activity characteristic of thrombin is activated in the complex, resulting in the conversion of fibrinogen to fibrin. This is the basis of the tube coagulase test, in which a clot is formed in plasma after incubation with the S. aureus broth-culture supernatant. Coagulase is a traditional marker for identifying S. aureus in the clinical microbiology laboratory. However, there is no evidence that it is a virulence factor, although it is reasonable to speculate that the bacteria could protect themselves from host defenses by causing localized clotting. Notably, coagulase deficient mutants have been tested in several infection models but no differences from the parent strain were observed. There is some confusion in the literature concerning coagulase and clumping factor, the fibrinogen-binding determinant on the S. aureus cell surface. This is partly due to loose terminology, with the clumping factor sometimes being referred to as bound coagulase. Also, although coagulase is regarded as an extracellular protein, a small fraction is tightly bound on the bacterial cell surface where it can react with prothrombin. Finally, it has recently been shown that the coagulase can bind fibrinogen as well as thrombin, at least when it is extracellular. Genetic studies have shown unequivocally that coagulase and clumping factor are distinct entities. Specific mutants lacking coagulase retain clumping factor activity, while clumping factor mutants express coagulase normally. Staphylokinase Many strains of S. aureus express a plasminogen activator called staphylokinase. The genetic determinant is associated with lysogenic bacteriophages. A complex formed between staphylokinase and plasminogen 8 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ activates plasmin-like proteolytic activity which causes dissolution of fibrin clots. The mechanism is identical to streptokinase, which is used in medicine to treat patients suffering from coronary thrombosis. As with coagulase there is no evidence that staphylokinase is a virulence factor, although it seems reasonable to imagine that localized fibrinolysis might aid in bacterial spreading. S. aureus can express proteases, a lipase, a deoxyribonuclease (DNase) and a fatty acid modifying enzyme (FAME). The first three probably provide nutrients for the bacteria, and it is unlikely that they have anything but a minor role in pathogenesis. However, the FAME enzyme may be important in abscesses, where it could modify anti-bacterial lipids and prolong bacterial survival. The thermostable DNase is an important diagnostic test for identification of S. aureus. Since the beginning of the antibiotic era S. aureus has responded to the introduction of new drugs by rapidly acquiring resistance by a variety of genetic mechanisms including (1) acquisition of extrachromosomal plasmids or additional genetic information in the chromosome via transposons or other types of DNA insertion and (2) by mutations in chromosomal genes. Many plasmid-encoded determinants have recently become inserted into the chromosome at a site associated with the methicillin resistance determinant. There may be an advantage to the organism having resistance determinants in the chromosome because they will be more stable. There are essentially four mechanisms of resistance to antibiotics in bacteria: (1) enzymatic inactivation of the drug, (2) alterations to the drug target to prevent binding, (3) accelerated drug efflux to prevent toxic concentrations accumulating in the cell, and (4) a by-pass mechanism whereby an alternative drug-resistant version of the target is expressed. Antimicrobial Drugs Ever since the first use of penicillin, S. aureus has shown a remarkable ability to adapt. Resistance has developed to new drugs within a short time of their introduction. Some strains are now resistant to most conventional antibiotics. It is worrisome that there do not seem to be any new antibiotics on the horizon. Any recent developments have been modifications to existing drugs. 9 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ Protocol № 34 Theme: Gramnegative cocci (gonococci, meningococci). I. Observe the smears below. Using appropriately colored pencils draw the following cells. Neisseria meningitidis Neisseria gonorrhoeae Neisseria gonorrhoeae (Gram stain) (Gram stain) (Methylene blue stain) II. Study scanning electron micrograph of Neisseria III. Study nutrient media for cultivation of meningococci and gonococci: a) serum agar; b) ascetic agar; c) chocolate agar, d) MTM (Martin-Thayer medium) contains antibiotics to inhibit normal body flora: – vancomycin to inhibit gram-positive bacteria; – colistin to inhibit gram-negative bacteria; – trimethoprim to suppress Proteus; – nystatin to inhibit yeast. IV. Identification of Neisseria: 1. Oxidase test. The oxidase test is based on the bacterial production of an oxidase enzyme. Cytochrome oxidase, in the presence of oxygen, oxidizes the para-amino dimetheylanaline oxidase test reagent in a Taxo-N® disc. In the immediate test, oxidase-positive reactions will turn a rose color within 30 seconds. Oxidase-negative will not turn a rose color. This reaction only lasts a couple of minutes. All Neisseria are oxidase positive. The oxidase test can be performed using a Taxo N® disc. A moistened Taxo N® disc can be placed on a growing culture and a blackening of the colonies surrounding the disc indicates a positive oxidase test. All oxidase-positive cultures would be gram stained to confirm gram-negative diplococci. The ability to grow on MTM chocolate agar and the positive oxidase test indicate the organism is most likely a pathogenic Neisseria. Identification of genus and species can be confirmed by carbohydrate fermentation. 2. Carbohydrate fermentation. The various species of Neisseria can be differentiated according to fermentation patterns using glucose and maltose media. If fermentation occurs, acid end products cause the phenol red pH indicator to turn yellow. N. gonorrhoeae ferments only glucose whereas N. meningitidis ferments glucose and maltose. Nonpathogenic neisseriae usually ferment only sucrose. 10 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ Carbohydrate fermentation by gonococci and meningococci: Species Glucose Maltose Neisseria gonorrhoeae acid - Neisseria meningitidis acid acid V. Differentiation of Neisseria meningitides and Neisseria catarrhalis on serum and MPA at different temperatures: Neisseria meningitidis Neisseria catarrhalis 370 С 220 С 370 С 370 С 220 С 370 С MPA serum serum MPA serum serum agar agar agar agar Result: Result: _______________________________ __________________________________ _______________________________ __________________________________ VI. Therapy of gonococcal and meningococcal infections: a third-generation cephalosporin: cefixime or ceftriaxone; penicillins: oxacillin, ampicillin, ampicillin+oxacillin (Ampiox); fluroquinolones: ciprofloxacine, lomefloxacin, moxifloxacin, levofloxacin. VII. Study the scheme of laboratory diagnosis of gonococcal and meningococcal infections. 1. Microscopy - intracellular gram negative diplococci. 2. Immunefluorescence. 3. Culture of specimen under increased CO2 tension. To isolate N. meningitidis, cultures are taken from the nasopharynx, blood, CSF, and skin lesions. To diagnose genital gonorrhea in males, the sample is taken from the urethra, in females - from the cervix and the rectum. Identification of colonies by: oxidase test, gram-staining, carbohydrate fermentation reactions: N. gonorrhoeae ferments only glucose whereas N. meningitidis ferments glucose and maltose. 4. Precipitation test, ELISA - detection of meningococcal antigen by antimeningococcal serum on cerebrospinal fluid or from skin lesions for rapid identification. 5. Serology: CFT (diagnosis of chronic gonorrhea). ADDING THEORETICAL MATERIAL The Neisseriaceae are a family of Beta Proteobacteria consisting of Gram- negative aerobic bacteria from fourteen genera (Bergey's 2005), including Neisseria, Chromobacterium, Kingella, and Aquaspirillum. The genus Neisseria contains two important human pathogens, N. gonorrhoeae and N. meningitidis. N. gonorrhoeae causes gonorrhea, and N. meningitides is the cause of meningococcal 11 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ meningitis. N. gonorrhoeae infections have a high prevalence and low mortality, whereas N. meningitides infections have a low prevalence and high mortality. Neisseria gonorrhoeae infections are acquired by sexual contact and usually affect the mucous membranes of the urethra in males and the endocervix and urethra in females, although the infection may disseminate to a variety of tissues. The pathogenic mechanism involves the attachment of the bacterium to nonciliated epithelial cells via pili and the production of lipopolysaccharide endotoxin. Similarly, the lipopolysaccharide of Neisseria meningitides is highly toxic, and it has an additional virulence factor in the form of its antiphagocytic capsule. Both pathogens produce IgA proteases which promote virulence. Many normal individuals may harbor Neisseria meningitides in the upper respiratory tract, but Neisseria gonorrhoeae is never part of the normal flora and is only found after sexual contact with an infected person (or direct contact, in the case of infections in the newborn). Neisseria gonorrhoeae Neisseria gonorrhoeae is a Gram-negative coccus, 0.6 to 1.0 µm in diameter, usually seen in pairs with adjacent flattened sides. The organism is frequently found intracellularly in polymorphonuclear leukocytes (neutrophils) of the gonorrhea pustular exudate. Fimbriae or pili, which play a major role in adherence, extend several micrometers from the cell surface Neisseria gonorrhoeae possesses a typical Gram-negative outer membrane composed of proteins, phospholipids, and lipopolysaccharide (LPS). However, neisserial LPS is distinguished from enteric LPS by its highly-branched basal oligosaccharide structure and the absence of repeating O-antigen subunits. For these reasons, neisserial LPS is referred to as lipooligosaccharide (LOS). The bacterium characteristically releases outer membrane fragments called "blebs" during growth. These blebs contain LOS and probably have a role in pathogenesis if they are disseminated during the course of an infection. N. gonorrhoeae is a relatively fragile organism, susceptible to temperature changes, drying, uv light, and other environmental stresses. Strains of N. gonorrhoeae are fastidious and variable in their cultural requirements, so that media containing hemoglobin, NAD, yeast extract and other supplements are needed for isolation and growth of the organism. Cultures are grown at 35-36 degrees in an atmosphere of 3-10% added CO2. The disease gonorrhea is a specific type of urethritis that practically always involves mucous membranes of the urethra, resulting in a copious discharge of pus, more apparent in the male than in the female. The first usage of the term "gonorrhea", by Galen in the second century, implied a "flow of seed". For centuries thereafter, gonorrhea and syphilis were confused, resulting from the fact that the two diseases were often present together in infected individuals. Paracelsus (1530) thought that gonorrhea was an early symptom of syphilis. The confusion was further heightened by the classic blunder of English physician John Hunter, in 1767. Hunter intentionally inoculated himself with pus from a patient with symptoms of gonorrhea and wound up giving himself syphilis! The causative agent of gonorrhea, Neisseria gonorrhoeae, was first described by A. Neisser in 1879, in 12 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ the pustular exudate of a case of gonorrhea. The organism was grown in pure culture in 1885, and its etiological relationship to human disease was later established using human volunteers in order to fulfill the experimental requirements of Koch's postulates. Gonorrheal infection is generally limited to superficial mucosal surfaces lined with columnar epithelium. The areas most frequently involved are the urethra, cervix, rectum, pharynx, and conjunctiva. Squamous epithelium, which lines the adult vagina, is not susceptible to infection by the N. gonorrhoeae. However, the prepubescent vaginal epithelium, which has not been keratinized under the influence of estrogen, may be infected. Hence, gonorrhea in young girls may present as vulvovaginitis. Mucosal infections are usually characterized by a purulent discharge. Uncomplicated gonorrhea in the adult male is an inflammatory and pyogenic infection of the mucous membranes of the anterior urethra. The most common symptom is a discharge that may range from a scanty, clear or cloudy fluid to one that is copious and purulent. Dysuria (difficulty in urination) is often present. Inflammation of the urethral tissues results in the characteristic redness, swelling, heat, and pain in the region. There is intense burning and pain upon urination. Endocervical infection is the most common form of uncomplicated gonorrhea in women. Such infections are usually characterized by vaginal discharge and sometimes by dysuria. About 50% of women with cervical infections are asymptomatic. Asymptomatic infections occur in males, as well. Males with asymptomatic urethritis are an important reservoir for transmission and are at increased risk for developing complications. Asymptomatic males and females are a major problem as unrecognized carriers of the disease. In the male, the organism may invade the prostate resulting in prostatitis, or extend to the testicles resulting in orchitis. In the female, cervical involvement may extend through the uterus to the fallopian tubes resulting in salpingitis, or to the ovaries resulting in ovaritis. As many as 15% of women with uncomplicated cervical infections may develop pelvic inflammatory disease (PID). The involvement of testicles, fallopian tubes or ovaries may result in sterility. Occasionally, disseminated infections occur. The most common forms of disseminated infections are a dermatitis-arthritis syndrome, endocarditis and meningitis. Ocular infections by N. gonorrhoeae can have serious consequences of corneal scarring or perforation. Ocular infections (ophthalmia neonatorum) occur most commonly in newborns who are exposed to infected secretions in the birth canal. Part of the intent in adding silver nitrate or an antibiotic to the eyes of the newborn is to prevent ocular infection by N. gonorrhoeae. Pathogenesis. Gonorrhea in adults is almost invariably transmitted by sexual intercourse. The bacteria adhere to columnar epithelial cells, penetrate them, and multiply on the basement membrane. Adherence is mediated through pili and opa (P.II) proteins. although nonspecific factors such as surface charge and hydrophobicity may play a role. Pili undergo both phase and antigenic variation. 13 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ The bacteria attach only to microvilli of nonciliated columnar epithelial cells. Attachment to ciliated cells does not occur. Most of the information on bacterial invasion comes from studies with tissue culture cells and human fallopian tube organ culture. After the bacteria attach to the nonciliated epithelial cells of the fallopian tube, they are surrounded by the microvilli, which draw them to the surface of the mucosal cell. The bacteria enter the epithelial cells by a process called parasite-directed endocytosis. During endocytosis the membrane of the mucosal cell retracts and pinches off a membrane-bound vacuole (phagosome) that contains the bacteria. The vacuole is transported to the base of the cell, where the bacteria are released by exocytosis into the subepithelial tissue. The neisseriae are not destroyed within the endocytic vacuole, but it is not clear whether they actually replicate in the vacuoles as intracellular parasites. A major porin protein, P.I (Por), in the outer membrane of the bacterium is thought to be the invasin that mediates penetration of a host cell. Each N. gonorrhoeae strain expresses only one type of Por; however, there are several variations of Por that partly account for different antigenic types of the bacterium. Neisseria gonorrhoeae can produce one or several outer membrane proteins called Opa (P.II) proteins. These proteins are subject to phase variation and are usually found on cells from colonies possessing a unique opaque phenotype called O+. At any particular time, the bacterium may express zero, one, or several different Opa proteins, and each strain has 10 or more genes for different Opas. Rmp (P.III) is an outer membrane protein found in all strains of N. gonorrhoeae. It does not undergo antigenic variation and is found in a complex with Por and LOS. It shares partial homology with the OmpA protein of Escherichia coli. Antibodies to Rmp, induced either by a neisserial infection or by colonization with E. coli, tend to block bactericidal antibodies directed against Por and LOS. In fact, anti-Rmp antibodies may increase susceptibility to infection by N.gonorrhoeae. During infection, bacterial lipooligosaccharide (LOS) and peptidoglycan are released by autolysis of cells. Both LOS and peptidoglycan activate the host alternative complement pathway, while LOS also stimulates the production of tumor necrosis factor (TNF) that causes cell damage. Neutrophils are immediately attracted to the site and feed on the bacteria. For unknown reasons, many gonococci are able to survive inside of the phagocytes, at least until the neutrophils themselves die and release the ingested bacteria. Neisserial LOS has a profound effect on the virulence and pathogenesis of N.gonorrhoeae. The bacteria can express several antigenic types of LOS and can alter the type of LOS they express by some unknown mechanism. Gonococcal LOS produces mucosal damage in fallopian tube organ cultures and brings about the release of enzymes, such as proteases and phospholipases, that may be important in pathogenesis. Thus, gonococcal LOS appears to have an indirect role in mediating tissue damage. Gonococcal LOS is also involved in the resistance of N. gonorrhoeae to the bactericidal activity of normal human serum. Specific LOS 14 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ oligosaccharide types are known to be associated with serum-resistant phenotypes of N. gonorrhoeae. N. gonorrhoeae can utilize host-derived N-acetylneuraminic acid (sialic acid) to sialylate the oligosaccharide component of its LOS, converting a serum- sensitive organism to a serum-resistant one. Organisms with nonsialylated LOS are more invasive than those with sialylated LOS but organisms with sialylated LOS are more resistant to bactericidal effects of serum. There is also antigenic similarity between neisserial LOS and antigens present on human erthyrocytes. This similarity to "self" may preclude an effective immune response to these LOS antigens by maintaining the immunotolerance of the host. N. gonorrhoeae is highly efficient at utilizing transferrin-bound iron for in vitro growth; many strains can also utilize lactoferrin-bound iron. The bacteria bind only human transferrin and lactoferrin. This specificity is thought to be, in part, the reason these bacteria are exclusively human pathogens. Strains of N. gonorrhoeae produce two distinct extracellular IgA1 proteases which cleave the heavy chain of the human immunoglobulin at different points within the hinge region. Split products of IgA1 have been found in the genital secretions of women with gonorrhea, suggesting that the bacterial IgA1 protease is present and active during genital infection. It is thought that the Fab fragments of IgA1 may bind to the bacterial cell surface and block the Fc-mediated functions other immunoglobulins. Occasionally, as described above, invading Neisseria gonorrhoeae enter the bloodstream causing a Gram-negative bacteremia which may lead to a disseminated bacterial infection. Asymptomatic infections of the urethra or cervix usually serve as focal sources for bacteremia. Strains of N. gonorrhoeae that cause disseminated infections are usually resistant to complement and the serum bactericidal reaction. This accounts for their ability to persist during bacteremia. In Gram-negative bacteremias of this sort, the effect of bacterial endotoxin can be exacerbated by the lyis of bacterial cells which may simply liberate soluble LOS. Treatment. The current CDC Guidelines recommend treatment of all gonococcal infections with antibiotic regimens effective against resistant strains. Currently recommended antimicrobial agents are ceftriaxone, cefixime, ciprofloxacin, or oflaxacin. Neisseria meningitidis The bacterium Neisseria meningitidis, the meningococcus, is identical in its staining and morphological characteristics to Neisseria gonorrhoeae. However, at the ultrastructural level, N. meningitidis has a prominent antiphagocytic polysaccharide capsule. N. meningitidis strains are grouped on the basis of their capsular polysaccharides, into 12 serogroups, some of which are subdivided according to the presence of outer membrane protein and lipopolysaccharide antigens. Neisseria meningitidis is usually cultivated in a peptone-blood base medium in a moist chamber containing 5-10% CO2. All media must be warmed to 37 degrees prior to inoculation as the organism is extremely susceptible to 15 http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6206a3.htm?s_cid=mm6206a3_e S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ temperatures above or below 37 degrees. This trait is rather unique among bacteria. Also, the organism tends to undergo rapid autolysis after death, both in vitro and in vivo. This accounts for the dissemination of lipopolysaccharide (endotoxin) during septicemia and meningitis. The organism tends to colonize the posterior nasopharynx of humans, and humans are the only known host. Individuals who are colonized are carriers of the pathogen who can transmit disease to nonimmune individuals. The bacterium also colonizes the posterior nasopharynx in the early stages of infection prior to invasion of the meninges. Most individuals in close contact with a case of meningococcal meningitis become carriers of the organism. This carrier rate can reach 20 percent of the contact group before the first case is recognized, and may reach as high as 80 percent at the height of an epidemic. Structure and Classification. The only distinguishing structural feature between N. meningitidis and N. gonorrhoeae is the presence of a polysaccharide capsule in the former. The capsule is antiphagocytic and is an important virulence factor. Meningococcal capsular polysaccharides provide the basis for grouping the organism. Twelve serogroups have been identified (A, B, C, H, I, K, L, X, Y, Z, 29E, and W135). The most important serogroups associated with disease in humans are A, B, C, Y, and W135. The chemical composition of these capsular polysaccharides is known. The prominent outer membrane proteins of N. meningitidis have been designated class 1 through class 5. The class 2 and 3 proteins function as porins and are analogous to gonococcal Por. The class 4 and 5 proteins are analogous to gonococcal Rmp and Opa, respectively. Serogroup B and C meningococci have been further subdivided on the basis of serotype determinants located on the class 2 and 3 proteins. A handful of serotypes are associated with most cases of meningococcal disease, whereas other serotypes within the same serogroup rarely cause disease. All known group A strains have the same protein serotype antigens in the outer membrane. Another serotyping system exists based on the antigenic diversity of meningococcal LOS. Pathogenesis. Infection with N. meningitidis has two presentations, meningococcemia, characterized by skin lesions, and acute bacterial meningitis. The fulminant form of disease (with or without meningitis) is characterized by multisystem involvement and high mortality. Infection is by aspiration of infective bacteria, which attach to epithelial cells of the nasopharyngeal and oropharyngeal mucosa, cross the mucosal barrier, and enter the bloodstream. If not clear whether blood-borne bacteria may enter the central nervous system and cause meningitis. The mildest form of disease is a transient bacteremic illness characterized by a fever and malaise; symptoms resolve spontaneously in 1 to 2 days. The most serious form is the fulminant form of disease complicated by meningitis. The manifestations of meningococcal meningitis are similar to acute bacterial meningitis caused by other bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, and E. coli. Chills, fever, malaise, and headache are the 16 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ usual manifestations of infection. Signs of meningeal inflammation are also present. Clinical manifestations of N. meningitidis infection. The onset of meningococcal meningitis may be abrupt or insidious. Infants with meningococcal meningitis rarely display signs of meningeal irritation. Irritability and refusal to take food are typical; vomiting occurs early in the disease and may lead to dehydration. Fever is typically absent in children younger than 2 months of age. Hypothermia is more common in neonates. As the disease progresses, apnea, seizures, disturbances in motor tone, and coma may develop. In older children and adults, specific symptoms and signs are usually present, with fever and altered mental status the most consistent findings. Headache is an early, prominent complaint and is usually very severe. Nausea, vomiting, and photophobia are also common symptoms. Neurologic signs are common; approximately one-third of patients have convulsions or coma when first seen by a physician. Signs of meningeal irritation such as spinal rigidity, hamstring spasms and exaggerated reflexes are common. Petechiae (minute hemorrhagic spots in the skin) or purpura (hemorrhages into the skin) occurs from the first to the third day of illness in 30 to 60% of patients with meningococcal disease, with or without meningitis. The lesions may be more prominent in areas of the skin subjected to pressure, such as the axillary folds, the belt line, or the back. Fulminant meningococcemia occurs in 5 to 15% of patients with meningococcal disease and has a high mortality rate. It begins abruptly with sudden high fever, chills, myalgias, weakness, nausea, vomiting, and headache. Apprehension, restlessness, and delirium occur within the next few hours. Widespread purpuric and ecchymotic skin lesions appear suddenly. Typically, no signs of meningitis are present. Pulmonary insufficiency develops within a few hours, and many patients die within 24 hours of being hospitalized despite appropriate antibiotic therapy and intensive care. Virulence Factors. For a time, the virulence of Neisseria meningitidis was attributed to the production of an "exotoxin" that could be recovered from culture filtrates of the organism. But when studies revealed that antitoxin reacted equally well with washed cells as culture filtrate, it was realized that the bacteria underwent autolysis during growth and released parts of their cell walls in a soluble form. Hence, the major toxin of N. meningitidis is its lipooligosaccharide, LOS, and its mechanism is endotoxic. The other important determinant of virulence of N. meningitidis is its antiphagocytic polysaccharide capsule. The human nasopharynx is the only known reservoir of N. meningitidis. Meningococci are spread via respiratory droplets, and transmission requires aspiration of infective particles. Meningococci attach to the nonciliated columnar epithelial cells of the nasopharynx. Attachment is mediated by fimbriae and possibly by other outer membrane components. Invasion of the mucosal cells occurs by a mechanism similar to that observed with gonococci. Events involved after bloodstream invasion are unclear and how the meningococcus enters the central nervous system is not known. 17 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ Epidemiology. The meningococcus usually inhabits the human nasopharynx without causing detectable disease. This carrier state may last for a few days to months and is important because it not only provides a reservoir for meningococcal infection but also stimulates host immunity. Between 5 and 30% of normal individuals are carriers at any given time, yet few develop meningococcal disease. Carriage rates are highest in older children and young adults. Attack rates highest in infants 3 months to 1 year old. Meningococcal meningitis occurs both sporadically (mainly groups B and C meningococci) and in epidemics (mainly group A meningococci), with the highest incidence during late winter and early spring. Whenever group A strains become prevalent in the population, the incidence of meningitis increases markedly. Treatment. Penicillin is the drug of choice to treat meningococcemia and meningococcal meningitis. Although penicillin does not penetrate the normal blood-brain barrier, it readily penetrates the blood-brain barrier when the meninges are acutely inflamed. Either chloramphenicol or a third-generation cephalosporin such as cefotaxime or ceftriaxone is used in persons allergic to penicillins. Control. Groups A, C, AC, and ACYW135 capsular polysaccharide vaccines are available. 18 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ Protocol № 35 Theme: Microbiological diagnosis of diseases caused by E. coli I. Observe the smears below. Using appropriately colored pencils draw the following cells. Escherichia coli (Gram stain) II. Study culture and biochemical properties of E. coli: Growth on MPA Carbohydrates fermentatiom Growth Growth on Sl an tcu kt ur e Pl ate cu ltu re Gl uc os e La cto s e M alt os e M an ni to l Su cr os e In do lon MPB Endo agar H2S Turbidity Colorles Colorless Red Acid, Acid, Acid, Acid, - + - bright smooth colonies gas gas gas gas bright colonies IV. Study antigenic properties E. coli. Strains E. coli that cause infections Infection Serological group О-Ag Н-Ag К-Аg Intestinal Enterotoxigenc О6, О8, О11, О15, Н4, Н7, Н9, Н11, Н12, - О20, 0114, О115, Н19, Н20, Н28, Н40 О153,О166 и др. Enteropathogenic О18, О26, О44, О55, Н2, Н6, Н7, Н11, Н12, - О111, О112 и др. Н14, Н18 Enteroinvasive О28, О29, О115, О136, - - О143 и др. Enterohemorrhagic О26, О157 Н6, Н7, Н8, Н11 - Urinary tract infections О1, О2, О4, О6, О7, - К1, К2, К5, К12, К13 О8, О9, О11, О18 и др. Bacterimia О1, О2, О4, О6, О7, - К1, К2, К5, К12, К15, О8, О9, О11, О18 и др. К23 Meningitidis О1, О6, О7, О16, О18, - К1 О83 19 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ While Escherichia coli is one of the dominant normal flora in the intestinal tract of humans and animals, some strains can cause infections of the intestines.  Enterotoxigenc E. coli (ETEC) produce enterotoxins that cause the loss of sodium ions and water from the small intestines resulting in a watery diarrhea. Over half of all travelers' diarrhea is due to ETEC; almost 80,000 cases a year in the U.S.  Enteropathogenic E. coli (EPEC) causes an endemic diarrhea in areas of the developing world, especially in infants younger than 6 months. The bacterium disrupts the normal microvilli on the epithelial cells of the small intestines resulting in maladsorbtion and diarrhea.  Enteroinvasive E. coli (EIEC) invade and kill epithelial cells of the large intestines causing a dysentery-type syndrome similar to Shigella common in underdeveloped countries.  Enterohemorrhagic E. coli (EHEC), such as E. coli 0157:H7, produce a shiga-like toxin that kills epithelial cells of the large intestines causing hemorrhagic colitis, a bloody diarrhea. In rare cases, the shiga-toxin enters the blood and is carried to the kidneys where, usually in children, it damages vascular cells and causes hemolytic uremic syndrome. E. coli 0157:H7 is thought to cause more than 20,000 infections and up to 250 deaths per year in the U.S.  Diffuse aggregative E. coli (DAEC) causes watery diarrhea in infants 1-5 years of age. They stimulate elongation of the microvilli on the epithelial cells lining the small intestines. Enteroaggregative E. coli (EAEC) is a cause of persistant diarrhea in developing countries. It probably causes diarrhea by adhering to mucosal epithelial cells of the small intestines and interfering with their function. V. Self-work of students: A. Isolation of pure culture from “feces of patient with enteric fever”: - study growth on Endo agar: red glistening smooth colonies; - prepare a smear, stain after Gram, microscopy Gram stain B. Seeding “blood of patient with enteric fever” on bile broth (5 ml blood in seering, bile broth (50ml)). VI. Study the scheme of laboratory diagnosis of E.coli infections. Specimen: feces. 1 step Seeding on Endo or MacConkey media 2 step culture properties slide AT with O-sera Gram stain Seeding on Ressel medium 3 step Identification: Hiss media, Tube AT with O- and K sera MPB (protein hydrolysis) 20 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ ADDING THEORETICAL MATERIAL Escherichia coli Theodor Escherich first described E. coli in 1885, which he isolated from the feces of newborns. The GI tract of most warm-blooded animals is colonized by E. coli within hours or a few days after birth. The bacterium is ingested in foods or water or obtained directly from other individuals handling the infant. The human bowel is usually colonized within 40 hours of birth. E. coli can adhere to the mucus overlying the large intestine. Once established, an E. coli strain may persist for months or years. Resident strains shift over a long period (weeks to months), and more rapidly after enteric infection or antimicrobial chemotherapy that perturbs the normal flora. The basis for these shifts and the ecology of Escherichia coli in the intestine of humans are poorly understood despite the vast amount of information on almost every other aspect of the organism's existence. The entire DNA base sequence of the E. coli genome has been known since 1997. E. coli is the head of the large bacterial family, Enterobacteriaceae, the enteric bacteria, which are facultatively anaerobic Gram-negative rods that live in the intestinal tracts of animals in health and disease. The Enterobacteriaceae are among the most important bacteria medically. A number of genera within the family are human intestinal pathogens (e.g. Salmonella, Shigella, Yersinia). Several others are normal colonists of the human gastrointestinal tract (e.g. Escherichia, Enterobacter, Klebsiella), but these bacteria, as well, may occasionally be associated with diseases of humans. Physiologically, E. coli is versatile and well-adapted to its characteristic habitats. It can grow in media with glucose as the sole organic constituent. Wild- type E. coli has no growth factor requirements, and metabolically it can transform glucose into all of the macromolecular components that make up the cell. The bacterium can grow in the presence or absence of O2. Under anaerobic conditions it will grow by means of fermentation, producing characteristic "mixed acids and gas" as end products. However, it can also grow by means of anaerobic respiration, since it is able to utilize NO3, NO2 or fumarate as final electron acceptors for respiratory electron transport processes. In part, this adapts E. coli to its intestinal (anaerobic) and its extraintestinal (aerobic or anaerobic) habitats. E. coli can respond to environmental signals such as chemicals, pH, temperature, osmolarity, etc., in a number of very remarkable ways considering it is a unicellular organism. For example, it can sense the presence or absence of chemicals and gases in its environment and swim towards or away from them. Or it can stop swimming and grow fimbriae that will specifically attach it to a cell or surface receptor. In response to change in temperature and osmolarity, it can vary the pore diameter of its outer membrane porins to accommodate larger molecules (nutrients) or to exclude inhibitory substances. With its complex mechanisms for regulation of metabolism the bacterium can survey the chemical contents in its environment in advance of synthesizing any enzymes that metabolize these compounds. It does not wastefully produce enzymes for degradation of carbon 21 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ sources unless they are available, and it does not produce enzymes for synthesis of metabolites if they are available as nutrients in the environment. E. coli is a consistent inhabitant of the human intestinal tract, and it is the predominant facultative organism in the human GI tract; however, it makes up a very small proportion of the total bacterial content. The anaerobic Bacteroides species in the bowel outnumber E. coli by at least 20:1. however, the regular presence of E. coli in the human intestine and feces has led to tracking the bacterium in nature as an indicator of fecal pollution and water contamination. As such, it is taken to mean that, wherever E. coli is found, there may be fecal contamination by intestinal parasites of humans. Escherichia coli in the Gastrointestinal Tract. The commensal E. coli strains that inhabit the large intestine of all humans and warm-blooded animals comprise no more than 1% of the total bacterial biomass. Pathogenesis of E. coli. Over 700 antigenic types (serotypes) of E. coli are recognized based on O, H, and K antigens. At one time serotyping was important in distinguishing the small number of strains that actually cause disease. Thus, the serotype O157:H7 (O refers to somatic antigen; H refers to flagellar antigen) is uniquely responsible for causing HUS (hemolytic uremic syndrome). Nowadays, particularly for diarrheagenic strains (those that cause diarrhea) pathogenic E. coli are classified based on their unique virulence factors and can only be identified by these traits. Hence, analysis for pathogenic E. coli usually requires that the isolates first be identified as E. coli before testing for virulence markers. Pathogenic strains of E. coli are responsible for three types of infections in humans: urinary tract infections (UTI), neonatal meningitis, and intestinal diseases (gastroenteritis). The diseases caused (or not caused) by a particular strain of E. coli depend on distribution and expression of an array of virulence determinants, including adhesins, invasins, toxins, and abilities to withstand host defenses. Summary of the virulence determinants of pathogenic E. coli: Adhesins Invasins Motility/ Toxins CFAI/CFAII Hemolysin chemotaxis LT toxin Type 1 fimbriae Shigella-like Flagella ST toxin P fimbriae "invasins" for Shiga toxin S fimbriae intracellular Cytotoxins Intimin (non-fimbrial adhesin) invasion and spread Endotoxin (LPS) EPEC adherence factor Genetic attributes Antiphagocytic Defense Defense against Genetic exchange by transduction and surface properties against immune conjugation Capsules serum responses Transmissible plasmids K antigens bactericidal Capsules R factors and drug resistance plasmids LPS reactions K antigens Toxin and other virulence plasmids LPS LPS Siderophores and siderophore uptake K antigens antigenic systems variation Pathogenicity islands 22 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ Urinary Tract Infections. Uropathogenic E. coli (UPEC) cause 90% of the urinary tract infections (UTI) in anatomically-normal, unobstructed urinary tracts. The bacteria colonize from the feces or perineal region and ascend the urinary tract to the bladder. Bladder infections are 14-times more common in females than males by virtue of the shortened urethra. The typical patient with uncomplicated cystitis is a sexually-active female who was first colonized in the intestine with a uropathogenic E. coli strain. The organisms are propelled into the bladder from the periurethral region during sexual intercourse. With the aid of specific adhesins they are able to colonize the bladder. The adhesin that has been most closely associated with uropathogenic E. coli is the P fimbria (or pyelonephritis-associated pili [PAP]). The letter designation is derived from the ability of P fimbriae to bind specifically to the P blood group antigen which contains a D-galactose-D-galactose residue. The fimbriae bind not only to red cells but to a specific galactose dissaccharide that is found on the surfaces uroepithelial cells in approximately 99% of the population. The frequency of the distribution of this host cell receptor plays a role in susceptibility and explains why certain individuals have repeated UTI caused by E. coli. Uncomplicated E. coli UTI virtually never occurs in individuals lacking the receptors. Uropathogenic strains of E. coli possess other determinants of virulence in addition to P fimbriae. E. coli with P fimbriae also possess the gene for Type 1 fimbriae, and there is evidence that P fimbriae are derived from Type 1 fimbriae by insertion of a new fimbrial tip protein to replace the mannose-binding domain of Type 1 fimbriae. In any case, Type 1 fimbriae could provide a supplementary mechanism of adherence or play a role in aggregating the bacteria to a specific manosyl-glycoprotein that occurs in urine. Uropathogenic strains of E. coli usually produce siderophores that probably play an essential role in iron acquisition for the bacteria during or after colonization. They also produce hemolysins which are cytotoxic due to formation of transmembranous pores in host cell membranes. One strategy for obtaining iron and other nutrients for bacterial growth may involve the lysis of host cells to release these substances. The activity of hemolysins is not limited to red cells since the alpha-hemolysins of E. coli also lyse lymphocytes, and the beta-hemolysins inhibit phagocytosis and chemotaxis of neutrophils. Another factor thought to be involved in the pathogenicity of the uropathogenic strains of E. coli is their resistance to the complement-dependent bactericidal effect of serum. The presence of K antigens is associated with upper urinary tract infections, and antibody to the K antigen has been shown to afford some degree of protection in experimental infections. The K antigens of E.coli are "capsular" antigens that may be composed of proteinaceous organelles associated with colonization (e.g., CFA antigens), or made of polysaccharides. Regardless of their chemistry, these capsules may be able to promote bacterial virulence by decreasing the ability of antibodies and/or complement to bind to the bacterial surface, and the ability of phagocytes to recognize and engulf the bacterial cells. The best studied K antigen, K-1, is composed of a polymer of N-acetyl neuraminic 23 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ acid (sialic acid), which besides being antiphagocytic, has the additional property of being an antigenic disguise. Neonatal Meningitis. It affects 1/2,000-4,000 infants. Eighty percent of E. coli strains involved synthesize K-1 capsular antigens (K-1 is only present 20-40% of the time in intestinal isolates). E. coli strains invade the blood stream of infants from the nasopharynx or GI tract and are carried to the meninges. The K-1 antigen is considered the major determinant of virulence among strains of E. coli that cause neonatal meningitis. K-1 is a homopolymer of sialic acid. It inhibits phagocytosis, complement, and responses from the host's immunological mechanisms. K-1 may not be the only determinant of virulence, however, as siderophore production and endotoxin are also likely to be involved. Neonatal meningitis requires antibiotic therapy that usually includes ampicillin and a third-generation cephalosporin. Intestinal Diseases Caused by E. coli. As a pathogen, E. coli is best known for its ability to cause intestinal diseases. Five classes (virotypes) of E. coli that cause diarrheal diseases are now recognized: enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroaggregative E.coli (EAEC). Each class falls within a serological subgroup and manifests distinct features in pathogenesis. Enterotoxigenic E. coli (ETEC). ETEC is an important cause of diarrhea in infants and travelers in underdeveloped countries or regions of poor sanitation. In the U.S., it has been implicated in sporadic waterborne outbreaks, as well as due to the consumption of soft cheeses, Mexican-style foods and raw vegetables. The diseases vary from minor discomfort to a severe cholera-like syndrome. ETEC are acquired by ingestion of contaminated food and water, and adults in endemic areas evidently develop immunity. The disease requires colonization and elaboration of one or more enterotoxins. Both traits are plasmid-encoded. ETEC may produce a heat-labile enterotoxin (LT) that is similar in molecular size, sequence, antigenicity, and function to the cholera toxin (Ctx). It is an 86kDa protein composed of an enzymatically active (A) subunit surrounded by 5 identical binding (B) subunits. It binds to the same identical ganglioside receptors that are recognized by the cholera toxin (i.e., GM1), and its enzymatic activity is identical to that of the cholera toxin. ETEC may also produce a heat-stable toxin (ST) that is of low molecular size and resistant to boiling for 30 minutes. There are several variants of ST, of which ST1a or STp is found in E. coli isolated from both humans and animals, while ST1b or STh is predominant in human isolates only. The ST enterotoxins are peptides of molecular weight about 4,000 daltons. Their small size explains why they are not inactivated by heat. ST causes an increase in cyclic GMP in host cell cytoplasm leading to the same effects as an increase in cAMP. ST1a is known to act by binding to a guanylate cyclase that is located on the apical membranes of host cells, thereby activating the enzyme. This leads to secretion of fluid and electrolytes resulting in diarrhea. 24 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ The infective dose of ETEC for adults has been estimated to be at least 108 cells; but the young, the elderly and the infirm may be susceptible to lower numbers. ETEC adhesins are fimbriae which are species-specific. For example, the K- 88 fimbrial Ag is found on strains from piglets; K-99 Ag is found on strains from calves and lambs; CFA I, and CFA II, are found on strains from humans. These fimbrial adhesins adhere to specific receptors on enterocytes of the proximal small intestine. Symptoms ETEC infections include diarrhea without fever. The bacteria colonize the GI tract by means of a fimbrial adhesin, e.g. CFA I and CFA II, and are noninvasive, but produce either the LT or ST toxin. Enteroinvasive E. coli (EIEC). EIEC closely resemble Shigella in their pathogenic mechanisms and the kind of clinical illness they produce. EIEC penetrate and multiply within epithelial cells of the colon causing widespread cell destruction. The clinical syndrome is identical to Shigella dysentery and includes a dysentery-like diarrhea with fever. EIEC apparently lack fimbrial adhesins but do possess a specific adhesin that, as in Shigella, is thought to be an outer membrane protein. Also, like Shigella, EIEC are invasive organisms. They do not produce LT or ST toxin. The primary source for EIEC appears to be infected humans. Although the infective dose of Shigella is low (in the range of 10 to few hundred cells), volunteer feeding studies showed that at least 106 EIEC organisms are required to cause illness in healthy adults. Unlike typical E.coli, EIEC are non-motile, do not decarboxylate lysine and do not ferment lactose. Pathogenicity of EIEC is primarily due to its ability to invade and destroy colonic tissue. The invasion phenotype, encoded by a high molecular weight plasmid, can be detected by PCR and probes for specific for invasion genes. Enteropathogenic E. coli (EPEC). EPEC induce a profuse watery, sometimes bloody, diarrhea. They are a leading cause of infantile diarrhea in developing countries. Outbreaks have been linked to the consumption of contaminated drinking water as well as some meat products. Pathogenesis of EPEC involves a plasmid-encoded protein referred to as EPEC adherence factor (EAF) that enables localized adherence of bacteria to intestinal cells and a non fimbrial adhesin designated intimin, which is an outer membrane protein that mediates the final stages of adherence. They do not produce ST or LT toxins. Adherence of EPEC strains to the intestinal mucosa is a very complicated process and produces dramatic effects in the ultrastructure of the cells resulting in rearrangements of actin in the vicinity of adherent bacteria. The phenomenon is sometimes called "attachment and effacing" of cells. EPEC strains are said to be "moderately-invasive", meaning they are not as invasive as Shigella, and unlike ETEC or EAEC, they cause an inflammatory response. The diarrhea and other symptoms of EPEC infections probably are caused by bacterial invasion of host cells and interference with normal cellular signal transduction, rather than by production of toxins. Through volunteer feeding studies the infectious dose of EPEC in healthy adults has been estimated to be 106 organisms. 25 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ Some types of EPEC are referred to as diffusely adherent E. coli (DAEC), based on specific patterns of adherence. They are an important cause of traveler's diarrhea in Mexico and in North Africa. Enteroaggregative E. coli (EAEC). The distinguishing feature of EAEC strains is their ability to attach to tissue culture cells in an aggregative manner. These strains are associated with persistent diarrhea in young children. They resemble ETEC strains in that the bacteria adhere to the intestinal mucosa and cause non-bloody diarrhea without invading or causing inflammation. This suggests that the organisms produce an enterotoxin of some sort. Recently, a distinctive heat-labile plasmid-encoded toxin has been isolated from these strains, called the EAST (EnteroAggregative ST) toxin. They also produce a hemolysin related to the hemolysin produced by E. coli strains involved in urinary tract infections. The role of the toxin and the hemolysin in virulence has not been proven. The significance of EAEC strains in human disease is controversial. Enterohemorrhagic E. coli (EHEC). EHEC are recognized as the primary cause of hemorrhagic colitis (HC) or bloody diarrhea, which can progress to the potentially fatal hemolytic uremic syndrome (HUS). EHEC are characterized by the production of verotoxin or Shiga toxins(Stx). Although Stx1 and Stx2 are most often implicated in human illness, several variants of Stx2 exist. There are many serotypes of Stx-producing E. coli, but only those that have been clinically associated with HC are designated as EHEC. Of these, O157:H7 is the prototypic EHEC and most often implicated in illness worldwide. The infectious dose for O157:H7 is estimated to be 10 - 100 cells; but no information is available for other EHEC serotypes. EHEC infections are mostly food or water borne and have implicated undercooked ground beef, raw milk, cold sandwiches, water, unpasteurized apple juice and vegetables. EHEC are considered to be "moderately invasive". Nothing is known about the colonization antigens of EHEC but fimbriae are presumed to be involved. The bacteria do not invade mucosal cells as readily as Shigella, but EHEC strains produce a toxin that is virtually identical to the Shiga toxin. The toxin plays a role in the intense inflammatory response produced by EHEC strains and may explain the ability of EHEC strains to cause HUS. The toxin is phage encoded and its production is enhanced by iron deficiency. 26 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ Diarrheagenic E. coli - virulence determinants and characteristics of disease: ETEC EIEC EPEC EAEC EHEC - fimbrial - nonfimbrial - non fimbrial - adhesins not - adhesins not adhesins e.g. adhesins, adhesin (intimin) characterized characterized, CFA I, CFAII, possibly outer EPEC adherence - non invasive probably K88. K99 membrane factor (EAF) enables produce ST-like fimbriae - non invasive protein localized adherence toxin (EAST) - moderately - produce LT - invasive of bacteria to and a hemolysin invasive and/or ST (penetrate and intestinal cells - persistent - does not toxin multiply within - moderately diarrhea in young produce LT or - watery epithelial cells) invasive (not as children without ST but does diarrhea in - does not invasive as Shigella inflammation or produce shiga infants and produce shiga or EIEC) fever toxin travelers; no toxin - does not produce - pediatric inflammation, - dysentery-like LT or ST; some diarrhea, copious no fever diarrhea reports of shiga-like bloody discharge (mucous, toxin (hemorrhagic blood), severe - usually infantile colitis), intense inflammation, diarrhea; watery inflammatory fever diarrhea with blood, response, may be some inflammation, complicated by no fever; symptoms hemolytic uremia probably result mainly from invasion rather than toxigenesis 27 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ Protocol № 36 The theme: Microbiological diagnosis of diseases caused by Salmonella. I. Observe the smears below. Using appropriately colored pencils draw the following cells. Salmonella typhi Salmonella paratyphi A Salmonella paratyphi B (Gram stain) (Gram stain) (Gram stain) Salmonella enteritidis Salmonella typhimurium Salmonela choleraesuis (Gram stain) (Gram stain) (Gram stain) II. Study culture and biochemical properties of Salmonella: а) growth on MPA: __________________________________________________________; b) growth on Endo agar: ______________________________________________________; c) growth on Ressel agar: _____________________________________________________; d) indol production: __________________________________________________________; g) H2S production: ___________________________________________________________. III. Study antigenic structure of salmonella (Caufmann and Wait scheme): Species S antigen H antigen 1st phase 2nd phase Group А a, b - S. paratyphi A 1, 2, 12 Group В S. paratyphi B 1, 4, 5, 12 - e, n, x S. abortus ovis 4, 12 c 1, 6 S. typhimurium 1, 4, 5, 12 i 1, 2 Group С S. paratyphic 6, 7, Vi c 1, 5 S. cholerae suis 6, 7 c 1, 5 S. newport 6, 8 eh 1, 2 S. dusseldorf 6, 8 z4, z24 - Group D S. typhi 9, 12Vi d - S. enteritidis 1, 9, 12 g, m - S. moscow 9, 12 gq - 28 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ Group Е S. anatum 3, 10 eh 1, 6 S. Cambridge 3, 15 eh i, w IV. Study biochemical properties of Salmonella typhi and paratyphi A and B: Glucose Lactose Maltose Mannitol Sucrose Indol H2S S.typhi Acid _ Acid Acid _ _ + S.paratyp Acid, gas _ Acid, gas Acid, gas _ _ _hi A S.paratyp Acid, gas _ Acid, gas Acid, gas _ _ +hi B V. Study Salmonella typhi pathogenesis: VI. Study the scheme of laboratory diagnosis of enteric fever. Specimen: blood (1st week), feces (from 5th day). 1 step Seeding on Endo or MacConkey media 2 step culture properties slide AT with O-sera and H-sera Gram stain Seeding on Ressel medium 3 step Identification: Hiss media (sugars fermentation), MPB (protein hydrolysis) Serology (from 2nd week): 1. Tube agglutination test (Widal test) with O- and H-antigens to diagnose acute infection. 2. Passive hemagglutination test (PHAT) with Vi-antigen to diagnose convalescence and carrier state. V. Self-work of students: A. Continue of isolation of pure culture from “feces of patient with enteric fever”: - study growth on Ressel agar: there is no change of color of medium; - prepare a smear of lactose negative culture, stain after Gram, microscopy: Gram stain 29 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ - perform slide agglutination test for differentiation of Salmonella: 1. On a clean glass slide, draw two circles (1 cm in diameter) heavily with a red wax marker. 2. Place the slide in an empty Petri dish. 3. With a Pasteur pipette place a drop of the cell suspension within each circle. Make sure the drops appear as even, cloudy suspensions. 4. Without touching the dropper to the cells, place a drop of the anti-O4 antiserum on one suspension and a drop of anti-O9 on the other. 5. After several minutes, hold up the Petri dish and observe the slide from the bottom. Gently tap the dish to effect some mixing of the cell suspension. Where there is a reaction between antibodies in the antiserum and their homologous antigens on the cell wall of the bacteria, the cells will agglutinate, and the drop will appear to contain many small particles. Where there is no agglutination, the cell suspension will maintain its original, evenly-cloudy (grainy) appearance. 6. Discard the slide appropriately (or clean it off for reuse) and discard the Petri plate into the proper container. (Do not place glass petri plates into the pails with the disposable plastics!) For S.typhi test is positive with Salmonella Serological reactions for Salmonella. group, O-9 and Hd sera. The negative reaction (-). The positive reaction (+). - seeding on Hiss media and MPA for detection indol and H2S production. B. Result of growth on bile broth: turbidity. VI. Study ingredients and scheme of Widal test Ingrediantds: Patient’s serum, dilution 1:50; Diagnosticums (antigens) О and Н (S.typhi, S.paratyphi А, S.paratyphi В); Physiological solution. Scheme of Widal test Row Ingredient Serum dilution 1:100 1:200 1:400 1:800 Contol 1 Physiological solution 1,0 1,0 1,0 1,0 1,0 Patient’s serum (1:50) 1,0 1,0 1,0 1,0 S.typhi (OH) 3 drops 3 drops 3 drops 3 drops 3 drops 2 Physiological solution 1,0 1,0 1,0 1,0 1,0 Patient’s serum (1:50) 1,0 1,0 1,0 1,0 S.typhi (O) 3 drops 3 drops 3 drops 3 drops 3 drops 3 Physiological solution 1,0 1,0 1,0 1,0 1,0 Patient’s serum (1:50) 1,0 1,0 1,0 1,0 S.paratyphi A 3 drops 3 drops 3 drops 3 drops 3 drops 4 Physiological solution 1,0 1,0 1,0 1,0 1,0 Patient’s serum (1:50) 1,0 1,0 1,0 1,0 S.paratyphi B 3 drops 3 drops 3 drops 3 drops 3 drops 30 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ VII. Study results of Widal test Serum dilution Antigenes 1: 10 0 1: 20 0 1: 40 0 1: 80 0 1: 10 0 1: 20 0 1: 40 0 1: 80 0 1: 10 0 1: 20 0 1: 40 0 1: 80 0 1: 10 0 1: 20 0 1: 40 0 1: 80 0 OH + + + + + + - - + + + - + - - - S.typhi O + + + + - - - - - - - - + - - - OH - - - - + - - - - - - - + - - - S.paratyphi A - - - - - - - - - - - - + - - - H OH - - - - + + - - + - - - + - - - S.paratyphi B - - - - - - - - - - - - + - - - O Type of Infection Postvaccination Immunity (enteric Need to repeat reaction fever) VIII. Study scheme and principle of passive Vi-hemagglutination test with erythrocytic diagnosticum for enteric fever. I. Components: 1. Serum from patient (unknown Ab) 2. Specific erythrocytes diagnosticum (RBCs+known Ag) 3. NaCl solution Delution of serum from patient 1:10 1:20 1:40 1:80 1:160 1:320 Control Agglutination Non agglutination IX. Therapy of enteric fever: a third-generation cephalosporin: cefixime or ceftriaxone; aminoglycosides; fluroquinolones: ciprofloxacine, lomefloxacin, moxifloxacin, levofloxacin. 31 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ ADDING THEORETICAL MATERIAL Salmonella is a Gram-negative facultative rod-shaped bacterium in the same proteobacterial family as Escherichia coli, the family Enterobacteriaceae, trivially known as "enteric" bacteria. Salmonella is nearly as well-studied as E. coli from a structural, biochemical and molecular point of view, and as poorly understood as E. coli from an ecological point of view. Salmonellae live in the intestinal tracts of warm and cold blooded animals. Some species are ubiquitous. Other species are specifically adapted to a particular host. In humans, Salmonella are the cause of two diseases called salmonellosis: enteric fever (typhoid), resulting from bacterial invasion of the bloodstream, and acute gastroenteritis, resulting from a foodborne infection/intoxication. Salmonella nomenclature. The genus Salmonella is a member of the family Enterobacteriaceae. It is composed of bacteria related to each other both phenotypically and genotypically. Salmonella DNA base composition is 50-52 mol % G+C, similar to that of Escherichia, Shigella, and Citrobacter. The bacteria of the genus Salmonella are also related to each other by DNA sequence. Salmonella nomenclature has been controversial since the original taxonomy of the genus was not based on DNA relatedness, rather names were given according to clinical considerations, e.g., Salmonella typhi, Salmonella cholerae- suis, Salmonella abortus-ovis, and so on. When serological analysis was adopted into the Kauffmann-White scheme in 1946, a Salmonella species was defined as "a group of related fermentation phage-type" with the result that each Salmonella serovar was considered as a species. Since the host-specificity suggested by some of these earlier names does not exist (e.g., S. typhi-murium, S. cholerae-suis are in fact ubiquitous), names derived from the geographical origin of the first isolated strain of the newly discovered serovars were next chosen, e.g., S. london, S. panama, S. stanleyville. Antigenic Structure. As with all Enterobacteriaceae, the genus Salmonella has three kinds of major antigens with diagnostic or identifying applications: somatic, surface, and flagellar. Somatic (O) or Cell Wall Antigens. Somatic antigens are heat stable and alcohol resistant. Cross-absorption studies individualize a large number of antigenic factors, 67 of which are used for serological identification. O factors labeled with the same number are closely related, although not always antigenically identical. Surface (Envelope) Antigens. Surface antigens, commonly observed in other genera of enteric bacteria (e.g., Escherichia coli and Klebsiella), may be found in some Salmonella serovars. Surface antigens in Salmonella may mask O antigens, and the bacteria will not be agglutinated with O antisera. One specific surface antigen is well known: the Vi antigen. The Vi antigen occurs in only three Salmonella serovars (out of about 2,200): Typhi, Paratyphi C, and Dublin. Strains of these three serovars may or may not have the Vi antigen. Flagellar (H) Antigens. Flagellar antigens are heat-labile proteins. Mixing salmonella cells with flagella-specific antisera gives a characteristic pattern of 32 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ agglutination (bacteria are loosely attached to each other by their flagella and can be dissociated by shaking). Also, antiflagellar antibodies can immobilize bacteria with corresponding H antigens. A few Salmonella entericaserovars (e.g., Enteritidis, Typhi) produce flagella which always have the same antigenic specificity. Such an H antigen is then called monophasic. Most Salmonella serovars, however, can alternatively produce flagella with two different H antigenic specificities. The H antigen is then called diphasic. For example, Typhimurium cells can produce flagella with either antigen i or antigen 1,2. If a clone is derived from a bacterial cell with H antigen i, it will consist of bacteria with i flagellar antigen. However, at a frequency of 10-3- 10-5, bacterial cells with 1,2 flagellar antigen pattern will appear in this clone. Habitats. The principal habitat of the salmonellae is the intestinal tract of humans and animals. Salmonella serovars can be found predominantly in one particular host, can be ubiquitous, or can have an unknown habitat. Typhi and Paratyphi A are strictly human serovars that may cause grave diseases often associated with invasion of the bloodstream. Salmonellosis in these cases is transmitted through fecal contamination of water or food. Gallinarum, Abortusovis, and Typhisuis are, respectively, avian, ovine, and porcine Salmonella serovars. Such host-adapted serovars cannot grow on minimal medium without growth factors (contrary to the ubiquitous Salmonella serovars). Ubiquitous (non-host-adapted) Salmonella serovars (e.g., Typhimurium) cause very diverse clinical symptoms, from asymptomatic infection to serious typhoid-like syndromes in infants or certain highly susceptible animals (mice). In human adults, ubiquitous Salmonella organisms are mostly responsible for foodborne toxic infections. The pathogenic role of a number of Salmonella serovars is unknown. This is especially the case with serovars from subspecies II to VI. A number of these serovars have been isolated rarely (some only once) during a systematic search in cold-blooded animals. Salmonella in the Natural Environment. Salmonellae are disseminated in the natural environment (water, soil, sometimes plants used as food) through human or animal excretion. Humans and animals (either wild or domesticated) can excrete Salmonella either when clinically diseased or after having had salmonellosis, if they remain carriers. Salmonella organisms do not seem to multiply significantly in the natural environment (out of digestive tracts), but they can survive several weeks in water and several years in soil if conditions of temperature, humidity, and pH are favorable. Isolation and Identification of Salmonella. A number of plating media have been devised for the isolation of Salmonella. Some media are differential and nonselective, i.e., they contain lactose with a pH indicator, but do not contain any inhibitor for non salmonellae (e.g., bromocresol purple lactose agar). Other media are differential and slightly selective, i.e., in addition to lactose and a pH indicator, they contain an inhibitor for nonenterics (e.g., MacConkey agar and eosin- methylene blue agar). 33 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ The most commonly used media selective for Salmonella are SS agar, bismuth sulfite agar, Hektoen enteric (HE) medium, brilliant green agar and xylose-lisine-deoxycholate (XLD) agar. All these media contain both selective and differential ingredients and they are commercially available. Media used for Salmonella identification are those used for identification of all Enterobacteriaceae. Most Salmonella strains are motile with peritrichous flagella, however, nonmotile variants may occur occasionally. Most strains grow on nutrient agar as smooth colonies, 2-4 mm in diameter. Most strains are prototrophs, not requiring any growth factors. However, auxotrophic strains do occur, especially in host-adapted serovars such as Typhi and Paratyphi A. Characteristics shared by most Salmonella strains belonging to subspecies I: - Motile, Gram-negative bacteria - Lactose negative; acid and gas from glucose, mannitol, maltose, and sorbitol; no Acid from adonitol, sucrose, salicin, lactose - ONPG test negative (lactose negative) - Indole test negative - Methyl red test positive - Voges-Proskauer test negative - Citrate positive (growth on Simmon's citrate agar) - Lysine decarboxylase positive - Urease negative - Ornithine decarboxylase positive - H2S produced from thiosulfate - Do not grow with KCN - Phenylalanine and tryptophan deaminase negative - Gelatin hydrolysis negative. Pathogenesis of Salmomella Infections in Humans. Salmonella infections in humans vary with the serovar, the strain, the infectious dose, the nature of the contaminated food, and the host status. Certain serovars are highly pathogenic for humans; the virulence of rather rare serovars is unknown. Strains of the same serovar are also known to differ in their pathogenicity. An oral dose of at least 105 Salmonella typhi cells are needed to cause typhoid in 50% of human volunteers, whereas at least 109 S. typhimurium cells (oral dose) are needed to cause symptoms of a toxic infection. Infants, immunosuppressed patients, and those affected with blood disease are more susceptible to Salmonella infection than healthy adults. In the pathogenesis of typhoid the bacteria enter the human digestive tract, penetrate the intestinal mucosa (causing no lesion), and are stopped in the mesenteric lymph nodes. There, bacterial multiplication occurs, and part of the bacterial population lyses. From the mesenteric lymph nodes, viable bacteria and LPS (endotoxin) may be released into the bloodstream resulting in septicemia. Salmonella excretion by human patients may continue long after clinical cure. Asymptomatic carriers are potentially dangerous when unnoticed. About 5% of patients clinically cured from typhoid remain carriers for months or even years. Antibiotics are usually ineffective on Salmonella carriage (even if salmonellae are 34 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ susceptible to them) because the site of carriage may not allow penetration by the antibiotic. Salmonellae survive sewage treatments if suitable germicides are not used in sewage processing. In a typical cycle of typhoid, sewage from a community is directed to a sewage plant. Effluent from the sewage plant passes into a coastal river where edible shellfish (mussels, oysters) live. Shellfish concentrate bacteria as they filter several liters of water per hour. Ingestion by humans of these seafoods (uncooked or superficially cooked) may cause typhoid or other salmonellosis. Salmonellae do not colonize or multiply in contaminated shellfish. Typhoid is strictly a human disease. The incidence of human disease decreases when the level of development of a country increases (i.e., controlled water sewage systems, pasteurization of milk and dairy products). Where these hygienic conditions are missing, the probability of fecal contamination of water and food remains high and so is the incidence of typhoid. Foodborne Salmonella toxic infections are caused by ubiquitous Salmonella serovars (e.g., Typhimurium). About 12-24 hours following ingestion of contaminated food (containing a sufficient number of Salmonella), symptoms appear (diarrhea, vomiting, fever) and last 2-5 days. Spontaneous cure usually occurs. Salmonella may be associated with all kinds of food. Contamination of meat (cattle, pigs, goats, chicken, etc.) may originate from animal salmonellosis, but most often it results from contamination of muscles with the intestinal contents during evisceration of animals, washing, and transportation of carcasses. Surface contamination of meat is usually of little consequence, as proper cooking will sterilize it (although handling of contaminated meat may result in contamination of hands, tables, kitchenware, towels, other foods, etc.). However, when contaminated meat is ground, multiplication of Salmonella may occur within the ground meat and if cooking is superficial, ingestion of this highly contaminated food may produce a Salmonella infection. Infection may follow ingestion of any food that supports multiplication of Salmonella such as eggs, cream, mayonnaise, creamed foods, etc.), as a large number of ingested salmonellae are needed to give symptoms. Prevention of Salmonella toxic infection relies on avoiding contamination (improvement of hygiene), preventing multiplication of Salmonella in food (constant storage of food at 4°C), and use of pasteurized and sterilized milk and milk products. Vegetables and fruits may carry Salmonella when contaminated with fertilizers of fecal origin, or when washed with polluted water. The incidence of foodborne Salmonella infection/toxication remains reletavely high in developed countries because of commercially prepared food or ingredients for food. Any contamination of commercially prepared food will result in a large-scale infection. In underdeveloped countries, foodborne Salmonella intoxications are less spectacular because of the smaller number of individuals simultaneously infected, but also because the bacteriological diagnosis of Salmonella toxic infection may not be available. However, the incidence of Salmonella carriage in underdeveloped countries is known to be high. 35 S u r n a m e _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ D a t e _ _ _ _ _ _ _ _ _ _ _ Salmonella epidemics may occur among infants in pediatric wards. The frequency and gravity of these epidemics are affected by hygienic conditions, malnutrition, and the excessive use of antibiotics that select for multiresistant strains. Salmonella enteritidis Infection. Egg-associated salmonellosis is an important public health problem in the United States and several European countries. Salmonella enteritidis, can be inside perfectly normal-appearing eggs, and if the eggs are eaten raw or undercooked, the bacterium can cause illness. Most types of Salmonella live in the intestinal tracts of animals and birds and are transmitted to humans by contaminated foods of animal origin. Stringent procedures for cleaning and inspecting eggs were implemented in the 1970s and have made salmonellosis caused by external fecal contamination of egg shells extremely rare. However, unlike eggborne salmonellosis of past decades, the current epidemic is due to intact and disinfected grade A eggs. The reason for this is that Salmonella enteritidis silently infects the ovaries of hens and contaminates the eggs before the shells are formed. A person infected with the Salmonella enteritidis usually has fever, abdominal cramps, and diarrhea beginning 12 to 72 hours after consuming a contaminated food or beverage. The illness usually lasts 4 to 7 days, and most persons recover without antibiotic treatment. However, the diarrhea can be severe, and the person may be ill enough to require hospitalization. The elderly, infants, and those with impaired immune systems (including HIV) may have a more severe illness. In these patients, the infection may spread from the intestines to the bloodstream, and then to other body sites and can cause death unless the person is treated promptly with antibiotics. Exotoxins. Salmonella strains may produce a thermolabile enterotoxin that bears a limited relatedness to cholera toxin both structurally and antigenically. This enterotoxin causes water secretion in rat ileal loop and is recognized by antibodies against both cholera toxin and the thermolabile enterotoxin (LT) of enterotoxinogenic E. coli, but it does not bind in vitr