‘STAPHYLOCOCCUS AUREUS Morphology Cuttural characteristics Biochemical reactions Resistance Pathogenicity and virulence Staphylococcal diseases Typing methods Epidemiology Laboratory diagnosis Treatment OTHER COAGULASE-POSITIVE STAPHYLOCOCCI COAGULASE-NEGATIVE STAPHYLOCOCCL MICROCOCCI INTRODUCTION Staphylococci are Gram-positive cocci that occur in grape-like clusters. ‘hep ae aE and are the ‘most common cause of localised suppurative lesions in humans, Their ability to develop resistance to penicillin and other antibiotics enhances their importance as a human pathogen, especially in the hospital environment. fercan ent robial suscepti i was identified as Sat sand ant Staphylococcus ‘Tum in diameter, arranged characteristicall Staphylococci were first observed in human pyo- genic lesions by von Recklinghausen in 1871. Pasteur (1880) obtained liquid cultures of the cocci from pus and produced abscesses by inoculating them into rab- bits. It was Sir Alexander Ogston who gave it the name Staphylococcus (staphyle in Greek, meaning ‘bunch of grapes’; kokkos meaning a berry) due to the typical occurrence of the cocci in grape-like clusters in pus and in cultures, Ogston noticed that non-virulent sta- phylococei were also often present on skin surfaces. Most staphylococcal strains from pyogenic lesions were found to produce golden yellow colonies, and the strains from normal skin, white colonies on solid media. Rosenbach (1884) named them S.aureus and S.albus, respectively. Passet (1885) described a third variety, S.citreus, producing lemon yellow colonies. Teresa ees O Morphology aphylococci are spherical cocci, approximately like clusters (Fig. 21.1). Cluster formation a fvision occurring in three planes, with daughter cells tending to remain in close proximity. They are non-motile and non-sporing. A few strains possess tigated fortis asthe possibilty of spread to cece a h © scanned with OKEN ScannerFig 21.2 Staphylococcus aureus in Gram stain jeroscopically visible capsules, particularly in young cultures, Many apparently non-capsulated strains have Fig.24.2 Growth on blood agar amounts of capsular material on the surface. They stain readily with aniline dyes and are uniformly ¢ Several selective media have been devised fo Gram positive. Under the influence of penicillin and ing S.aureus from specimens such as feces contin, certain chemicals, they may change to L forms, ing other bacteria. These include media contain 8-10% NaCl (saltemilk agar, salt broth, ijn Cultural characteristics chloride and tellurite (Ludtam’s: medium) ac _They grow readily on ordinary media within a tempera: polymyxin. C,MB,UP =p far i Cw 2h onium ESS) pase eaions Sct and a pH of 7.4.7.6. They are aerobes and facultativO2) Sind anaerobes, emma They are catalase positive (unlike streptococci) ang © Onnnutrient agar, after incubation for 24 hours, the usually hydrolyse urea, nitrates t0 nitrites ae QoP in aeolen ae liquefy gelatin and are MR and VP positive but ix me colonies are large (24 mm in diameter), jreular, See mea negative. ‘TGREX Smowih, shiny, opaque and easily emulsifi- ; : able. Most strains produce golden yellow pigment, The two important species causing infections in though ost sain produce golden yelow pigment] may Be WT, orange or yellow. The shumans ate differentiated by the characteristics shown pigment does not diffuse into the medium. Pigment in Table 21.1. production occurs optimally at 22°C and only in ‘The other human pathogen S.saprophyticus aerobic cultures, Pigment production is enhanced lar to S.epidermidis except that the former is resistant when 1% glycerol monoacetate or milk is incorpo- _'© Novobiocin, whereas the latter is not. rated in the medium. The pigment is believed to be, G} 4 lipoprotein allied to carotene. On nutrient agar Resistance slope, confluent growth presents a characteristic Staphylococci are among the more resistant non-spor- ‘oil-paint’ appearance. ing bacteriajDried on threads, they remain viable Tor '* On blood agar, the colonies are similar to those on 7 a iF-Most strains are hemolytic, especially Table 23.1 Differences between S.aureus and S.epidermidis ¢) jon_dioxide, is marked on rabbit or sheep blood and weak on horse blood agar. For primary isolation, | Coagulase sheep blood agar is recommended. Human blood Mannitol fermentation should not be used as it may contaitf anlibodjes'F— Heat stable nuclease “other tah Por 2 ‘Phenolphthalein phosphata: * In MacConkey agar, they produce smaller colonies Beta hemolyis on blood agar shat are pink due to lactose fermentdtion,f————_ Golden yellow pigment * In liquid media, uniform turbidity is produced. ‘Sensitivity to lysostaphin © scanned with OKEN ScannerGe ted Pu daw es ao S-canantls. They have been isolated fi al lated fron dei atte 2:3.snonths They may withstand orc ininotes, Theie thermal death yy Tape : Mts 62°C for 30 mine/2) utes, Some staphylocove’ require heating at 80" ins Pathogenicity and virulence cone hour to be killed, Hea tesistant strains have the ability 1 grow at a higher temperatures o are Most strains grow in the presence of 104% NaCl ang some even in 15% NaCl These features are of signif cance in food preservation, Theyresist 1% phenol for 15 minutes/ Mercury pe; chloride TO solution kills then a Ners ian aniline dyes are strongly bacter crystal violet being lethal at ration of 1 in 500,000 and brilliant green at 1 in 10,000,000, Staphylococci are uniformly resistant to lysozymes but some micrococei are sensitive to it. They are gen- erally sensitive fo Iysostaphin—a mixture of enzymes produced by a particular strain of S.epidermidis, Staphylococci were uniformly sensitive to penicilin in the pre-antibiotic era, with very few strains capable of producing penicillinase. Soon after penicillin came to be used clinically, resistant strains began to emerge, first in hospitals and then in the community at lage. Penicillin resistance is of three type * Production of beta Inctamase (penicillinse), which inactivates penicillin by splitting the beta lactam rin, 7 Ato D. Hospital strains usually form type A penicil- linase. Penicillinase is an inducible enzyme and its production is usually controlled by plasmids which are: i i conju tion. The same plasmid may carry genes for resistance to a range of other antibiotics and heavy metals. ® Alterations in the penicillin binding protein PBP2a and changes in bacterial surface receptors, reduc- ing binding of beta lactam antibiotics to cells. This change is normally chromosomal in nature and is expressed more at 30°C than at 37°C. This resist- ance also extends to cover beta lactamase-resist- ant penicillins such as methicillin and cloxacillins (called MRSA). Some of these strains may show resistance to other antibiotics like erythromycins, tetracyclines, aminoglycosides and heavy metals and ‘cause outbreaks of hospital infection. These strains have been called ‘epidemic methicillin-resistant Staphylococcus aureus’ or EMRSA (as methicillin is an unstable drug, oxacillin was used as a marker for MRSA in sensitivity tests, but now cefoxitin is recommended). camiagejTn Intoxications, the disease is caused by the * Development of tolerance to penicillin, by which the bacterium is only inhibited but not killed. Staphylococci produce two types of diseases; infections ‘and infoxieutions. In the former, the cocci gain access to damaged skin, mucosal or tissue sites, colonise by ‘idhering to cells or extracellular matrix, evade host defence mechanisms yand multiply and_cause tissiie bacterial toxins produced either in the infected host or preformed in vitro. ‘Xnumber of staphylococcal factors, both cell associ- ated and extracelfular, have been identified, which may influence virulence. However, apart from the exotoxins which cause specific clinical syndromes, no other fac- tor has a decisive role in pathogenesis. ‘The_virulenee factors described include the following: Cell associated polymers: © The cell, ide.p an confers Figidity and structural integrity to the bacterial cel. “@) It activates the complement and induces release of “inflammatory cytokines. Ae "Teichoie acid, an antigenic component of the cell wall, facilitates adhesion of the cocci to the host cell surface and protects them from complement-medi- ated opsonisation, © The capsular polysaccharide surrounding the cell ‘all inhibits opsonisation, Cell surface protein Protein A, present in most S.aureus strains, has many Biological properties, including chemotactic, anti-phagocytic and anti-complementary effects. Italso induces platelet damage and hypersensitivity. Protein A binds to the Fc terminal of IgG molecules (except IgG3), leaving the Fab region free to com- bine with its specific antigen. Protein A-bearing staphylococci coated with any IgG antiserum will be agglutinated if mixed with the corresponding antigen. This procedure, known as co-agglutina- tion, has many applications such as streptococcal grouping and gonococcal typing. Protein A is a B cell mitogen. It has also been used as a ligand for isolation of IgG. * Clu factor, another surface protein, is the oagulase’ which is responsible for the slide congulase test. © scanned with OKEN ScannerExtracellular enzymes: © Coaguase isan enzyme which brings about clotting: a or rabbit plasina, Wacts with a coat reacting factor (CRE) present i plasma, binding 60 prothnombin and converting fibrinogen to fibrin, His te Nisis of the tubs coagulase test, Coagulase does pigs and some other species because they lack CRE Caleium or other ing factorsare not required for coagulase action, Eight ¢ypes of coagullase have been identified, Most human strains form coagulase type A. Coagulase and clumping factor differ in many respects, # Staphylococci produce a number of lipid hydro- lases or lipases which help them infect the skin and subcuranoous tissues, * Hyaluronidase breaks down the connective tissue. Staphylokinase (fibrinolysin), fatty acid modifying enzymes and proteases help in initiation and spread of infection. ‘A heat stable nuclease is a characteristic feature of S.aureus ‘* Staphylococci possess protein receptors for many mammalian proteins such as fibronectin, fibrinogen, IgG and Clg. These facilitate staphylococcal adhe- sion to host cells and tissues. Toxins: —S Ciiolitic toxins are membrane-active substances, consisting of fou Hemolysins and a leucocidin: (®_Alpha hemolysin (alpha toxin, lysin) is the most ‘J “Fmportant among them. It is a protein inactivated at 70°C| but reactivated paradoxically at 100°C. This is because at 60-70°C, the toxin combines with a heat labile inhibitor which is denatured at 100°C, leaving the toxin free. Alpha toxin lyses rabbit erythrocytes, but is less active against sheep and human red cells. It is also leucocidal, cyto- toxic, dermonecrotic (on intradermal inoculation in rabbits), neurotoxic and lethal (on intravenous inoculation in rabbits). It is toxic to macrophages, lysosomes, muscle tissues, the renal cortex and the circulatory system. OQ Beta hemolysin is a sphingomyelinase, hemolytic Tor sheep cells, but not for human or rabbit cells. “Trani heel phenomenon, the hemoly- sis being initiated at 37°C, but becoming evident only after chilling. @)Gamma hemolysin is composed of two separate proteins, both of which are necessary for hemo- “Tie activity, | Parti) BACTERIOLOGY = Delin hemolysin as Asters e eet the call membranes of erythrocytes, Jeu inaerophages and platelets + # Leucocidin (called the Panton—Valenting PVL after its discoverer) is 80 0 tone op toxin, like gamma Iysin, being composed Po° $ and F components. Such bi-componeny ©" 4 ey brane-actie toxins have been grouped ge Anymenotropic toxins, Enterotoxin is responsible forthe manifesta staphylococcal food poisoning—nausea, ygor and diarthea 2-6 hours after consuming fogg taminated by preformed toxin, The toxin is ran heat stable, resisting 100°C for 10 10 40 mie? depending on the concentration ofthe toxin nae’? nature ofthe medium. About two-thirds of sat strains, growing in carbohydrate and protein secrete the toxin, Meat and fish or milk and mp products cooked and lft at room temperate sk contamination with staphylococci, for enough tin forthe toxin to accumulate, are the common ign? responsible, The soutce of infection is usually afoot handler who isa carrier. The illness is usually se limited, with recovery in a day or so. Eight antigenic types of enterotoxin are currenly known, named A, B,C, D, E and H. They an formed by toxigenic strains, singly or in combina, tion. The toxin i believed to act directly on the aut. nomic nervous system to cause the illness, rather than on the gastrointestinal mucosa. The toxin is antigenic and neutralised by the specific anttaxn, ‘Type A toxin is responsible for most cases. Sensitive serological tests such as latex agglutination and ELISA are available for detection of the toxin, The toxin is potent—micrograms can cause il ness. Some cases of post-antibiotic diarrhea are caused by enterotoxin-forming _staphylococi The toxin also exhibits pyrogenic, mitogenic, hypo- tensive, thrombocytopenic and cytotoxic effects. Toxic shock syndrome (TSS) is a potentially fatal rmulisystem disease presenting with fever, hypoten- sion, myalgia, vomiting, diarrhea, mucosal hypet- emia and an erythematous rash which desquamates subsequently. This is associated with infection of mucosal or sequestered sites by toxic shock sy drome toxin (TSST)-producing S.aureus strats usually belonging to bacteriophage Group 1. TSST type 1 (formerly also known as enterotoxin type ‘or pyrogenic exotoxin C) is most often responsible, © scanned with OKEN Scannerthough enterotoxins Bor © may also e; syndrome. TSST-1 antibody is seen in convalescents, This ig protetiveand is absence sa ecto in the pathoge esis of the condition, ‘Though tampon-related TSS is now rare, the syndrome occurs in other infections ofthe skin, mucosa and other sites and also in some surgical wounds. Staphylococcal enterotoxins and TSST-1 are superantigens which are potent activators of lymphocytes. Being VB-restricted T cell mitogens, such superantigens stimulate very large numbers of T calls, without relation to their epitope specificity. This leads to an excessive and dysregulated immune response, with release of cytokines interleukins 1, 2, tumour necrosis factor and interferon gamma, This explains the multisystem involvement and florid manifestations in staphylococcal food poison- ing and TSS. ¢ Exfoliative (epidermolytic) toxin, also known as ET or ‘exfolatn’, is responsible for the staphylococcal scalded skin syndrome (SSSS), an exfoliative skin disease in which the outer layer of the epidermis gets separated from the underlying tissues. The severe form of SSSS is known as Ritter’s disease in the newborn and toxic epidermal necrolysis in older patients. Milder forms are pemphigus neonatorum and bullous impetigo. Staphylococcal infections are among the most com- ‘mon bacterial infections and range from the trivial to the fatal, They are characteristcally localised pyogenic lesions, in contrast to the spreading nature of strepto- coocal infections. The common pyogenic staphylococcal infections areas follows (Case 1): © Skin and_soft tissue: Folicultis, furuncle (boil), abscess (particularly breast abscess), wound infec-, tion, carbunele, impetigo, paronychia, less often ‘elulitis * Musculoskeletal; Qsteomyelitis, arthritis, bursitis, Pyomyositis © Respiratory: Tonsillts, pharyngitis, sinusitis, otitis, bronchopneuinranta, Tang abscess, empyema, rarely pneumonia © Central nervous system: Abscess, meningitis, intracranial thrombophlebitis ‘use the ndovascula endocarditis : © Urinary: Staphylococci are uncommon in routine urinary tract infections, though they do cause infection in association with local instrumenta- tion, implants or diabetes. Urinary isolates of staphylococci are to be considered significant even with low colony counts, as they may be related to bacteremia, The common toxin-mediated staphylococcal dis- cases are as flldws (Case © Food poisoning Toni shock drome Toate Shock syndrome © Scalded skin syndrome (as described above) Bacteremia, septicemia, pyemiay ‘Typing methods Phenotypic-Bacteriophage typing: Staphylococci may be typed based on their susceptibility to bacteri- ‘ophages. An internationally accepted set of phages is used for typing. Staphylococcal phage typing is done by a pattern method (Fig. 21.3) Not all cultures are typeable by this procedure, and the susceptibility patterns of circulating strains vary by time and locality, Hence, phages in the reference set require periodic revision Molecular typing: Due to lack of discriminatory power of phenotyping methods, molecular typing is currently being carried out. For example, DNA finger- printing, ribotyping and PCR-based analysis for genetic pleomorphism is used. Pulse field gel electrophoresis (PFGE) and sequence-based typing methods are now used in outbreak investigations. Epidemiology ( f+~ = Staphylococci are primary parasites ofhumanbeings and animals, colonising the skin, skin_ glands and” mucous “Imembranef. The most common sources of infection are human patients and carriers, animals and inanimate objects being less important. Patients with superfi infections and respiratory infections disseminate large numbers of staphylococet into the environment. About 10-30 per cent of healthy persons carry staphylococci nthe nose and about 10 per cent in the perineum and ar, ‘Vaginal carriage is about 5-10 per cent, hich rises greatly during menses, a factor relevant in the pathogenesis of TSS related to menstruation, Staphylococcal carriage starts early in life, colo sation of the umbilical stump being very common in © scanned with OKEN ScannerTH RACTE@OROGY | oS es Fig 223 Bacteriophage typing of staphylococci ies born in hospitals. Some carriers, called ‘shed- ers’, disseminate very large numbers of cocci for prolonged periods. The cocci shed by patients and car- contaminate fomites such as handkerchiefs, bed linen and blankets and may persist on them for days or weeks. Staphylococci may also come from infected domestic animals such as cows. Staphylococcal disease may follow endogenous or exogenous infection. The modes of transmission may be by contact, direct or through fomites, by dust or by sirborne droplets. MRSA: Hospital infections by _staphylocoe« deserve special attention because of their frequency and because they are caused by strains resistant to various antibiotics. MRSA or methicilin- S.aureus strains are a common cause of postop- erative wound infection and other hospital cross- infections, Most of these are due to certain strains of staphylococci present in the hospital environment, the ‘so-called ‘hospital strains’, They are resistant to all beta lactam antibiotics as well as other antimicrobial agents. They belong toa limited number of phage types and are commonly resistant to penicillin and other antibiotics routinely used in hospitals, Some ‘epidemic strains’ cause epidemics of hospital cross-infections, The main cause of concern now is the emergence of community acquired MRSA strains (CA MRSA) which were earlier restricted to the hospital seting (HA MRSA). Outbreak control measures: Measures for the control of staphylococcal infection in hospitals include: ce folation of pationts With Open staphy, lesion of staphylococcal lesions = Detection of staphy : MOng su + ies and other hospital stall and kein n, away from work till the lesions are healed then, « Following strict aseptic techniques in thegtgs 5 Hand washing, the oldest, simplest and mone tive method of checking hospital cross. in which unfortunately is often neglected If an outbreak of staphylococcal sepsis goa, search may be made for caries among the hy ‘taf. Those detected should be treated with Io, ‘cation of muprocin or chlorhexidine, tions, Pit al app, 4) Laboratory diagnosis T Specimen: The specimens to be collected ea ype lesion (for example, pus from suppin a eT nee te k Tod poisoning, feces and the remains of suspr food should be collected. For the detection of cane the nasal swab is the usual specimen, Swabs from re perineum, pieces of hair and the umbilical stun may be necessary in special situations. 2. Microseopy: Direct microscopy with Gram, stained smears is useful in the case of pus, Wen cocci in clusters may be seen {THIS OF no valu fy Specimens like sputum where mixed bacterial flora ae normally present. 3. Culture: Diagnosis may readily be made by ‘aulture. The specimens are plated_on blood agar. Staphylococcal colonies appear after overnight inci Baton: Specimens where staphylococt are expect “fo be scanty and outnumbered by other bacteria (or example, swabs from carriers, feces in food poisoning cases) are inoculated on selective media like Ludlam’s or salt-milk agar or Robertson’s cooked meat medium containing 10% sodium chloride. Smears are examined from the cultures and the coagulase test caried out when staphylococci are isolated. 4. Mdentification: «The cougulase test can be done using two methods: tube and slide. “The tube congulase test detects free coagulase About 0.1 ml of a young broth culture or weit culture suspension of the i added to about_0,5 ml_of human or rabbit plasma.in-® be used as the anticoagulant for ing.the © scanned with OKEN ScannerCitrate is no ye uty pet Seed because itised by some contaminant f ria, causing false posi a Roa ea causing false postive results, Postve and ns S are also set up. The tubes are xe Tots. ma clots A clots and does not Now od. Continued incubati sot recommendad the et mag ee fibrinolysin formed by some strains (Fig. 214 ~ Theslide tes detecting bound congulase ie rch ually gives results parallel with the st. When there is divergence, the tube test will be the deciding factor. For the slide test, th isolate is emulsified in a drop of saline on Hlde, After checking for abserice of autoagélutination a drop of human or rabbit plasma is added to the emulsion and mixed. Prompt clumping of the indicates a positive test. Positive and nega- tive controls are also set up (Fig. 21.5). ze Aniblttcsensltvny tess should be performed as quide To weatment. This is important as staphylo- cocci readily develop resistance to drugs «Typing may be done if the information is desired for epidemiological purposes. Other typing methods include antibiogram pattern, plasmid profile, DNA fingerprinting, ribotyping and PCR-based analysis for genetic pleomorphism 5, Serological tests: These may sometimes help in the @agnosis of hidden deep infections. Anti-staphy- Fig 21.4 Tube coagulase test: positive, negative Positive (clumps) Nogativo (no chimps) ‘Side coagulase test Fig. 215. Slide coagulase test: negative and positive lolysin (anti-alphalysin) titres of more than two units per ml, especially when the ttre is rising, may be of value in the diagnosis of deep-seated infections such as bone abscess. (099 bose ‘Treatment a: ‘As drug resistance isso common among staphylococci yh the appropriate antibiotic should be chosen based on antibiotic sensitivity tests. Benzyl penicillin The most “Miective antibiotic, Whe sain is sensitive, Methicillin was the first compound developed to combat resist ance due to penicillinase (beta lactamase) production by staphylococci. Due to limitations in clinical use of methicillin, cloxacillins are used instead against peni- cillinase-producing strains. However, MRSA became common—resistant not merely to penicillin, but also to all other beta lactam antibiotics and many others besides. For life-threatening staphylococcal infections, vancomycin is the drug of choice. Strains resistant to vancomycin and teicoplanin are still not common. For mild superficial lesions, systemic antibiotics may not be necessary. Topical application of drugs not used systemically, such as bacitracin, chlorhexidine or mupirocin, may be sufficient. ‘Some strains show the phenomenon of drug toler- ance. These strains will be found to be susceptible in the disc sensitivity test but their minimum bactericidal concentration will be very much higher than their mini- mum inhibitory concentration, They are not killed by antibiotics in the usual doses and persist, leading to failure in eradicating the infection. Carriers: The treatment of carriers is by local appli- cation of suitable antibiotics such as mupirocin and antiseptics such as chlorhexidine. [In resistant cases posing major problems, Ptmprcin along with another oll © Scanned with OKEN Scanner" © in the long-term sup- S.aureus, a few other staphylococcal species Se coagulase posite for example, S.ntermedius and SFicus. These are animal parasites and do not infect humans, eT elected] Coagulase-negative staphylococei constitute a major component ofthe normal loraof the human body, Some Species can produce human infections —S epidermidis, Schaemolyticus and S.saprophyticus. S epidermidis is invariably present on normal human skin, It is non-pathogenic ordinarily but can cause disease when the host defences are breached, It is ¢ common cause of stitch abscess. It has a predilection {ot growth on implanted foreign bodies such as ant portant factor in the S.opidermidis. 1 is. an matrix which protects ial agents and helps in coloni. infections, us may be present on norma hy daa reat area and can cause inet skin ‘fection, particularly in sexually active ; eae The infecting strains are usually seni to most common antibiotics, except nai i S.saprophyticus is novobiocin resistant. as ay ‘These are Gram-positive cocci which occur most ix pairs, tetrads or irregular clusters. They are Catalase and mealfed oxidase positive and_ aerobic. Thy are ordinarily non-pathogenic. They resemble staphylococci, but in stained smears the cells are ‘gener. ally larger and more Gram variable than staphylocoe and appear in tetrads. In cultures, they form smaller colonies. The common laboratory test used to differ. entiate between micrococci and staphylococci is Hugh and Leifson’s oxidation—fermentation test in which mictococci show oxidative and staphylococci show fermentative patterns (Fig. 21.6). Pe non-motite, ANd Facuttatinn - © scanned with OKEN ScannerMophotoey a) chvactenstcs Biochemie reactons esstonce songenk sure fei Tours and other vtence feos Parrogenicty Sreptococcal diseases Epidemiology Laboratory diagnos Prophyants Treament OTHER HEMOLYTIC STREPTOCOCCI Group 8 Groupe Group F Group G The Enterococcus species Group D Virdans group INTRODUCTION Streptococei_are Gram-positive cocci arranged in chains or pairs (Fig. 22.1). They ate partof trenormal {Tora oF fumans and animals] Some of them are human Pathogens. The most important is Streptococcus pyo- ‘genes causing pyogenic infections, with a characteris- tic tendency to spread, as opposed to staphylococcal lesions, which are typically localised. It is also respon- sible for the non-suppurative lesions, acute rheumatic fever and glomerulonephritis which occur as sequelae to infection. Cocci in chains were first seen in erysipelas and wound infections by Billroth (1874), who called them streptococci (streptos, meaning twisted or coiled), Fig. 22.1 Streptococci (Gram-positive cocci in chains) Ogston (1881) isolated them from acute abscess, distinguished them from staphylococci and estabishe, their pathogenicity by animal inoculation, Rosenbach (1884) isolated the cocci from human suppurativlesions and gave them the name Streptococcus pyogenes. Classification Several systems of classification have been used, but in medical bacteriology the following method is use, Based on hemolysis on blood agar: Aerobic and facultative anaerobic streptococci are classified on the basis of their hemolytic properties (Fig. 22.2). Brown (1919) categorised them into three varieties based on their growth in 5% horse blood agar. Alpha (a) hemolytic streptococci produce a greenish discolouration with partial hemolysis around the colo- nies. The zone of lysis is small (I or 2 mm wide) wit indefinite margins, and unlysed erythrocytes can be made ut microscopically within this zone, ‘These are know 4s viridans streptococei. The alpha streptococci are no" ‘mal commensals in the throat, but may cause opportune ist infections rarely. (Pneumococcus [S,pnewmoniaé is also an alpha hemolytic streptococcus.) © scanned with OKEN Scannerstreprococc! | 0, requtement - “nou Cbkgate onoocbes oes se + tere + % asap san aces 2 cag te | pysobgeal and ) esos rou) | ae mea See eee jroup-specific C ‘by physiological and biochemical propertos MNOPORSTUY) Seco ig ren) n Fig 22.2 Classification of streptococci Beta (8) hemolytic streptococci produce a sharply defined, clear, colourless zone of hemolysis, 2-4 mm wide, within which red cells are completely lysed. The term ‘hemolytic streptococci’ applies strictly only to beta lytic strains. Most pathogenic streptococci belong 10 this group. Gamma (}) or non-hemolytic streptococci produce no change in the medium and include the fecal strepto- cocci which are classified as the Enterococcus species. Based on carbohydrate antigen or Lancefield groups: Hemolytic streptococci were classified by Lancefield (1933) serologically into groups based on the nature of a carbohydrate (C) antigen on the cell wall, These are known as Lancefield groups, twenty ‘of which have been identified so far and named A~V (without I and J). The great majority of hemolytic streptococci that produce human infections belong to group A. They are subdivided into types based on the M proteins, called Griffith typing. About 80 types of Sipyogenes have been recognised so far (types 1, 2, 3 and so on). Table 22.1 shows the medically important strepto- cocci and their characteristics, and Figure 22.2 shows their classification. rec) aera Morphol ‘ New Morphology Gry +ve Creu, Wen Metle The individual cocct are spherical or oval, 0.5-1.0 pm = Pert in diameter. Size variations result from cultural con- | ditions, for example, when grown anaerobically, they mm are somewhat smaller. They are arranged in chains, ) the length of which vary within wide limits and is influenced by the nature of the culture medium, chains being longer in liquid than in solid media. Chain for- mation is due to the cocci dividing in one plane only and the daughter cells failing to separate completely (S.salivarius forms the longest chains). Streptococci are non-motile and non-sporing, Some strains of S,pydgenes and some group C strains have, capsules composed of hyaluronic acid, while polysaccharide capsules are encountered in members of groups Band D. These capsules are best seen in very young cultures, } Cultural characteristics ItLis_an_acrobe and a facultative angerobe, growi ive anaerobe, growing, Best at a temperature, of 37°C (range 22-42°C). Iris exacting in nutritive requirements, growth occurring © scanned with OKEN ScannerPetit BACTERIOLOGY Ay important streptococc! and thelr characteristics “Specievorcomman Lencefield — Hemolysis Habitat fn Laboratory tests Comm rome group ‘human hosts a pyogenes a Throat, skin Bacitracin sensith Per res, oo . be : YR tost positive; tract infect Y Ribose not fermented Pyoderma, , theumatic f ‘ slomerlonepr, Sogalactise 8 Bera Female CAMP est hippurate Neonatal meg genital tract, hydrolysis septicemig "et, rectum | Seguisimiis c Beta Throat ribose and tehalose Pharyngitis fermentation endocarditis Sanginesus ACRG — Betafalpha, Throst.colon, Group Astrains bacitracin Pyogenic infec vuntypable gamma) female genital resistant PYR negative; ions tract Minute colony variants of i ather groups Enterococeussp Gamma Coton Growth in 6.5% NaCl; PYR Urinary tract (Sfoecatis and (alpha, beta) positive infections, endocay. | ‘other enterococci) ditis, suppurative infections | Endocarditis Non-enterococcal D Gamma Colon No growth in 6.5% NaCl i Group D species (S.bovis} Endocarditis Viridens strepto- Nottyped Alpha Mouth, colon, Optochin resistant, (S.sanguis) dental cocci (gamma) _female genital species classification on caries (Simutang). (many species) tract biochemical properties 5 z a Streptococcus pyogenes ‘Clinical Case 1A seven-year-old gil presented with severe sore throat and fever up to 39°C for the previous three days. She denied coryza or earache. On examination, she was found to have bilateral tender submandibular \ympk ‘enopathy, enlarged tonsils and pharyngeal exudates. A throat swab culture was performed and, pending the esul, she was started on oral penicillin. The culture after 24 hours was positive for beta hemolytic colonies, which were | Gram-positive cocci in short chains on smear examination and sensitive to peniclin, The girl was diagnosed With streptococcus pharyngitis. 3 isl Clinical Case 2_Annine-year-old girl developed fever and sore throat, and group A streptococci were cultured fro throat. She developed pain and tender swelling of both knees and ankles; @ palpable’effusion of the right knee ‘drained, which yielded straw-coloured fluid. Culture ofthe aspirate was negative. A few days later, the gil began exp riencing shortness of breath. Physical examination and chest x-ray suggested mild congestive heart failure. The gi Wa diagnosed with acute rheumatic fever, A murmur was audible at the cardiac apex on follow-up after one-year. linical Case 3A seven-year-old boy developed skin infection but his mother did not seek medical help, he pass coloured urine. Four days later, he developed pitting edema in both ankles. On examination, the boy was found to nave elevated blood pressure, 4+ proteinuria, red blood cell casts and elevated serum creatinine, The boy Was diagnosed with acute post-streptococcal glomerulonephritis. asi only in media containing fermentable carbohydrates or hemolysis around them, Growth and hemolysis are enriched with blood or serum, promoted by, CO, (Figs 22.3 and 224 © On blood agar, after incubation for 24 hours, the Virulent strains, on fresh isolation from ea colonies are small (0.5-1.0 mm), circular, semi- produce a ‘matt’ (finely granular) colony, ti ‘Tansparent, low, convex discs with amare oT lear” avirulent strains form ‘glossy’ colonies. Strains Wi x discs with Ar area oT clear © scanned with OKEN Scannerfig 223. Blood agar: Spyogenes showing beta hemolysis Fig 22.4 Blood agar: Spyagenes magnified to show small colonies surrounded by zones of clear hemolysis well marked capsules produce ‘mucoid’ colonies, corresponding in virulence to the matt type. «In liquid media, such as glucose or serum broth, growth occurs as a granular turbidity with @ pow: dery deposit. No pellicle is formed. Biochemical reactions = Bigchemicn ee Sire ‘vera sugars producing acid but FB gas. They are catalast nega and are not sovble Pe Hydrolysis of pyr ' a 0% bre,punt rode Ppeta-naphthylamide (PYR tes) and fire to ferment ribose help differentiate S.pyogenes from olher streptococci. Resistan ‘Sipyogenes is a deli heat (54°C for 30, cate organism, easily destroyed by minutes) it dies in a few days in cultures, unles stored at «low temperature (4°C)» preferably in Robertson's cooked meat medium It ca However, survive in dst for several wees if protected from sunlight tis rapidly inactivated by antiseptic tis more resistant to crystal violet than many bacteri including S.aureus, Crystal violet (I mg/L) nalidixic acid (15 mg/L) and colistin sulphate (10 mg/L) added {0 blood agar provide a good selective medium for the isolation of streptococci, including pneumococci tis susceptible to sulphonamides and many antibiotics, but unlike Saureus does not develop resistance to drugs Sensitviy to bacitracin is employed as a convenient method for diferentating. S.pyogenes from other hemolytic streptococci Antigenic structure Figure 2255 illustrates the disposition of the various antigens in S.pyogenes. The eapsule, when present inhibits phagocytosis, It is not antigenic in hue beings. The,cell wall is composed of an outer layer of protein and lipoteichoic acid, a middle layer of grou specific carbohydrate and an inner layer of peptidog- Iycan, The peptidoglycan (mucoprotein) is responsible for cell wal rigidity. It also has some biological proper ties such as pyrogenic and thrombolytic activity. Carbohydrate antigen: On the basis of the C ca¥- hydrate antigen, Sypyogenes is classified under LLanefild group A. This antigen shows cross-react, ity with some human tissues (see Post-streptococcal infection sequelae). As this antigen is an integral part the cell wal it has to be extracted for grouping BY 4 precipitation test with group antisera. For the test streptococci are grown in Todd-Hewitt broth and cntected with hydrochloric acid (Lancefield’s acid capsule a Pentdogyean ‘Cytoplasm Cytoplasmic membrane Cal wall Fimbi (Pus) Fig. 22.5. Antigenic structure of S pyogenes ee © scanned with OKEN Scannerseaction method), formamide (Puller’s method). J by Singwonnyees ibis (Mantes cving (Rants ancl Randall's act aint the specific antisera x fibes, Drocipi interface Between the extract Grouping may also be nis have been voter part of the cell wall. Sypyogenes be typed based on: : + -M proteins: This is the most important protein J for typing as well as for virulence. It acts as by inhibiting phagocytosis. It is . The antibody to the M protein promotes of the coccus and is therefore pro- tein is heat and acid stable but yptic digestion. It can be extracted id's scid extraction method and typing done with type-specific sera. About 80 M protein types have been recognised. + T proteins: The T protein is an acid-labile,trypsin- resistant antigen present in many serotypes of S.pogenes. It may be specific but many different M types possess the same T antigen. It is usually demonstrated by the slide agglutination test using trypsin-treated whole streptococci * R proteins: Some types of S.pyogenes (2, 3, 28 and 48) and some strains of groups B, C and G contain « third antigen, the R protein. The T and R proteins have no relation to virulence. A non-type-specific protein, associated with the M protein, known as the ‘M-associated protein (MAP), has been identified. Hairdike pili (fimbria): These project through the capsule of group A streptococci. The pili consist partly of M proteins and are covered with lipoteichoic acid ‘which is important in the attachment of streptococci to epithelial cells. Various structural components of S,pyogenes exhibit antigenic cross-reaction with different tissues of the human body. Antigenic relationships have been dem- onstrated between capsular hyaluronie acid and human synovial fluid, cell wall proteins and myocardium, ‘group A carbohydrates and cardiac valves, cyloplasmic ‘membrane antigens and vascular intima, and peptidog- lycans and skin antigens. It has been postulated that these antigenic cross-reactions may account for some of the manifestations of rheumatic fever and other sireptoysin 'O" and 'S", Sueptolvsin Op soe streptococcal ise immunological natures Toxins and other virulence factors S pyogenes forms several exotoxins and enzym., oe Fife to its virulence. Besides these, the Moro ich alsoacts asa viru! ieeTactorby inhibiting hago ein “The C polysaccharide has been shown to hayg 2%. eflect on connective issue in experimental ang tococci produce two he Hemolysins: bin it i oxygen labile, It is inactive in gy’ Sed fom bit may Be tated by eam mild reducing agents. On blood agar, strepay activity is seen only in pour plates and notin gue cultures tay be obtained in the active state by gee® ing streptococci in broth containing reducing agen such as sodium hydrosulpit. Iti also heat age Itappears tobe important in contributing to vrten Itis lethal on intravenous injection into animals ang has speciticcardiotoxc activity. It also has leucotos activity. In its biological action, streptolysin O regen bles the oxygen-labile hemolysins of C perfing C.tetani and S.pneumoniae. : @ Streptolysin O is antigenic, and antstreptelyg O appears in sera following streptococcal infe. tion, Estimation of this antibody (ASO titre) is, standard serological procedure for the tetrospc. tive diagnosis of infection with S.pyogenes, The Iysin is inhibited by cholesterol but not by normal sera, Following certain chemical treatments or ba. terial contamination, sera may develop inhibitory activity due to some changes in the lipoproteins, Such sera are unfit for the ASO test. Because of the complexity of the hemolysis inhibition tes, the ‘ASO test is now done by the serological method of latex agglutination. An ASO titre in excess of 200 units is considered significant and suggests ether recent or recurrent infection with streptococti Streptolysin S$ and © are produced by groups A, Cand G also, © Streptolysin S is an oxygen-stable hemolysin and s9 isresposbefor ho enol sat round te coceal colonies on the surface of blood agar plates {is called streptolysin $ since itis soluble in serum. {isa protein but is not antigenic, Convalescent se dlo not neutralise streptolysin activity, It is itib- ited non-specitically by serum lipoprotein —_ © scanned with OKEN Scannerryogenis exotorin (erytheogenis, Dick, seatlattnal joviad: This tovin was named “erythnogenie® because « iowtadterimal injoction inter susceptible individuals sioced an erythematous rtction (Dik test, 1924), ys test was used t0 identify children susceptible to a type of acute pharyngitis: ‘etbematous fish, sting this toxin, The primary effect of the toxin is induction of fever sexiso twas renamed streptococcal pyrogenic exotoxin {SPE}. Three types of SPE have been identified—A, Band C. Types A and C are coded for by bacterioph es, while type B gene is chromosomal. SPEs are “superantigens” (like staphylococcal enterotoxins and the TSS toxin), T cell mitogens that induce a ma jease of inflammatory cytokines, causing fever, shock sue damagi Streptokinase (fibrinolysin): This toxin promotes the ipsis of human fibrin clots by activating a plasma pre- cursor (plasminogen). It is an antigenie protein, and neutralising antibodies appear in convalescent sera, ntistrepokinase antibodies provide retrospective evi- dence of streptococcal infection. Fibrinolysin appears to play a biological role in streptococcal infections by breaking down the fibrin barrier around the lesions and facilitating the spread of infection, Streptokinase is ven intravenously for the treatment of early myocar- dial infarction and other thromboembolic disorders. Deoxyribonucleases (streptodornase, DNAase): Te tause Cepolymerisation of DNA. Pyogenic exudates contain large amounts of DNA, derived from the nuelei of necrotic cells. Streptodornase helps to liquefy the thick pus and may be responsible for the thin serous character of streptococcal exudates, Thi property has been applied therapeutically in liquefying localised collections of thick exudates, as in empyema ‘A preparation containing. streptokinase and strepto- dornase is available for this purpose. Four antigenically distinct DNAases, A, B, Cand D, have been recognised, of which type B is the most anti- genic inhuman beings. Demonstration of anti-DNAase B antibody is useful in the retrospective diagnosis of S.pyogenes infection, particularly in skin infections, where ASO titres may be low, Streptodornase B and D also possess ribonuclease activity. Nicotinamide adenine dinucleotidase (NADase, formerly diphosphopyridine nucleotidase, DPNase): This acts on the co-enzyme NAD and liberates i ith extensive used by the S.pyogenes steains ‘Streptococcus namie from the molecule. It is antigenic and is speeii- cally noaralised by th seri. ‘The biological signific though iis believed to be leucotoxie. Hynluronidase: ‘This enzyme breaks down hyaluronic acid of the tissues. This might favour the spread of infection along the intercellular spaces. Streptococei possess a hyaluronic acid capsule and also claborate a hyaluronidase—a seemingly self-destrus tive process. It has, however, been found that strait that form hyaluronidase in large quantities (M types 4 and 22) are non-capsulated. The enzyme is antigenic and specific antibodies appear in convalescent sera Serum opacity factor: Some M types of S.pyogenes produce alipoproteinase which results in opacity when applied to agar gel containing horse or swine serum. This is known as serum opacity factor (SOP). Other enzymes: Many strains also produce proteinase, phosphatase, esterases, amylase, N-acetyl glucosami- nidase, neuraminidase and other toxins or enzymes. It is not known whether, and to what extent, these contribute to pathogenesis. Pathogenicity the “The diseases cause by S.pyogenes can be suppurative oF ioesianmaiie whch ratty, whicl cli the sequelae to post: Sireptococcal infections. + Sipyogenes produces pyogenic infections with a ten- ay read ol Toeally, along [ymphaticg and through ‘Teploadstrean— Streptococeal diseases 1, Suppurative: fections: The primary site of invasion Respiratory infections: Of the human body ‘rSeapenesctevoat Soke throat_is_the_most_common, streptococcal disease. it may be localised fas (gnsillits or_may involve the pharynx more itso) group A streptococci adhere to the pharyngeal epithelium by ‘ans of the lipoteichoic acid covering the surface pili he glycoprotein fibronectin on the epithelial cells serves as the receptor to the lipoteichoic acid ligand. ‘Tonsillitis is more common in older children and adults than in younger children, who commonly develop dif- fuse pharyngitis. Localisation is believed to be favoured by hypersensitivity due to prior contact (Case 1), From the throat, streptococci may spread to the sur- rounding tissues, leading (o suppurative complications © scanned with OKEN Scannercvastoiitie. quinsy. Link's ay rarely lead t0 & Smenmeness! prcuimonia sckfom follows cation of aupporetive a a may occur ae a compl causes FTAA Skin and soft tissue infootionyy S a TST tis, Infection of minor Peepticemi 1 infections of the skin ss and gol kat OF fection involving the affected skin, which is Surated, is sharply demarcated nealthy area. While erysipelas young ssod by Sprogenes belonging to a lim- ter of serotypes. Usually the higher numbered of the lower numbered M types which infexion of scabies lesions are the main causes o jomerdlonephritis in children in the tropics. tibody response to streptolysin gh and ASO estimation does not have as | significance as in pharyngeal infections. 1o DNAase B and hyaluronidase are more the retrospective diagnosis of pyoderma ante- to acute glomerulonephritis. Streptococcal subcutaneous infections range from cellulitis to necrotising fasciitis. The latter condi- tion is more commonly caused by a mixed acrobic and enacrobic bacterial infection, but some strains of S poogenes (more particularly M types 1 and 5 forming pyrogenic exotoxin A) may alone be responsible. This is ordinarily « sporadic condition and has been known since 1883, but small outbreaks in the UK and the USA have recently caused much alarm because of their fatality. These strains have earned severity and hig notoriety under the name ‘flesh eating bacteri In such cases, extensive necrosis of subcutaneous and muscular tissues and adjacent fascia is associated with a severe systemic illness—a toxic shock-like syndrome with disseminated intravascular coagulation and mul- tiple system failure, S pyogenes can be isolated from the affected site and rising titres of antistreptolysin and anti-DNAase B demonstrated. Though the isolates penicillin sensitive in vitro, treatment with penicili LYST TYRANT TI Genital infections: may not be effective, Vancomycin is the in life-threatening: infections, Softtssucinfections with some M types ory 1.312, 28) may sometimes cause a toxig 4 2°R Arne resembling staphylococcal TSS, gy {TSS and necrotising fascitis occur only in pee immune to the infecting M types. jth aerobic and anacrob tococed are normal inhabitants of the female yo". S.pyogenes Wi portant cause of puerpeys ta withtheinfetion usually beingexogenous, Thee SP, demonstration by Semmelweis in 1847 that pea outbreaks of puerperal fever could be prevented Pt simple measure of hand washing by those atten! labour wards remains a landmark in clinical mio ogy. Puerperal fever is now much more comma to endogenous infection with anaerobic streprn Streptococcal puerperal sepsis used to take jotics became available on Persons tl Re ny he Poco, aheayay ve infections: S.pyogenes may cay abscesses in internal organs such as the brn, lange liver and kidneys, and also septicemia and pyemia, 2. Non-suppurative _post-streptococeal sequelag S.pyogeres infections Tead to two important nonsup _-Durative sequelae—acute_theumatic fever, (Case 2) ‘and acdte glomerulonephritis (Case 3). These com, plications ensue T=3 WEEKS aT the acute infections) that the organism may not be detectable when sequelae set in, They differ in their natural history in @ number of respects (Table 22.2). The pathogenesis of these complications is not clearly understood. The essential lesion in rheumatic fever is carditis, including connective tissue degener tion of the heart valves and inflammatory myocardial lesions characterised by Aschoff nodules. Typical, rheumatic fever follows persistent or repeated step tococeal throat infections with a strong antibody response, The lesions are believed to be the result of hypersensitivity to some streptococcal component. thas also been suggested that an element of autoin- munity may be involved, and antigenic cross-reactions have been demonstrated between streptococci and heart tissues. Lesions resembling rheumatic fever have been produced experimentally in rabbits by repeated fection with S,pyogenes and in mice by injection of sonic lysates of the cocci. While rheumatic fever may follow infection with an serotype of S.pyogenes, nephritis is caused by only a © scanned with OKEN Scannerand glomerutonephitis Comporison of theumatic fev oper SeTaTaTION Eirehthl serotype of Spyogenes Any anmune response Marked Complement level Unaffected Genetic suscepti Present Repeated attacks Common Penicillin prophylaxis Essential course Progressive or static Prognosis: Variable “pephritogenic’ types. Inthe topics, skin infections are perhaps more important inthis respect than throat infec- tions. The nephorits is usually a self-limited episode that resohes without any permanent damage. The pathogen- ‘esis may be due to antigenic cross-reactions between the lomerular membrane antigen and cell membranes of nephritogenic streptococci, or more often it may be an immune complex disease. This condition has been pro- duced in monkeys and rabbits by repeated infection with ‘ype 12 Spyogenes or injection of bacterial products, ‘nd in mice with soluble streptococcal products. Epidemiology The main source of S.pyogenes is the human upper #espiatory tracl—ihroal, nasopharynx and nose—ot “Patents and carriersyCarnier rates of up to 2U'per cent fave Been observed. ‘observed. Symptomless infection is common and helps maintain the organism in the community. ‘Transmission of infection is either by direct contact or yugh contaminated fingers, dust or Tommesgtm the tropics, streptococcal infection of the skin is common ‘and may be spread by non-biting insects, particularly the eye gnat Hippelates. Spc on fan in more frequent in children at 58 years of age than in children below the age of two years or in adults. They are more common in winter in the temperate countries. No seasonal distribution has been identified in the trop- ics. Crowding is an important factor in the transmission of infection, Outbreaks of infection may occur in closed ‘communities such as boarding schools or army camps. Immunity is type specific and appears to be associ- ‘ated with the antibody to the M protein. Re infections ‘occur because of the multiplicity of serotypes. Throat or skin atnecessany” Pyoderma types 49, 53-55, 59-61 and pharyngitis strains 4 and 12 Moderate Lowered Not known Absent Not indicated Spontaneous resolution Good Laboratory diagnosis In acute infections, diagnosis is established by culture while in the non-suppurative complications, diagnosis is mainly based on the demonstration of antibodies. C%Specimen:, Throat svab, pus swab or exudes "are collected. In rheumatic fever and glomerulonephri- tis, serum is collected for serology, 2. Microscopy: Presumptive information may be obtained by an examination of Gram-stained films from pus, The presence of Gram-positive cocci in chains is indicative of streptococcal infection. However, smears are of no value in infections of the throat or genita; lia, where streptococci may form part of the resident flora 5, Culture: For cultures, swabs should be collected | _ ee O/T rat they ate more sensitive to bacitraclty digg of 0.040 sreploconei_A filler By onthe surtawe or aM NON dovbon. a. wade zon. ef inhibition iy S prvgenes but not with other streplocecct a cheumatic fever and glomeruoneph 5. Serology: rospective diagnosis of streptococcal infection may be os smonsteating high levels of ablishod by eptococeal toxins ‘© The Standard test is antistreptolysin O tat ASO titres higher than 200 are indicative of prior sptoceecal infection. High levels are usually found rheumatic fever but in glomerulonephritis, mation is also commonly than 300 are taken as si B and antihyaluronidase tests are very useful for the retrospective diagnosis of streptococcal pyoderma, for which ASO is of much less value. + The streptozyme test, a passive slide hemagglutina- tion test using erythrocytes sensitised with a crude preparation of extracellular antigens of streptococci, is a convenient, sensitive and specific screening test. Ik becomes positive after nearly all types of strepto- coceal infections, whether of the throat or the skin. Typing MTR protein-based typing of S.pyogenes is required only for epidemiological purposes and may be done by precipitation or agglutination techniques using specific antisera. __ Prophylaxis The indication for prophylaxis in streptococcal infec- tion is only in the prevention of sheumatic fever. This is achieved by long-term administration of penicillin in children who have developed early signs of rheumatic fever. ‘This prevents streptococcal re-infection and further damage to the heart, Antibiotic prophylaxis is ‘not useful for glomerulonephritis as this complication follows a single streptococcal infection, and re-infec- tions do not occur. Treatment Allbeta hemolytic Group A streptococci are sensitive to ind most are sensitive to erythromycin. In 10 penicillin, erythromycin orcephalexin nay be used Strains resin 10 er hromyaiy lev apy ™ hen reported, "Flracytines and sulphona hy eonmended. Antinncrobial APS Nave efor vatablished glomerulonephritis and rheum MH eee Tee} Resides Sipyoqenes, streptococci belonging wy y, BC, D, FG and rarely H, K , O and ® may a iw infection. Of these, B C and G a = cause hun common. Group B ‘These are important pathogens of cattle, produ ovine mastitis. Streptococcus agalactiae is an imp tant human pathogen responsible for the folly. infections. : Neonatal infections: From the 1960s, group sg, tococeus has assumed great clinical importance ashe single most common cause of neonatal meningitis, the West. Infection in the newborn is classified as the ‘Early onset type, occurring within a week of binh «Late onset type, developing between the second and twelth weeks of life ‘The, more common early onset type presents as ‘meningitis or septicemia, and is often fatal. Infections acquired from the maternal vagina during birth. In the Jate onset type, infection is more often obtained from the environment and presents as septicemia, Other group B infections in neonates include anh. tis, osteomyelitis, conjunctivitis, respiratory infections, peritonitis, omphalitis and endocarditis, Group B streptococci may also cause adult infee- tions, including puerperal sepsis and pneumonia. Their ability to hydrolyse hippurate acts as a presumpine identification method. They may be identified by the CAMP test (Christic, Atkins and Munch-Peterson), which can be demonstrated as an accentuated zone of hemolysis when S.agalactiae is inoculated perpen dicular 10 a streak of S.aureus grown on blood ager (Fig. 22.6). Human pathogenic group B strains pos 5 a polysaccharide capsule which appears to conf virulence. Nine capsular serotypes have been identified, antibodies to which confer type-specific protection. Group Streptococei of this group are predominantly ans! pathogens, Group C strains isolated from hums © scanned with OKEN Scanner_—— J Group 8 stroptocoect Group A stroptocoeet yig.22.6 CAMP test sources ustally belong to the Swequisimitis species, Trean cause upper respiratory infections, as well as esp infections such as endocarditis, osteomyelitis trun abscess, pneumonia and puerperal sepsis. Strains tne often tolerant to pencilin and serious infections stay not respond to penicilin treatment, The addi ton of gentamicin is recommended in serious cases. Tr resembles Sipyogenes in fermenting trehalose but differs in fermenting ribose. It produces streptolysin ©, streptokinase (antigenically distinct from that produced by Sipyogenes) and other extracalllarsub- Frances, S.equisimilis is the source of streptokinase ted for thrombolytic therapy in patients, Group F These grow poorly on blood agar unless incubated under CO,, They have been called the ‘minute strep- tococci’”. They are sometimes found in suppurative lesions. One member of this group is Streptococcus MG which is an alphalytic strain isolated from cases of primary atypical pneumonia, Demonstration of agglutinins to Streptococcus MG in the sera of patients had been used as a diagnostic test for primary atypical pneumonia. Group G “These are commensals in the throats of human beings, “They may occasionally cause ton and urinary infections in human Other groups Groups H and K sometimes cause infective endocardi- xd mainly from the healthy human tis. Group Q isis throat and may cause acute tonsillitis and endocarditis. Group R strains are natural pathogens of pigs: They hhave beon reported from occasional eases of mening tis, septicemia and respiratory infection in persons 19 ‘contact with infected pigs oF contaminated meat. cocci hus been reclassified asa separate genus called Enterococcus and contains different species, for exam ple, faecalis, E.fuecium and Exdurans. Enterococe! inctive features that distinguish possess several di them from streptococ of 40% bile, 6.5% sodium chloride, at pH 9. and in 0.1% methylene blue milk. ‘© On MacConkey medium, they produce pink colonies. © They are relatively heat resistant, su for 30 minutes. © They pically appear as pairs cells in a pair arranged at an any (Fig. 22.7). ¢ They are usually non-hemol may show alpha or beta hemolysis. Tdontifiation of the Enterococcus species is made ‘on biochemical grounds. E,faecalis is the enterococ- us most often isolated from human sources. It cam Be identified by its ability to ferment mannitol, sucro%ts ‘Cebitol and esculin, and to grow” on teliurite blood agar producing black colonies. Enterococci are present in the intestine, genital tract and saliva, They ate frequently isolated from cases of urinary tract infection and wound infection. ‘They may dso cause endocarditis, infection of the biliary tract septicemia and intra-abdominal abscess complicating diverticulitis and peritonitis. ‘Antimicrobial resistance: Strains resistant to penicillin and other antibiotics occur frequently, soit s essential to perform antibiotic sensitivity for proper therapy. Enterococci are intrinsically resistant to cephalosporins and offer low level resistance to aminoglycosides. In penicillin-sensitive strains, synergism occurs with combination treatment with penicillin and aminoglyco- side, However, ifthe strain shows high-level resistance to aminoglycosides, this synergism does not occur. ‘The choice of drug for infections due to such strains is vancomycin. Recently, VRE (vancomycin-resistant enterococel) have begun to emerge. The phenotypes “They can grow in the presence at 45°C tiny deep- ing at 60°C of oval cocci, the gle to each other Jytic, though some strains © scanned with OKEN Scanneree. mene erm Oval cells arranged in pairs at mn short chains. reponsibh ein resistance could be Van A, B.C. DE. The mechanism is the alteration of D-aln- nyl-D-alanine chain in the cell wall Group D Members of this group (S.boris, S.equinus) are gener- ally susceptible to penicillin and are inhibited by 6.5% sodium chloride or bile. They may cause urinary infee= tion or endocardit Viridans group This group, formerly called Streprococcus viridans, is 4 miscellany of streptococci normally resident in the mouth and upper respiratory tract, and typically pro- ducing greening (alpha lysis) on blood agar—hence the name viridans Some of them may be non-lytic. They cannot be categorised under the Lancefield antigenic groups, However, based on sugar fermentation, cell wall coms poston a prueton of dexteans and ean, fave been classified into many SPECS, For gy its, Somutans, Ssalvars and S.sangus, ‘They are ordinarily non-pathogenic: but cq, on ceasion enuse disease, In persons with pre-eyiy i carne lesions, they may cause bacterial endoeagi Scans boing, most often responsible, Poly tooth extraction or oer dental procedures, they cae transient bacteremia and get implanted on damage op prosthetic valves o in a congenitally diseased hap, tnd grow to form vegetation, Prophylactic aniigge cover is advisable in such persons before tooth extrge, tion or similar procedures, While Viridans streptocgg are generally penicillin sensitive, some strains may he resistant, is therefore essential that in endocardit, the causative strain is isolated and its antibiotic soy, sitivity determined so that appropriate antibiotics ig axlequale bactericidal concentration can be employed for treatment, Sireplococeus mutans is an alpha hemolytic step. tococcus which is part of the normal flora of the oral ewvily, This Gram-positive coccus is commonly found in the mouth, from where it can spread to cause dena caries or endocarditis in individuals with risk fac tors (dental extraction in people with damaged heat valves), ‘The bacterium has a polysaccharide coat (glycocalyx) that allows it to stick to teeth and also to damaged heart valves; it can invade the bloodstream, also produces acid from sugar in saliva, and this pro: motes erosion of tooth enamel. Normal body defences are usually adequate to prevent disease, In microscopy, Gram-positive cocci in chains are noted. It cannot be grouped by the Lancefield scheme and is resistant to ‘optochin and bile, Disease due to this bacterium can be prevented by maintenance of good oral hygiene and by regular dental check-up; prophylactic antibiotics may be needed prior to major dental work on people with damaged heart valves, they imple RECAP + Bacteria belonging to the genus Streptococcus are Gra facultative anaerobes. They occur in pals or short a (require enriched media such as blood) for growth, AP MED sg Im-positve, oidase and catalase negative, and ind long chains ': Some are nutritionally fastidious © scanned with OKEN ScannerStreptococcus Pneumoniae and : between the organism and pneumonia was esa by Fraenkel and Weichselbaum inde ently in 1886. Morphology S pneumococci are typically small (1 jm), gated cocci, with one end broad or rounded an other pointed, presenting a flame-shaped or la appearance. They occut i 2 pair, the capsule enclosing each pi The capsules are best seen in material taken diredy eee from exudates and may be lost on repeated cultivation a Gram-positive, lan- Inculture, the typical morphology may not be apparent (ommonly"FelerFS and the cocci are more rounded, tending to occur in specific polysaccharide of the human are Gram p demonstrated as a ‘Ginicat Case 1A 60-year-old man was brought in with a history of hi ‘ous twa days He also had mild chest pain and productive cough. The sp ‘showed consolidation in the right lower lobe. The direct smear observed after Gram: positive, lanceolate-shaped diplococcl, Sputum culture after 24 hours was positive 25 greenish colonies on blood agar. 1 ‘was dlagnoSed wit after another 24 hours showed the organism to be sens to which he responded. Clinical Case 2 A five-year-old child was brought to the Emergency department high-grade fever, He had also had 1~ eae one to have altered sensorivm and neck ri showed high counts of the presence of Gramp. hemolytic colonies on sheey and optochin sensi f the polymorphs. The culture was positive for alpha ‘agar which had the typical draughtsman colonies’ appeara Se catalase suggestive of Spneumeniae, The patient was started on penicilin and responded '© @ scanned with OKEN Scanner‘Streptococcus Pneumoniae colonies on blood ‘of beta hemolysis res do not undergo autolysis. ions CL 1e are catalase and oxidase negative. They 1% sodium deoxycholate solution Ta few drops of catrare, the are added to 1 ml of an over culture clears due to lysis of the ¢ Iysesuithin.a few minut a constant iae and hence is of diagnostic importance. The test should be carried out at neutral pH using deoxycholate and live young cel solubility test is based on the pres- mmoniae of an autolytic amidase that Fig 232 Spneumoniae, India ink preparation to show capsules Cultural characteristics SS , ‘Smeumoniae have complex growth requirements and of the organisms, ‘grow only in enriched media. They are aerobes and facultative anaerobes, the optimum temperature being 37°C (range 25-42°C) and pH 7.8 (range 6.5-8.3). Growth is improved by 510% C incubation for psumoriae jee by bla gi pha hemolysis) around them, resembling colonies lans streptococci. On further incubation the raised edges and central roncentric rings are seen on the surface when viewed fro or carrom coin appearanc develop abundant capsular material (types 3 and 7) form large mucoid colonies. Fig 23.3. Bile solubility test @ scanned with OKEN ScannerPec ACTEROOEY Bem Capsulates lanceol Qeetiung Tes Positive Gatonies on Nood ager later draughtsman" Uniform turbidity Positive Positive Positive Fatal infection Growth in guid mest Bae studeiny ns and are readily th point 52°C for 15 due to sn accumulation of xy be maintained on semi- ve 10 most antibiotics, beta lactams Almost all strains were began to appear. Optochin sensitivity: The sensitivity of S.pneumoniae wl hydrocuprein) 1/500,000 is useful g them from streptococci. When a dise sar inoculated with S.preumoniae, a wide zone wn appears on incubation (Fig. 25.4). Antigenic properties, The most important antigen of the Spneumoniae is the type-specific capsular polysac- As this polysaccharide diffuses into the 323.4 Spneumoniae colonies sensitive to optochin Initially dome-shaped, ture. they die on pro 1 Dleesnonon Draenor S pneumonise and Viridans streptococel ate dploeneel Non pines. ol orend cay Negative Dome-shaped colonies Granular turbidity, powdery depos, Negative 4 Negative j Negative | Non-pathogenic ; i culture medium or infective exudates and tis is also called the ‘specific soluble substance im it Sppneumoniae are classified based on the any: SS), nature of the capsular polysaccharide, and now than 90 different serotypes are recognised. 1, 2, 3 and so on. Serotyping based on capsular antigens may ried out by: @ Agglutination of the cocci with the type. speci antiserum Precipitation of the SSS with the specific serum By the capsule swelling or ‘quellung’ react (quellung = swelling), described by Neufeld (1999, Here, a suspension of S.pneumoniae is mixed on slide with a drop of the type-specific antiserum ang aloopful of methylene blue solution. In the presenge of the homologous antiserum, the capsule becomes apparently swollen, sharply delineated and refrac. The quellung test can be done directly with sputum from acute pneumonia cases. It used to be a routine bedside procedure in the past when the specie antiserum was used for the treatment of pneumonia (Fig. 25.5). © PCR-based tests have shown higher sensitivity in the detection of infections, especially meningitis a, it can detect the presence of a small number of the specific DNA sequences of the bacteria which can- not be cultured by conventional methods due to the administration of prior antibiotics or because of a smaller bacterial load in the body fluids. org Named deca ic Other antigens: S.preumoniae contain other ant- gens as well—a nucleoprotein deep inside the cell and a somatic ‘C’ carbohydrate antigen, both of which are species specific. ‘An abnormal protein (beta globulin) that precipitates with the somatic ‘C’ antigen of S.pnewmoniae appea's in the acute phase sera of pneumonia but distp- © scanned with OKEN ScannerFg 235 Quellung reaction srs during convalescence. It also occurs in some cal conditions. This is known as the Cereactive protein (CRP). Its apparent antibody-like relationship to the ‘C’ antigen of S.pneumoniae is only fortuitous. It is not an antibody produced as a result of pneumococcal infection. Its an ‘acute phase’ substance, produced in hepatocytes. Its production is ulated by bacterial infections, inflammation, ma- Tignancies and tissue destruction. It disappears when the inflammatory reactions subside. Variation On repeated subculture, S.pnewmoniae undergo smooth-to-rough (S-R) variation. Inthe R form, the colonies are rough and the cocci are non-capsulated, autoagelutinable and avirulent. R forms arise as spon- taneous mutants and outgrow the parental $ form: artificial culture; in tissues, such R mutants are elimi- nated by phagocytosis, Rough S.pneumoniae derived from the capsulated cells of one serotype may be made to produce capsules of the same or different serotypes, on treatment with DNA from the respective serotypes of S.preumioniae. This transformation, which may be demonstrated in vivo or in vitro, was discovered by Griffith (1928) and is of considerable historical interest as the first demonstration of genetic exchange of information in bacteria. Toxins and other virulence factors The virulence of S pneumoniae depends on: © The eapsular polysaccharide, because of its acidic and hydrophilic properties, protects the cocci from pneumoniae are not media or exudates. ce phagocy- such as Streptococcus Pneumoniae Phagocytosis. Capsulated [phagocytosed efficiently in th “They are, however, susceptible to ‘surla tosis’, being engulfed against a firm surface, ‘a fibrin clot ot epithelium. ‘The enhanced virulence of type dlue to the abundance ofits capsular material. Non- capsulated strains are avirulent. The antibody to the capsular polysaccharide affords protection against infection. © Pacumolysin, duced by S,pneumoniae, ‘ment activating properties factor. It is immunogenic. Pneumolysin- ‘mutants show reduced virulence in experimental animals. © Autoly “Bacterial components in infected tiss contribute fo virulence. . Sipreumoniae produce an oxygen labile hemolysin and a leucocidin but these are weak and make no con- tribution to virulence 3 S.pneumoniae is a membrane damaging toxin pro- has eytotoxic and comple~ ‘and so may be a virulence negative + Pneumococcal autolysins, by releasing ues, may als Pathogenicity Experimentally, (onion _can_be_ross, in_mice_or_rabl by_intraperitoneal inoculation rein 1-3 days, an of _S.pneumoniae, 1 S pneumoniae can be demonstrated in large nuimbers in the peritoneal exudate and heart blood. ‘S.pneumoniae colonise the human nasopharynx and may cause infection of the middle ear, paranasal sinuses and respiratory tract by direct spread. Infection ‘of the meninges may also occur, by contiguity or through blood. Pneumococcal bacteremia may also lead to distant infections in the heart, peritoneum or joints. Infection is commonly endogenous, but exog- ‘enous infection may also occur, especially with highly virulent strains. ‘S,pneumoniae are one of the most common bacteria causing pneumonia, both lobar and bronchopneu- monia. They also cause acute tracheobronchitis and ‘empyema (Case 1). Aspiration of nasopharyngeal secretions containing ‘S.pneumoniae into the lower respiratory tract is a com- ‘mon event and may occur even in sleep, Normal mu- cosal defence mechanisms such as entrapment, expul- sion and the cough reflex, aided by the ciliary escalator effect, prevent the establishment of infection. When the normal defences are compromised by viral infection, © scanned with OKEN ScannerSACTERIONOGY Paces ara ape the penteochial tssttes the pend is common dharing the is de to diflusion Tower Je into Nood and f of symptoms ticapsular i slmast always a second= nay be caused by any serotype he damage to the respiratory ve bronchial secretions on facilitate the size along the bronchial tree. frequently a terminal event in are commonly associated with acute exacerbations in chronic bronchitis. The copious secretions in chronic bronchitis aid pneu- casion. Another bacterium commonly asso- this condition is Haemophilus influenzae. Meningitis isthe most serious of pneumococcal in- fections. In is usually secondary to other pneumococcal infections such es pneumonia, otitis media, sinusitis or conjunctivitis but in 2 proportion of cases, other foci of infection may not be demonstrable. Pneumococcal meningitis occurs at all ages. Untreated cases are al- most invariably fatal, Even with antibiotic therapy, the case fatality rate is about 25 per cemt (Case 2). S.pneumoniae may zlso produce suppurative lesions in other parts of the body—empyema, pericar- itis, otitis media, sinusitis, conjunctivitis, suppurative arthritis and peritonitis. It is also responsible for ocular infections like keratitis and dacryocystitis. Epidemiology The source of human infection is the respiratory tract of carriers and, less often, of patients. S,pneumoniae occur in the throats of approximately half the popula- tion sampled at any one time. They are transmitted by contaminated droplets or droplet nuclei. Dissemination is facilitated by crowding, Infection usually leads only to pharyngeal carriag Disease results only when host resistance is lowered by contributory factors such as respiratory viral infec tions, pulmonary congestion, stress, malnutrition, im- munodeficiency or alcoholism. Splenectomy and sickle cell disease are important predisposing conditions, TS Sypmeumoniae serotypes Vary Brey jn jy The ease fatality rates of pneumonia tay vayy Re, ing to the virulence of the infecting serotyp yen the most vielen, The commonest pret? Fi feetions are otis media and sinusitis. Prior yyotl i infection or allergy eausing congestion and py predispose to these conditions. Serotypes 6, 14 and 25F are commonly encountered in theye ¢ tions in the West. In adulis, types 1-8 are responsible for 75 per cent of eases of pneumococcal preumon st for more than 50 per cent of all fatalities due 5° mocaccal bacteremia. In children, types 6, 14, yg 23 are frequent causcs. nd Inindia, lobar pneumoniaisusually asporadicqisy but epidemies may occur among closed commune” a in army camps. The incidence of bronchopneys nia increases when an epidemic of influenza or gn viral infection of the respiratory tract occurs, Casas more common in winter and affect the two extreme age groups more often. Laboratory diagnosis The clinical diagnosis of pneumonia is easy but as ty disease may be caused by several different mictootgan, isms, etiological diagnosis should be made by labors tory tests. This is of great importance in treatment, 1, Specimen: Sputum, CSF, blood for culture ang fine are used for antigen detection 2Mltssany’ Inthe acute phate of bar reno i, the rusty sputum contains S.pneumoniae in lage numbers, with hardly any other kind of bacterium. They may be demonstrated by Gram stain, In acute ots media, S.pneumoniae may be demonstrated in the fid aspirated from the middle ear. In meningitis, presump- tive diagnosis may be made from Gram-stained films of CSE. Gram-positive diplococei can be seen both inside the polymorphs and extracellularly, 3. Cultuss; The sputum, after homogenisation if Tecessary, is inoculated on blood agar plates and incubated at 37°C under 5~10% CO,. Growth occurs after overnight incubation. Isolation from respiratory secretions is facilitated by using blood agar containing ‘gentamicin 5 ug/ml Blood culture: In the acute stage of pneumonia, the organism may be obtained from blood culture in slucose broth. Isolation of S,pnewnoniae from blood indicates a bad progno: “i Mage + 19p ‘Ong. © scanned with OKEN Scannerinoculation: tn ‘ J ate expected ta be Mouse r : Pecimens — where scanty, tsolations may eritoeal inoculation in ice, es ale negative, Inoculated mie die hn se may be sheomonstrat peritoneal exudate and heart Mood fe olsiaunet by tntray ¥ slays, andl S prreume The test mu may ye with occasional strains that are avirulent nice (YPC HY stains) 4 Antigendetection: Although diagnosisisconfirmed ty ale meting nes hich ae dure, it may be possible to establish the di r 1¢ diagnosis by strating the SSS in CSE by precipitation with fontiveta oF the latex agglutination test, : an be demonstrated in too, rine and cerebrospinal fluid by counterina poslectrophoresis, Now, an Capsular polysaccharide wnochromatography- tue testis available for the detection of polysite ride antigen in urine 6. Biomarker using latex py snostic procedure, Procaleitonin i CRP testing, by passive agglutination another /e pneumococ- disease, and the levels are monitored to determine prognosis and response to treatment, 7. Molecular_methods: PCR-based methods have suet potential where the patient hus taken antibiotics, Prophylaxis. ‘Yinmunity is type specific and associated with antibod- ies to the capsular polysaccharide. ‘The existence of some 90 serotypes makes a complete polyvalent vac- cine impracticabl. + A polyvalent polysaccharide vaccipe represent- “ing the capsular antigens GF TET ost prevalent a _—_—_—_—— nccus Preumaniae werotypes fay heen stated to give 0-90 an, Hip not meant for general use, but only in petions nt enhanced risk of pneumococeal infection ‘such as those with absent or dysfunctional spleen, slekle vell d celine diseare, chronic. renal, nd liver diseases, diabetes mellitus and ciey including HIV infection. It is ‘ot recommended in children under the age of two years and those with Iymphoreticular malignancies nd immunosuppressive therapy. . njugate va Tphitheriae) is now available which can be used in children from two months to two years, However, protection would depend on whether the serotypes included in the vaccine are Iso prevalent in the community where the vaccine is used. Treatment ¢ antibiotic of choice is parenteral penicillin in se- vere cases and amoxycillin in milder ones, provided the infecting strain is penicillin sensitive. Many, penicillip- resistant strains are also resistant 4o other antibioties like erythromycin and tetracycline. The resistance may be intermediate (MIC 1 pig) or high (2 jug or more) and due to mutation or gene transfer. The mode of resistance is not production of beta lactamase, but alteration in the penicillin binding proteins on the bac- {crial surface. Such strains are also resistant to multiple drugs, A drug-resistant S,pneumoniae (DRSP) strain originating in Spain has spread to most parts of the ‘world, posing problems in treatment. A third-generation cephalosporin is indicated in such ‘cases, Vancomycin is to be reserved for life-threatening illnesses with highly resistant strains. © scanned with OKEN ScannerCORYNEBACTERIUM DIPHTHERIA Somphaiogy choracteristics OTHER PATHOGENIC CORYNEBACTERIA ULCERANS OTHER CORYNEFORM BACTERIA INTRODUCTION Corynebacteria are Gram-positive, non-acid_fast, non-motile zads with irregularly stained segments, and Sometimes granules. They frequently show club-shaped swellings—hence the name Corynebacterium (from coryne, meaning club). The most important member lose siblings and completion of vaccinatlon was advised, bacterium Corynebacterium diphtheriae Bat eed) dULate | | of the genus is C.diphiheriae, the causative g | iphtheria ; oben hhas been known since Bet gy | chen ‘The disease was first recognised as a clinical egy Bretonneau (1826) who called it “diphtheritg wy diphiheros, meaning leather). The name ig erie from the tough, leathery pseudomembrane fot! the disease. The diphtheria bacillus was first byes and described by Klebs (1885) but was first cut by Loeffler (1884). It is hence known as the Kia! Loeffler bailus or KLB. Locler studied the eft thebacilusin experimental animals and concluded the disease was due to some diffusible product of te bacillus. Roux and Yorsin (1888) discovered the dg theria exotoxin and established its pathogenic ety ‘The antitoxin was described by von Behring (1890, Ceo) a ae Uc) Morphology The diphtheria bacillus is aslender rod witha tendency ta clubbing at one or both ends, measuting approximately 3-6 x 0.6-0.8 um, The bacilli are pleomorphic, They are non-sporing, non-capsulated and non-motile, They are Gram positive but tend to be decolourised easily, ‘The granules are often situated at the poles of the bacilli and are called polar bodies. They are more strongly Gram positive than the rest of the bace- rial cell. Stained with Loeffler's methylene blue, the granules take up a bluish-purple colour and are hence called metachromatic granules. They are also called nical Case A five-year-old child presented to the Pediatrics Outpatient department with a history of pain in the throat and difficulty in swallowing. He had had high-grade fever for the previous two days, On examination, he was found to have cervical lymphadenopahy, and the tonsillar pillars were covered by a white discharge. The mother was informed. | that her child's immunisation was not complete, A throat swab was collected and submitted for microscopy and cultue. Albert's stain showed the presence of rod-shaped bacteria, green in colour, along with bluish-black granules. A diagnosls Of diphtheria was made and passive immunisation along with antibiotics started. The antibiotics were also prescribed to | © scanned with OKEN Scanneri nast_granuie a Babes oF sot Soot polmtaphosph ye fe These granules te and serve as stor- es. such as Albert's, ; 4 + Neisser's an Se have beet devised for demonstrating i tor leary. The baci sve arranged in a cha sf ‘They are usually seen in ies (resembling the stakes of a fence) or the bacil being at various angles o each ing the letters V or L. This has been oat, FS fa the Chinese letter Or cuneiform arrangement sis due t0 incomplete separation of the daughter Tag afer binary Hission. Cultural characteristics Growth i scanty on ordinary media, Enrichment with food. serum oF exg 8 necessary for good growth, ‘The optimum temperature for growth is 37°C (range 15-40°C) and the optimum pH is 7.2. Cdiphiheriae is snaerobe and a facultative anaerobe, ‘The usual media employed for the cultivation of the diphtheria bacillus arc: Loeffler’s serum slope: Diphtheria bacilli grow ot [pelfers Serum slope very rapidly and colonies can Fe wen in 6-8 hours, long before other bacteria Frow. Colonies are at first small, circular white opaque dscgpput enlarge on com Zequire a distinct yellow tint. « Telurite blood agar: Several modifications of tellur- ite bloed agar have been utilised, such as McLeod’s and Hoyle's medig. Tellurte (0.04%) inhibits the growth of most other bacteria, acting as a selective agent, Diphtheria bacilli reduce tellurite to metal- lic tellurium, which is incorporated in the colonies, giving them a grey or black colour. The growth of diphtheria bacilli may be delayed on the tellurite medium and colonies may take two days to appear. Based on colénial morphology on the tellurite medium and other properties, McLeod classified theria bacilli into three types: gravis, intermedius and nitis. The names were originally proposed to relate to the nicl severity ofthe disease produced by the three types—gravis, causing the most serious, and mitis the mildest variety, with intermedius being responsible for disease of intermediate severity. However, this associa tion is not constant. is and intermedius types are associated case fatality rates, while mitis infections are less lethal. ° ' 'ralytic complications are most common in gravis, h iemorrhagic complications in gravis and interme= dius, and obstructive lesions in the air passage in imitis infections, Ingener is the predominant strain in endemic ‘areas, while gravis and intermedius tend to be epi- demic, The mitis type is better able than the more virulent types to establish a commensal relationship with the host. Wide variations have been noted in the frequency ofthe different types in different places at different times, Evidence shows that the gravis and, to a lesser extent, the intermedius strains are able to spread more readily than mitis in populations naturally immune or artificially immunised, Table 25.1 lists the characteristics ofthe three types. Rivchemical reactions Hiss’s~-serum™-sugars Diphtheria_bacilli_ferment glucose, galactose, maltose and dextrin with the pro wn __dustion of acid but without gas) they do not ferment lactose, mannitol GF sucrose. Some strains of virulent diphtheria bacilli have been found to ferment sucrose. Itis necessary to use Hiss's serum sugars for fermenta~ tion tests. Proteolytic activity is absent. They do not hydrolyse urea or form phosphatase. Toxin it strains of diphtheria bacilli produce a very powerful exotoxin. The pathogenic effects of the bacillus are due to the toxin, Almost all strains of gravis and intermedius (about 95-99 per cent) are toxige only about 80-85 per cent of mitis strains are so. ‘The proportions vary with the origin of the cultures tested, Strains of all three types are invariably virulent when isolated from acute cases. Avirulent strains are common among convalescents, contacts and carriers, particularly in those with extrafaucial infection. There is considerable variation in the amount of toxin produced by the different strains, some produc~ ing it abundantly and others only poorly. But the toxins produced by all strains of the diphtheria bacilli are qualitatively similar. The standard strain almost univer- sally used for toxin production is the Park-Williams 8 strain, which has been variously described as a mitis (Topley and Wilson) and intermedius (Cruickshank). © scanned with OKEN Scanner‘Usually short rods, with uniform staining few or no granules, Some degree of pleomorphism, with Xy barred, snow-shoe and & 0 Dlood ager periphery and commencing erenat 2-3 days, 3-5 mm in size, Nat Consistency of colonies Hemotysis Growth in broth Glycogen and starch Positive Tehas a molecular weight of about 62,000. It potent and the lethal dose for a 250-g guinea 0.0001 mg. It consists of two fragments, A and B, MW 24,000 and 538,000, respectively. Both fragment ‘re necessary for the tonic effect. When released by t bacterium, the toxin is inactive because the active site on fragment A is masked. ion is probably accom- plished by proteases present in the culture medium and infected tissues. All the enzymatic activity of the toxin is present in fragment A. Fragment binding the toxin to the cells. The antibody to fragment is extremely cells. The toxin is labile. Prolonged storage, i 37°C for 46 weeks, treatment with 0,2-0.4% formalin |. Toxoid is toxin that has toxin production are as follows: * The toxigenicity of the diphtheria baci on the presence is depends of corynephages (tox+), s may be rendered beta or some other lysogenic or phage conver- greyish black centre, paler, ‘and crenated ‘Long barred forms with clubbed ends; poor granu- lation, very pleomorphic 18-hour colony mi a cedge— frog's ege' Intermediate between Soft, buttery, emulsifiable easily fine granular sediment Negative sion. The toxigencity remains only as long g bacillus is lysogenic. When the bacillus is cure phage, as by growing it in the presence Of antiphage serum, it loses its toxigenic capacity, = ‘Toxin production is also influenced by the concen tion of iron in the medium. The optimum level ofan for toxin production is 0.1 mg/l, wi tion of 0.5 mgy/ inhi i ion: The diphtheria top protein synthesis, Specifical @ concentra. s the formation of the myocardium, adrenals and nerve endings. @ scanned with OKEN ScannerVery pro URT ted vaginal or Bacteriophage typing © cutaneous. "afte ‘inf. fAbout 15 bacteriophage types have been described. wns are usuall sn to-fhe Types I and I IV and Vi intermedius, ions. Sometimes, VII avirulent gravis and the remainder virulent gravis. The phage pes are appre sable. sytem of jough pseudomembrane forma- ies occur. tion may somet ial diphtheria the MOST COMMON PE ANE Thay sry ffom mild catarshal inflammation to very wide read invohement (Case). According tothe clinica with DNA probes. fi kes oratory diagnosis, there is severe nia with marked adeni neck). Death is!“he due to tory failure. There is a high incidence ~ Mogiéal purposes but not for th of paralytic sequelae in those who recover. cases. Specific treatment should be insti ‘© Septic, which leads to ulceration, cellulitis and even gangrene around the pseudomembran Hemorrhagic, which is characterised by bleeding from the edge of the membrane, epistaxis, conjunc- tival hemorrhage, purpura and generalised bleeding tendency. laboratory tests. Any delay may be fatal men: _One or two swabs from the lesions are ‘The common compl : Asphyxia due to mechanical obstruction of the respi- ratory passage by the pseudomembrane for which an emergency tracheostomy may become necessary. Acute relatory failure, which may be peripheral @ scanned with OKEN Scannermn, progressing to necr al and no change do not die. As many as ten strane tested at atime on a rab can by ‘which is more specific. In vitro tests © Elek’s gel preci Sctztian: For culture, the swabs are THOSaEe oa LoefMer’s serum (1000 units/ml) is placed on the sur normal horse serum agar in a pet ot aval serum may be used. When the w= the surface is dried and nerrow stele the strains are made at ri strip. Positive and negative controls should be pa up. The plate is incubated at 37°C for 24-48 howe ‘Toxins produced by the bacterial growth wil ram slope may show growth ive, will have to be incubated for 24 hours. Tellurite plates will have to be incubated for atleast vo days before being considered but some brands of peptone and some samples of serum do not give diagnosis to be © Tissue culture test: The t may be by the in vivo or Baga -be-temonst the former by the subcutaneous or the latter by the preci xin produced diffuses tissue culture t them, In vivo tests “ZS Subéuianeoiis test; The gro: night culture on Loeffler's slop. 2-4 ml of broth and 0.8 ml of the emul: A @ scanned with OKEN Scannerthousands, with a mortality rate of up to 20 per cent ‘of what can befall countries that neglect _Active_immunit ‘ions deteriorate. done using a a disease of child- hood. Its rare in the first year of life due to the passive immunity obtained from the mother, reaches a peak between 2 and 5 years, falls slowly between 5 and 10 years, and ra for- is myoh-more immunogenic than z Mm ay ecH om iphl hundredfold or more in endemic areas are the most fection. In the temperate regions, the nose and throat, Nasal carriers for longer periods than pharyngeal fect the skin diphtheria toxoid given in a dose of 10-25 Lf u more often than the respiratory tract, Tox recommended. Smaller doses (1~2 Lf units) are used in the skin for over three years. ren and adults to preparations, the lower dose of toxoid carriage is mainly harbour the ba to diphtheria but may also lead to faucial dip s do not seem to play an In nature, diphtheria is vit gs, though cows may on occasion be found to @ scanned with OKEN Scannerssoee, falloweal by a fourth dose about a year after i Dowster dose is given at school entry Pescive imovonisation: This is an emergency mear ible persons are exposed ofstunhtberia ja.odited iS Teeonsists of the subcutas nistration of 500-1000 units of serum, ADS), As this is a horse against hypersensitivity should be observed, Combined immunisation: This consists of adminis dsorbed toxoid on one gr, siher am, to be continued al receive ADS prophylactically should 1 sation. 1 of diphtheria consists of antitoxic rapy. Antitoxin should be given imme- uspected, asthe fatality rate yy in starting antitoxic treatment. recommended dose is 20,000-100,000 units for ses, half the dose being given intravenously. xin treatment is generally not indicated in cuta- phtheria os the causative strains are usually Spee and antibiotic th is fe to penicillin and can be cleared from the throat witht Saar by penicil- lin treatment. Diphtheria patients are given a course cf penicilin though it only supplements and does not coxin therapy. Erythromycin is more active than penicillin in the treatment of carriers. ois Pee A Sera aN CORYNEBACTERIUM ULCERANS ‘This bacillus related to C.diphtheriae can cause diph- theria-like lesions. It resembles the gravis type of the diphtheria bacillus but it liquefies gelatin, ferments trchaluse slowly and does not reduce nitrate to nti It produces two types of toxins, one probably identi- cal to the diphtheria toxin and the other resembling the toxin of C,pseudotuberculosis. It is pathogenic to guinea pigs, the lesions produced resembling those caused by C.liphtheriae. It has been found to ~ cause infection in cows, and human infec, toe transittedthronigh cow's ai, 1p 5g 6 ORs i, erythromycin, Diphtheria antitoxin j, rt It has been suggested that Cuuleerans be comet subgroup of diphtheria bacilli rather than ged, vies Pa species. at “areanobacteriwm — (formerly Coryney, acmolyticum can cause pharyngitis and ny akelum can cause cuLAMeOUS and blood jgy th, in immunocompromised hosts. It is usual resistant, responding only to vancomycin, nary importance Preisz-Nocard bacilli (C.pseuotubercutessy "the cause pseudotuberculosis in sheep and sy) “hich lymphadenitis in horses, C.renale causing one pyelonephritis in cattle and C.egui, which i 2 from pneumonia in foals. at et Iy mutigg ray CORYNEBACTERIA CAUSING SUPERF) ‘SKIN INFECTIONS Enythrasma, a localised infection of the stratum g rneum usually affecting the axilla and groin, is enc by Cminutissimum. Ths is a lipophilic cornet? rium and can be grown readily in media contain, 20% fetal calf serum. "s Catenuis has been associated with trichomes axillaris, characte -d by the formation of Pigmented nodules around axillary and pubic hair shafis CIAL DIPHTHEROIDS Corynebacteria resembling C.diphtheriae occur as normal commensals in the throat, skin, conjunc and other areas. These may sometimes be mistaken or diphtheria bacilli and are called diphtheroids. In gen- eral, diphtheroids stain more uniformly than diphtheria bacilli, possess few or no metachromatic granules and tend to be arranged in parallel rows (palisades) rather than in a cuneiform pattern. However, some diphtheroids may be indistinguishable from diphtheria bacilli microscopically. Differentiation is by biochemi- cal reactions and more reliably by virulence tests. The common diphtheroids are C.pseudodiphtheriticun (C.hofmannii) found in the throat and C.xerosis found in the conjunctival sac. The former is urease positive tind does not ferment glucose, while the latter is wrest negative and ferments glucose, Both are pyraz dase positive, unlike diphtheria bacilli © scanned with OKEN ScannerCorynebacterium are non-motile, aerobic, Gram-positive bacilli, some species being commensals in the human body. Corynebacterium diphtheriae causes diphtheria in the upper respiratory tract. Skin infections may ‘occur due to toxigenic and non-toxigenic strains in skin lesions of individuals in poor socioeconomic Grcumstances. ‘The bacterium attaches to the back ofthe throat of (m diphtheria toxin, which kills celts and causes inflammation and fibrin accumulation tea tion of the characteristic pseudomembrane (which may break off and lead to asphyxi Corynebacterium diphtheriae grows slowly on selective media containing tellurite (colon but rapidly on enriched media such as Loeffer’s serum slope. Toxin production can be detected by precipitation with antibody in an Elek plate. Active immunisation during childhood ing antibodies that protect against the effects of active toxin secreted during infection. children, Ths is followed by secretion of the to the forma- the diphtheria toxoid stimulates the production of neutralis- @ scanned with OKEN Scannery @ scanned with OKEN Scanner hove 75°C and the minimum from Fe yo 45°C. Their salt tolerance varies from leg 2% to 25% NaCl. “ ibi Their spores are Meter and air and constitute the com, pacteriological culture medi bo, BACILLUS ANTHRACIS Morphology Resistance Pathogenicity ANTHRAX, Epidemiology Laboratory diagnosis Prophylaxis Treatment ANTHRACOID BACILLI BACILLUS CEREUS ‘own to possess spores (Koch 1876) and the fin, ium used for the preparation of an attenuated INTRODUCTION are classified into Sporogenous, rod-shay genera, the aerobic Bacil idia, THe genv' and virulence They are generally motile with peritrichous flagella, the anthrax bacillus being a notable exception. yin Morphology Baths. ‘Members of this group exhibit g o anthrax bacillus is one of the-fakgest ofA their properties. The genus includes psychrophilic, mesophilic and thermophilic species, the maximum temperatures for vegetative growth ranging from about swelling aro a history of injur Culture was positive for Bac amoxicillin.Commencing Tram the Top agar, the colonies are non-hemol 2 produce a narrow zone of hemolysis. occurs as floccular deposits, with use bulging of the veget Cultural characteristics 25-30°C. Good growth occurs on ordinary media. In cultures, the bacilli are arranged end to end in long chains. The ends of the bacilli are truncated or often concave and somewhat swollen so that a chain of bacilli presents a ‘bamboo stick’ appearance (Fig. 26.1). ——_ 20 ularly round colonies are Gimed-2=3 mm in diameter, raised, dull, opaque, —<~°8°" Re Fig. 263 Inverted fir tree appearance in gelatin stab Fie. 26 Bamboo stick appearance on Gram staining Fig. 26 vulture from cultures @ scanned with OKEN ScannerBiochemical reactions { .o8e end sucrose are fermented, produc 2s are reduced 10 fli rats Peluclm pe: ative bacilli are not particularly resistant and i Cin. 50 minutesJIn the carcasses : Fax, the bacilli mizin vigble in the hone marrow for a week and in icin for two weeks, Normal heat fixation of smears ¢ bacilli in blood films. The spores are to physical and chemical agents. They anthrax spores in less than 70 hours, Four per cent potassium permanganate kills them in 5 . Destruction of the spores in animal prod- 2% solution of wool and as hair and bristh at 30-40°C for 20 0.25% at 60°C for six hours for animal Pathogenicity 3 Jn nature, anthya aril sheep, and fess often of horses and si thentally most animals are suse or lesser degree. Ral ie bul exper to a greater flow of blood, vo virulenee factors have been identifeg « The_capsular polypeptide, which aig ‘a ing phagocylO5is. Loss of the x 02) which controls capsule prod, to loss of virulence. This is how the | anthrax spore vaccine (Sterne strain) was gp The anthrax toxin, which is encoded by «e388 forin was identified by the (rit Tea that injecting the sterile plasma of guinea pig ga? of anthrax into healthy guinea pigs ing d that death could be prevented by immanent The toxin is a complex of three fractions, "™™ — Edema factor (OF or Factor 1) = Protective antigen factor (PA or Factor [1 thal factor (LF or Factor Il} Thay are not oxic individually but the whole compl produces local edema and generalised shock. Thee three factors have been characterised and cloneh binds tothe receptorson the tage cell surface, and in turn provides attachment ses OF or LE, fac s binding to target cus, OF is an adenyl cyclase which @ scanned with OKEN Scanner‘occurs, with Epidemiology Anthrax is india, the number of ani the tens of thousands annu some countries such as imported through conta iiser and other animal carrying loads of hides and skins and hence was known as the hide porter’s An epizo the Andhra Pradesh-Tamil Nadu border, cau cutaneous and meningoencephalitic huma with high mortality rate, There have been outbreaks Anthrax infection in used to be commom-amiong worker - ries, due to inhalation of dust from infected wool © This is a hemorrhagic pneumonia with a high fatal- ity rate, Fig. 264 Cutaneous lesions oe @ scanned with OKEN Scanner3. Animal imocelation: The anthrax bacillus can 5. Serology for antibodies: Acute and convalescent * be obtained, since antibodies to sm can be demonstrated by gel diffusion, jon and ELISA techniques. &, Molecular methods: For further confirmation, rutile, non-hemolytic on blood agar and catalase posi- “e—can be given a presumptive report of anthrax. F initial confirmation, lysis by gamma phage and fluorescent antibody test (DFA) for capsule-spe —_ It was Pasteur’s convincing demons protective effect of his anthrax vaccine in experiment at Pouilly-le-Fort in 1881 that may beginning of sci ic immunoprophylaxis, the vaccine was the anthrax becillus attenuated bye at 42-45°C. BTR AS the spore is the common infective fon : nature, vaccines consisting of spores of anenm | strains were developed. The Sterne vaccine ¢ spores of a non-capsulsted, avirulent, mutan ‘The Mazzucchi vaccine contained attenuated Carbazoo strain vaccines have been used exter ing a single injection. They are not considered safe yg human use, though they have been ws immunisation in Russia. Alum precipitated toroid prepared from the protective antigen has been shown to be a safe and effective vaccine for human use. has been used in persons occupationally exposed 19 anthrax infection. Three doses given intramuscularlyat intervals of six weeks between the first and second, and six months between the second and third doses induce good immuni hh can be reinforced if necessary with annual chlorampheni strains resistant to pe longer used for treat floxacin are cot ae @ scanned with OKEN Scanneree TTC acetal Pemeee ALIPTT scophylavis and teat pots nd treatment. However, antihiot poreecT OA THe TOMI OEE HLH format ewes have ANTHRACOID BACILLI Many members of the genus Bacilly me acillus, othe ° anthrax bails, have oeeasionally wecdhuranter (Of them, the most important is B.cereus, which tions from 1970 has been recognised as a f food-borne vis with sep en troenteritis, It has also been ies emia, meningitis, endocarditis, pneumo- ive lesik as an opportunist pathogen, Bosbet iformis and a few other species have also occa, sionally been isolated from such lesions, These and a large number and variety of non-pathogenic aerobic spore bearing bacilli that appear as common contami. nants in cultures and have a general resemblance to the anthrax bacilli have been collectively called pseudoan- thrax or anthracoid bacilli. Table 26.1 lists the main

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