Morphology culture Resistance Classification CLOSTRIDIUM PERFRINGENS Morphology Cultural characteristics Virulence factors pathogenicity CLOSTRIDIUM SEPTICUM CLOSTRIDIUM NOVY! (C. OEDEMATIENS) CLOSTRIDIUM HISTOLYTICUM Pathogenesis GAS GANGRENE Clinical presentation Laboratory diagnosis Prophylaxis and treatment CLOSTRIDIUM TETANI Morphology Cuttural characteristics Cassifcation Pathogenicity TETANUS LOSTRIDIUM BOTULINUM Morphology Cultural characteristics Resistance Classification Pathogenicity BOTULISM Control, Clostridium perfringens. Clinical Case So-year-old man had a road trafic accident while travelling in a remote vilage area. He sustained multiple fractures with open wounds in the left leg. On reaching the nearest hospital two days late he was found ta bein shock. The wound was contamni- ‘nated with soil and blood: the local muscles appeared to have been crushed, He was started on supportive therapy and antibiotics, but after two days, the edema ‘and pain at the site increased and a serous discharge developed. When the area around the wound was pal- pated, crepitations were felt. Microscopic examination ‘of the wound discharge showed the presence of thick brick-shaped, Gram-positive bacilli along with Gram= positive cocci, Based on a provisional diagnosis of gas gangrene, immediate surgical treatment was carried Out. The exudate was also inoculated into Robertson's ‘cooked meat medium and cultured for anerobic bacte- ‘a, which was positive for Clostridium perfringens and peptostreptococci. Extensive excision of the local part had to be carried out to prevent further spread. INTRODUCTION cconsists_of Grat bic, spor The spores are The genus Clost ana ler than the bacillary bodies, giving the bacillus a swollen appearance, resembling a spindle—hence the name Clostridium (from Kloster, meaning spindle). The genus contains bacteria responsible for gas gangrene, food poisoning and tetanus. Some of the pathogens, for example, C.perfringens and C.tetani, are found normally in human and animal intestines. Many spe- ‘cies are pathogenic but most are saprophytes found in soil, water and decomposing plant and animal matter. Intestinal clostridia rapidly invade the blood and tissues of the host after death and initiate decomposition of the cadaver. Some (for example, C.acetobutylicum) are of industrial importance, and used for the production of chemicals such as acetone and butanol. Morphology Clostridia are highly pleomorphic, Gram positive and usually 3-8 x 0.4-1.2 um in size; in older a ooo_—= EP i Anaerobic Bacteria |: Clostridiumcultures, cells and. involution ‘with pertrichate 1 motile meh as Cperfingens 2 suet motile, Motility S10 fl None Cperfingens and COUN uma ie others are Not a grote th ae Srency in different sPeeie os pe EO das the baciliary body (8 7), some CuCl 8 teragenes) sporuate ead? “fi on as ceingens) do $0 incOnStT orulation a Cpe ee in the animal body. TPS shape and re gary indifferent species an hese are o to of pores a in picaton of eos win the identification and cies i) ‘Spores may be: i © Central or equator shape (C.bifermentans) | Sutcrmina, te bos i ial, giving the bacillus @ spindle s appearing club shaped (C perfringens) ‘e Oval and terminal, js racket (Cerium) “e Spherical and terminal, giving @ drut ance (C.tetani) Clie ae as “The sensitivity to oxygen Var~ aifgent spectes-Ssome (for example, Cnovyi) xygem, er exacting anaerobes and die on €xDOSUre [0 © nile others (for example, C:histolyticum) are aerotol- resembling @ tenn! imstick appear ROE ina cern “spherical and terminal spores Oval and terminal spores (eg. Clostridium tetan) (e9., Clostridium tertur) (val and central spores. subterminal spores {eg ostraum biermentans) (eg. Clestndum portngens) Fig 27.1 Types of spores a ily. OF serobically. OF MOTE impor en 81 F oxygen isthe provsi a i se potential (EA) the medium, f redor by adding reducin substance eved BY Ms, ascorbic acid, gluta, ed fatty @ a cane acid, alkaline glucose, sul Ape a nial concentra of CO, pce eure opts ember Re saprophytie hermophilic and others psychrophilc { His 7-7 ely stow on solid media. Colonial are hemolytic ie, Some species a 1 medium is Robertson's Y Septains unsaturaced fatty ais he reaction being catalysed by ‘iso sulphydryl compounds ation reduction (OR) iabl oid, Most species produce 8 The sac ces turns meat pink. T the proteolytic spe ck and produces foul and pervasie ik medium, the production of acid, charolytic spect ns meat wus mil be detected cies tu odours. In lit clot and gas ca Resistance The vegetative cells of clostridia do not differ from th of non-sporing bacilli in their resistance to physical an Chemical agents. The spores exhibit a pronounced but variable resistance to, heat fectants Spores of C.botulinumn 1 a fling after 34 hours Srrroven ai 4Q32C are not Killed completely in ess than 100 minutes. Spores of most strains of C.perfringens are “Tesiroyed by boiling for less than five minutes but spores ‘of some Type A strains that cause food poisoning sui" for several hours, C4etani spores persist for yearsin died earth, Spores of some strains of C.tetani resist boiling fo 15-90 minutes, hough in most cases, they are destroyed apa fie minutes. All species are killed by autoclaving aa comin 20 minutes. Spores are particularly as fae disinfectants. Formaldehyde is not pease ee may sometimes survive immersion pea 2 selon ot vp to five days. Halogens are effec: a ae iodine solution kills spores withi reef Chie Jdehyde (2% at pH 7.5-8.5) is ved ig spores. In general, clostridia are sus: = : ptible to metronidazole, penicillin, cephalosporins am* chloram I gis a less so to tetracyclines; and resistant © ioglycosides and quinolones. :losriia can produce disease only when the condi- sons are appropriate, Their invasive powers are limited towsahogenic clostridia. form powerful exotoxins Cootulinum is. virally non-invasive and non- jnfectious. Botulism is caused by the ingestion of preformed toxin in food. Eretani has litle invasive property and is confined to the primary site of lodgement. Tetanus results from the action of the potent exotoxin it produces, « Cperfringens, besides being toxigenic, are also inva- sive and can spread along tissues and even cause septicemia. Classification Many methods have been adopted for the classification ofelostridia. These include morphological features such fas the shape and position of spores and biochemical features such as saccharolytic and proteolytic capaci ties (Table 27.1). Clostridia of medical importance may ‘also be categorised based on the diseases they produce (see below). Pett deta (Cuwelehit, Bacillus aerogenes capsulatus, B phlegmonis emphysematosae) “The bacillus was originally cultivated by Achalme (1891) but was first described in detail by Welch and Nuttall (1892), who isolated it from the blood and organs of a cadaver. This is the most important of the clostridia causing gas gangrene. It also produces food poisoning and necrotic enteritis in human beings and ‘many serious diseases in animals. Ar im CReringens is a normal inhabitant of the large intestines of humans and animals tis found in the feces and it contaminates the skin of the perineum, buttocks and thighs. The spores are commonly found in soil, dust and air. Morphology It is a plump, Gram-positive bacillus with straight, parallel sides and rounded or truncated ends, about 4-6 X 1 jum in size, usually occurring singly or in chains or small bundles. It is pleomorphic, and filamentous and involution forms are common, It is ‘capsulated and non-motile. Spores are central or sub- terminal but are rarely seen in artifical culture or in ‘material from pathological lesions, and their absence is one of the characteristic morphological features of C perfringens. Cultural characteristics Ivis an anaerobe but can also grow under microaer- ophilic conditions. Oxygen is not actively toxic to the bacillus and cultures do not die on exposure to air, as happens with some fastidious anaerobes. It grows over a pH range of 5.5-8.0. and temperature range of 20-30°C. Though usually grawnat 57°C, a temper {ure of 45°C is optimal for many strains. The genera tion time at this temperature may be as short as ten minutes, This property can be utilised for obtaining pure cultures of C.perfringens. Robertson's cooked meat broth inoculated with mixtures of C.perfringens ‘and other bacteria and incubated at 45°C for 4-6 hours serves as enrichment, Subcultures from this transferred to blood agar plates yield pure or predominant growth of C:perfringens. Table 27.1 ‘Amorphological and biochemical classification of clostridiaParti! BACTERIOLOGY cooked mi owth occurs in Robertsor Siam is turned pink But Ts not digest The meat ear hasanacid econ anda sour oder ‘In litmus milk, fermentation of lactose leads to the formation of acid, which is indicated by the change in colour of the litmus from blue to red. The acid ‘coagulates the casein (acid clot) and the clotted milk is disrupted due to vigorous gas production. The paraffin plug is pushed up and shreds of clot are seen stick ing to the sides of the tube. This is known as stormy fermentation. ‘After overnight incubation on rabbit, sheep or human blood agar, colonies of most strains show target hemolysis, resulting from a narrow zone of ‘complete hemolysis due to theta toxin and a much ‘wider zone of incomplete hemolysis due to the alpha toxin. This double zone pattern of hemolysis may fade con longer incubation, Glucose, maltose, lactose fand sucrose are fermented with the production of and gas. It is indole negative, MR positive and VP negative. H,S is formed abundantly. Most strains reduce nitrates. Spores are usually destroyed within five minutes by boiling but those of the ‘food poisoning” strains of Type A and certain Type C strains resist boiling for 1-3 hours. Autoclaving at 121°C for 15 minutes is lethal. Spores ‘are resistant tisepties MT TiaaTCeants in common we. Virulence factors Cperfringens strains are classified into five types, A to E, based on the toxins they produce. Typing is K-40 Ken - Toews cade) as a aH MRA Che WRE , er lL ; fetal S (urotyst done by neutralisation tests with specific antvoxing {ntracutaneous injections in guinea pigs or intravenoyy injection in mice. ‘Toxins: Cperfringens is one of the most prolific of tonin-producing bacteria, forming at least 12 distin toxins, besides many other enzymes and biologically fetive soluble substances. The four ‘major toxins eta, epsilon and iota, are predominantly alpha, responsible for pathogenicity (Table 27.2) a (a) toxin is produced by all types of Cherfringens find most abundantly by Type A strains Sea Tnost important toxin biologically and jg responsible for the profound toxemia of gas gangrene Itis lethal, dermonecrotic and hemolytic. It is a phos. pholipidase (lecithinase C) which, in the presence of Ca** and Mg** ions, splits lecithin into phosphoryl choline and diglyceride. This reaction is seen as opal- ‘escence in serum or egg yolk media and is specifically neutralised by the antitoxin. Nagler reaetion: When C.perfringens is grown on a ‘medium containing 6% agar, 5% Fildes’ peptic digest of sheep blood and 20% human serum, with the antitoxin spread on one half of the plate, colonies on the other half without the antitoxin will be surrounded by a zone of opacity. There will be no opacity around the colonies on the half of the plate with the antitoxin, due to the specific neutralisation of the alpha toxin. This specific lecithinase effect, known as the Nagler reaction, is a useful test for the rapid detection of C.perfringens in clinical specimens. The incorporation of neomycin sul- phate in the medium makes it more selective, inhibiting coliforms and aerobic spore bearers. Human serum may be replaced by 5% egg yolk. The opalescence inaie the ving bacteria also (Cnovyi, C bifermenn a Mprios,some aerobic spore bearers). Inthe ang sore bacteria, the reaction i not neutralised by the tegen anti, exept with Ciena pa roduces 2 serologically related leithinase z ‘The alpha toxin is hemolytic for the red cells of most ies except horse and goat, due to its action on the srecpolipds on the erythrocyte membranes, Lysis ig Fiche hot-cold variety, being best seen after incuba. oa at 37°C followed by cooling at 4°C. Its relatively tow able and is only partially inactivated by botling for five minutes. . Beta (9), epsilon (c) and iota (\) toxins have lethal 4 ising properties. Gamma (j)-and eta (n) ‘pa rave mi Action. The delta (8) toxin has ‘etka effect and is hemolytic for the red cells of even feed ungulates (sheep, goats, pigs, cattle). The theta (@) toxin is an oxygen labile hemolysin antigenically related to streptolysin O. It is also lethal and a general ctolytic toxin, The kappa (x) toxin is a collagenase The lambda (A) toxin is a proteinase and gelatinase ‘The mu ()) toxin is a hyaluronidase, and the nu (v) toxin a deoxytribonuclease. Enzymes: Besides the toxins, C.perfringens also alee soluble substances. These include: ‘ enzymes that destroy the blood group substance, Aand H + a neuraminidase which destroys myxovirus recep- tors on red blood cells, + a substance that renders red blood cells panagglu- tinable by exposing their T antigens «a hemagglutinin active against the red blood cells of human beings and most animals fibrinolysin hemolysin distinct from the alpha, theta and delta toxins * histamine * a bursting factor which has a specific action on ‘muscle tissue and may be responsible for the char- acteristic muscle lesions in gas gangrene ‘circulating factor’ which can cause an increase in the adrenaline sensitivity of the capillary bed and ‘iso inhibit phagocytosis Pathogenicity Cperf. i a the following human tera Conialom yolk media may be produced by other lecithi : Gas g ‘dominan eens LPettringens Type A is the pre 2 the sole ot, SUSiNB BAS gangrene. It may occur Sative agent BUTS more commonly seen is only wh ate invaded that _gas_ganj ar ) se £28_gangrene (anaerobic Tivos) Food poisoning: 1 4 eae ng aot strains of Type A can produce pe patie mn ha are characterised by the marked Rte, ieir spores and the feeble produc- ol theta toxins. They have been shown 10 produce a heat labile gnterotoxin which, like the enterotoxins of Veholerae and enterotoxigenic E.coli, leads to fluid accumulation in the rabbit ileal loop. * Food poisoning by C.perfringens is usually caused by Ve a cold or warmed up meat dish. ‘When contaminated meat is cooked, the spores in fewr the interior may survive. During storage ot warm- ing, they germinate and multiply in the anaerot environment of the cooked meat. Large numbers of clostridia may thus be consumed, which may pass. unharmed by gastric acid due to the high protein content of the meat and reach the intestines where they produce the enterotoxin. After an incubation period of 8-24 hours, abdominal pain, diarshea and vomiting set in. The illness is self-limiting and recovery occurs in 24-48 hours. © Diagnosis is made by isolating heat-resistant perfringens Type A from feces and food. As this ‘may be present in normal intestines, isolation from feces, except in large numbers, is not meaningful. Isolation from food has to be attempted by direct plating on selective media, as the bacillus is present in food mainly as vegetative cells. Gangrenous appendicitis: C.perfringens Type A (and occasionally Type ae ‘ins Pe abies from gangrenous appendicitis. Demonstr et in these patients and the beneficial effects of the administration of antitoxin also suggest the causa tive role ofthe bacillus in this condition. It has been that the toxemia and shock in some cases of proposed ven and peritonitis may be due to the intestinal obstruction and peritonit toxins of C.perfringens. is is a severe and often fatal Necrotising enteritis: This i 9 sere oc trains mteritis It is caused by C.perfringens Type C EEBee g mucosal necrosis. The eae nan it New Guns pln abdominal pain and diarrhea fllowingunaccustome cae Immunisation with the type C toxoid icin shvn potest oa i coon, ilar tractinfection: Cperfing eestor burstios incon orien tect sete emphysematous choleysis and. pos cholecystectomy septicemia. Endogenous gas gangrene of _intra-abdor 1: Gas gangrene of the abdominal wall has been reported as an infrequent complication of abdominal surgery. The infection is endogenous, the organism being derived from the gut and contaminating the abdominal wall during surgery. Gas gangrene of the thigh as a result of infection tracking from the abdo. men has also been reported. Brain abscess and meningitis: Brain abscess and meningitis due to C.perfringens have been reported very rarely, Panophthalmitis: Panophihalmitis due to C perfringens has occasionally followed penetrating eye injuries, ian situations, Urogenital infections: Infection of the urinary tract may occasionally follow surgical procedures such as nephrectomy. Clostridial infection of the uterus is s cate and not infrequent condition, commonly asso. Guted with septic abortion. Septicemia is common ty this condition, ran .OSTOUNSEPTICUN This bacillus was first described by Pasteur and Joubert (1887) and called Vibrion septique. It is a pleomorphic bacillus, about 3-8 X 0.6 jum in size, forming oval, cen- tral ot subterminal spores. It is motile by peritrichate flagella. Growth occurs anaerobically on ordinary media, The colonies are initially irregular and transparent, turn- ing opaque on continued incubation, Hemolysis occurs pn horse blood agar. Growth is promoted by glucose, {tis saccharolytic and produces abundant gas, Parcll) BACTERIOLOGY the inc v factors: — resistant spores which germinate in th # Six groups have been recognised, based on gg and flagellar antigen ona * Cusprcum produces at lest four dig The alpha toxin is hemolytic dermonecrogie ct bl ox ecto eon lg ase, the gamma toxin a hyaluronidase an toxin an oxygen labile hemolysin, he * It produces a fibrinolysin, C.septicum is found in the soil or in animal intestn Itis associated with gas gangrene in humans, usualy Association with other clostridia, I also case re in sheep and ‘malignant edema’ in catte and sheep.» Seay OEDEMATIENs) This is a large, stout, pleomorphic, Gram-postng bacillus with large, oval, subterminal spon? itis widely distributed in soil. Iris a strict anacrobe, ready reeognn bY exposure to air. Four types (Aw Dye) Tne a based on the production of toxin, Ost ‘pe Ais of medical importance, as it eauses pro gan. Frrfe_ as gangrene caused by C:novyis characte by high mortality and large amounts of eden fhid Fe itle ono observable gas in infected tissue Ok crer® Produce veterinary disease, There was a leny Stet of Crtoryi Type A infection among here addicts in Britain in 2000, forming This is an actively oval, Pathogenesis The infection can be exogeneous or endogencous ® Exogeneous: Clostridia usually enter the wound along with implanted foreign particles such as soil (particularly manured or cultivated soil) road dust, bits of clothing or shrapnel. They may also be Present on normal skin, especially on the perineum and thighs, * Endogeneous: Infection may seen after clean surg cal procedures (especially amputations for vascular disease) and even injections (especially adrenaline). z isoi ning rant ures ently und so 4 vnc oud id contamination with no ps h no invasion ¢ pete ing Gace realing in tine wen the wieay in wound healing, a athe ellis i which cos nade th Amerians, wth minimal toxin productions esto of muscle tissues. The disease is gradual fe ine and may Vary fom a limited “gas fensive involvement of a limb, iserobie myositis OF £25 gangrene, Which isthe : rious, associated with clostridial invasion abscess’ to most se fealihy muscle tissues and abundant formation of exotoxins Gas gangrene results only if the conditions favour able for clostridial multiplication exist in the wound ‘The most important of these is low oxygen tension, ‘The ionised calcium salts and silicic acid in the soil cause neorosis. Crushing tissue or tearing of the arter- ies produces anoxia of the muscle, Extravasation of blood increases the pressure on the capillaries, reduc- ing blood supply still further. The Eh and pH of the damaged tissues fall, and these changes along with the chemical changes that occur within the damaged and anoxic muscles, including breakdown of carbohydrates and liberation of amino acids from proteins, provide ideal pabulum for the proliferation of anaerobes Exravasated hemoglobin and myohemoglobin are reduced and cease to act as oxygen carriers. As a result, aerobic oxidation is halted and anaerobic reduction of pyrwate to lactate leads to a further fall in Eh, The clostridia multiply and elaborate toxins which «cause further tissue damage. The lecithinases damage cell membranes and increase capillary permeability, leading to extravasation and increased tension in the affected muscles, causing further anoxic damage. The hemolytic anemia and hemoglobinuria seen in perfringens infections are due to the lysis of erythro- fi "6272 Gas gengrene es a lagen barriers in thoi, TM collagenases destroy cok down t=? inthe tlaves and teshanes eet spe he itera sabstncas gee of gas recuge, Comtidla: The, abundant. producti 5 reduces bn undant. production ‘ supply still urd foe The at? lil further by pressure of anoxic damage. It thus tion to spread from the 4 progressive one, extending becomes possible for the infea Original site, making the lesion GAS GANGRENE Oakley (1954) defin spreading, ede acteristically j ed_gas gangrene as a rapidly tmatous myonecrosis, occurring. cha rs in assoration with severe wounds of *xtensive muscle masses contaminated with path ee a th pathogenic cb Particularly with Cperingens. The disease) ls been referred to in the past as malignant edema. expt" Oke dessipve tome nae sah ‘myositis and clostridial myonecrosis (Fig, 27.2). 2 Gas gangrene is characterisically a disease of War, J in which extensive wounds with heavy contamination ,“) | are very common, In civilian life, the disease generally P74 follows road accidents or other types of injury involving xy v crushing of large muscle mass. Rarely, it may follow surgical operations. The bacteriology of gas gangrene is varied. Rarely is this due to infection by a single clostridium. Generally, several species of clostridia are found in association with anaerobic streptococe! and facultative anaerobes such as E.coli, proteus and staphylococci. Among. the pathogenic clostridia, C.perfringens is the most frequently encountered (approximately 60 per cent), with C.novyi and C.septicum next (20-40 per cent) and C.histolyticum less often. Other clostridia usu- ally found are C.sporogenes, Cfallax, C.bifermentans, Cssordellii, C.aerofoetidum and C.tertium, These may not be pathogenic by themselves. The relative incidence of the different species varies in different series of cases ‘and may be a reflection of the distribution of the spe: cies in different soils Clinical presentation The incubation period may be as short as seven hours ‘wound was created, Spas Tong as sik weeks after the woun r the average being 10-48 hours with C.perfringens, 2-5 days with Csepticum and 5-6 days with C.noryt ith i ising a sion, The disease develops wit pee cand-edema-of the affected part along wit systemic signs of toxemia. There is @ thin watery dis- pet Srylley bir,Parcit_ BACTERIOLOGY are guinous. Accumulation of gas tissues erepitant (Case). In untreated ¢ makes the ases, the disease Process extends rapidly and inexorably. Profound toy craaiatt Prostration develop and death oceurs due ¢ circulatory failure. Laboratory diagnosis ihe diagnosis of gas gangrene must be made Primarily on clinical grounds, and the function ofthe is only to provide confirmation of the and identification and enumes organisms. The mere presence of clostr does not constitute idia in wounds Bacteriological ing with the scanty seen in films from gas gangrene) Specimens: — 1 44.) be Cadf Eiseant wre: Ims from the muscles at the Sage or tig fess fhe tom the tissue inthe necrore eee the exudate in the deeper Léa a 7 Nagler 18 24-48 hours, to differ especially in C.perfri 84S gangrene. The i z characters, of Cperfringens infection (Fig. 27.5) with oval, subterminal spores indicate Crnowyi. Slender bacilli with round, terminal spores may be C.tetani or Ctetanomorphum. ah \erobic and anaerobic cultures are made Ser ot blood a Fig. 27.3 Gram stain of Clostridium perfri ‘gengrene: note the absence of spores However, C.perfringens kCitom bodies! ry reverse CAMP test is used and boat- or leaf:shaped pleomorphic baci vith CpertingerageN irregular staining suggest C.septicum, Large ba ‘antioxn entiate the organisms with hea. resistant spores. Blood Cultures are often. postive, ingens and C.septicum infection, s bacteremia may occur without solates are identified based on thei morphological, cultura, biochemical and toxigenic (Fig. 27.5) gar, preferably on 564 BENE rors Swarming. A plat of serum or cop laxis and treatment jleO% yolk agar, with C.perfringens antitoxin spread on one ' Nagler reaction (Fig. 27.4) Fou: roth are inoculated 5 and 20 minutes, od agar plates after all, is used for th tubes of Rober and heated at 100°C for 5, 10, 1 incubated and subcultured on blo ag Surgery is the most im, Tere ne ne Al damage’ tissue should be removed promptipans the wounds irrigated to rem, lots, necrotic tissue and foreign materials. In teas ‘gas gangrene ingens ingcat. ms, out eit nic ify is "P of ms Streplococcus pg275. Reverse CAMP test uncompromising excision of all affected parts may elife-saving. Where facilities exist, hyperbaric oxy gen may be beneficial in treatment Hl + Aging effective in prophylaxis, in combina. on with surgical methods, The drug of choice is metronidazole given intravenously before surgery and repeated eight hourly for 24 hours. As mixed aerobic and anaerobic infections are usual, a more broad-spectrum. antibiotic prophylaxis, such as a combination of metronidazole, gentamicin and amoxicillin, is advisable Passive immunisation with ‘anti~gas gangrene fequine polyvalent antitoxin in-@-dose-ol- 10,000 TU C.perfringens, 10,000 IU C.novyi and 5,000 IU C.septicum antitoxin given IM ot in emergencies IV) used to be the common practice in prophylaxis. However, in view of its uncertain _-sificagy and availability, its use has become rare, 4 La Cian is the causative organism of tetanus. Tetanus tas been known since very early times, having been ‘scribed by Hippocrates and Aretacus. The final rool of the etiological role of the bacillus in tetanus as furnished by Kitasato (1889) who isolated it in Pere culture and reproduced the disease in animals by ‘neculation of pure cultures. nla is widely distributed in soil and in the intes- ** of humans and animals. It is ubiquitous and has “recovered from a wide variety of other sources, a y aerobic Bacterial Closviglowy Morphology ation in length. It has roundedends.Itoccur The spores are spheri 4 straight axis, parallel sides and ssinglyand occasionallyin chain the bails the characteristic ramntoe Sreceae (Fig. 27.6). The morphology of the spore depends on its stage of development and the young spore may be oval rather than spherical. It is non-capsulated and motile by peritichate flagella, Young cultures are strongly Gram positive but older cells show variable Staining and may even be Gram negative Cultural characteristics It is an obligatory anacrobe that grows only in the absence of oxygen. The optimum temperature is 37°C and pH 7.4. It grows on ordinary media, Growth is improved by blood and serum but not by ghicose. Surface colonies are difficult to obtain as the growth has a marked tendency to swarm over the surface of the agar, especially if the medium is moist. An extremely fine, translucent film of growth is produced that is practically invisible, except atthe delicately filamentous advancing edge. This property enables the separation of Ctetani from mixed cultures. If the water of con- densation at the bottom of a slope of nutrient agar is inoculated with the mixed cell culture, after incubation anaerobically for 24 hours, subcultures from the top of -_ Fig. 27.6. Ctetan drumstick appearance on Gram sainthe tube will yield a pure growth of the tetanus baci (Fildes technique), In deep agar shake cul spherical Mufly balls, 1—3 mm in diameter, made up of filaments with a radial arrangement, In gelatin stab cultures, a fir tree type of growth occurs, with slo liquefaction. I¢ grows well in Robertson's cooked meat broth, with turbidity and some gas formation, ‘The meat is not digested but is turned black on prolonged incubation. On blood agar, o hemolysis is produced, which later develops into hemolysis, due to the pre duction of hemolysin (tetanolysin) Biochemical reactions: C.ietani has feeble protec Iytic but no saccharolytic properties, It does not attack any sugar. It forms indole. It is MR and VP negative H,S is not formed. Nitrates are not reduced. Gelatin liquefaction occurs very slowly. A greenish fluores ence is produced on media containing neutral red (as ‘on MacConkey medium) Resistance: The resistance of tetanus spores to heat appears to be subject to strain differences. Most are Killed by boiling for 10-15 minutes but some resist boiling for up to three hours. When destruction of spores is to be ensured, autoclaving at 121°C for 20 minutes is recommended. On the other hand, when heat is applied to free cultures of C.tetani from non sporing contaminants, it is important not to exceed 80°C for 10 minutes, as even this mild treatment can erable destruction. Spores can survive in soil for years, and are resistant to most antisep tics. They are not destroyed by 54 phenol or 0.1% mercuric chloride solution in two weeks or more. Todine (1% aqueous solution) and hydrogen peroxide (10 volumes) kill the spores within a few hours, Classification Ten serological types have been recognised based on agglutination (Types I to X). Type VI contains non. flagellated strains. All other types possess type-specific flagellar antigens All the types produce the same toxin, whic is neutralised by anttoxin produced against any ‘one type, Toxins: Ctetani produces at least hemolysin (tetanolysin) and two distinct 4 powerful a nether this plays inthe p n-labile hem zenlabile het pathogenesis OF Tata toxin. The toxin has been crystallised. Ii a sing © The purified toxin is active in extremely smal amounts and has an MLD for mice of about 50- % 10° mg. The amount of toxin produced depen. on the strain of the bacillus and the type of cult medium used. Its MLD for human beings is abou 130 nanograms. There is considerable variation in the susceptibility of different species of animals t the toxin. The horse is the most susceptible, Guinea Pigs, mice, goats and rabbits are susceptible in tha descending order. Birds and reptiles are highy resistant. Frogs, which are normally insusceptibe may be rendered susceptible by elevating their body temperature ; 5PG MD fen AY poe Pathogenicity Cetani has little invasive power. Washed injected into experimental animals do not germina and are destroyed by phagocytes. Germination and toxin production occur only if favourable conditio exist, such as reduced OR potential, devitalised tissues, foreign bodies or concurrent infection, The toxin pr duced locally is absorbed by the motor nerve endings and transported to the central nervous system intrax onally. The toxin is specifically and avidly fixed by Bangliosides of the grey matter of the nervous tissue. Mechanism of action: 1 smin resembles strychnine in its effects. The tetanus toxin specificall) blocks synaptic inhibition in the spinal cord, presum ably at inhibitory terminals that use glycine and GABAfhnine whi en magia ee eiieienicne ee enna ane a ag ii This results in muscle rigidity and we system she simultaneous contraction of nt, SP*™s arias cts ce TETANUS i ete Ses ie muscular spasm cnr tat sr, ses, becomi the mildest cases, beco alised, involving th ofthe somatic muscular system. Most freautayn’ th Most frequently See trivial sture wounds are particularly vulner io be noticed. Pi thleas they favour the growth of the anaerobic bacilh Rael, it may follow surgical operations, usually due to epsis. Sometimes, the disease may be due lapses in a : to kcal suppuration, such as otitis media (otogenic Sea aoe mt ee ticabortion. It may be caused by unhygienic praction, suchas application of cow dung on the umbilical stump wr riuals such as ear boring or circu may also be caused by unsterile injections i incubation period is variable, from two days tos aT weak Bar comnTnonly 6-12 days. This is influenced by several factors, such asthe siteand nature ofthe wound, the dose and toxigenicity ofthe contami: nating organism and the immune status of the patient, The incubation period is of prognostic significance the prognosis being grave when it is short. Of similar significance is the interval between the appearance of the fist symptom of the disease, usually trismus, and the onset of spasms (period of onset). Tetanus was @ serious disease with a high rate of mortality, 80-90 per cent, before specific treatment became available. Even with proper treatment the case fatality rate varies from 15 to 50 per cent. Tetanus neonatorum and uterine tetanus have very high fatal ity rates (70-100 per cent), while otogenic tetanus is much ess serious. Tetanus is more common in the developing coun- tres, where the climate is warm, and in rural areas where the soil is fertile and highly cultivated, where buman and animal populations are substantial and live inclose association and where unhygienic practices are common and medical facilities poor. In rural India, tet- ‘nus was a common cause of death, particulary in the ision. Tetanus ct onisation of infants Laboratory diagnosis H tetans sh clin ground, (orl lays be made on imation Net bateY tet oly hep eon 10 estaba ent, it may not be pose diagnosis may be mad by de ge rato ae be made by demonstration of Cetant nicroscopy, culture or toxigenicity Rees igeniciy tests, Microscopy is unreliable and d TTR nelabe and demonstration of the demenictrnae i fy wounds in sls not some wounds without teams developing may ot albo be posible to dsnguish by mlcroncony nee" Clan’ and morphology sna baci Tats Cteanomorpum and Cspheades ‘culture is more dependable, Isolation is more likely from excised bits of tissue from the necrotic depths of wounds than from wound swabs The material is inoculated on one half of a blood ‘gar plate. Cetani produces swarming growth which may be detected on the opposite half of the plate after 1-2 days of anaerobic incubation. The tubes of cooked meat broth, one of which is heated to 80°C for 15 minutes, the second for five minutes, and the third left unheated. The purpose of heating for diferent periods is to kill vegetative bacteria, while leaving undamaged tetanus spores, which vary widely in heat resistance, The cooked meat tubes are incu bated at 37°C and subcultured on one half of blood agar plates daily for up to four days. C.tetani may be isolated in pure culture by subculturing from the swarming edge of the growth. The incorporation of polymyxin B, to which clostridia are resistant, makes the medium more selective. «© For in vitro identification and toxigenicty testing blood agar plates (with 4% agar to inhibit swarming) having tetanus antitoxin (1500 units per mi) spread ‘over one half of the plate are used. The C.tetani strains are stab-inoculated on each half of the plate, Auhich is then incubated anaerobically for two days. snic C.etani strains show hemolysis around the half without the anttoxin fhe antitoxin on the other half lentification of the culture a8 le as a test of toxigenicity material is also inoculated into thr Toxiger the colonies, only on Lysis is inhibited by This may help in Cuetani but is unreliablsince it indicates the production only of tetanolysin and not necessarily of tetanospasmin, whieh te the * In vivo toxigenictyis best tested in animals, A 24 day-old cooked meat culture (0.2 ml) is imoculeied into the root of the tal ofa mouse. A second mouse that has received the tetanus antitoxin (1000 units) an hour earlier serves as the control. Symplome develop in the test animal in 12-24 hours, beginning ith stiffness in the til. Rigidity proceeds tothe leg On the inoculated side, the opposite leg, trunk and forelimbs, in that order. The animal dies within two days but may be killed earlier as the appearance of ascending tetanus is diagnostic Prophylaxis Jetanus is a preventable disease. As the spores are Ubiquitous, wound contamination is unavoideble: The disease is caused by the action of the toxin, Therclons the obvious and most dependable method of preven tion isto build up active anttoxie e immunisation of children and b appropriate. ‘The nature of prophylaxis depends largely on the {ype of the wound and the immune status ofthe patient, The prophylaxis methods available are: * Surgical prophylaxis aims at removing foreign bod. is, necrotic tissue and blood clots, to prevent an anaerobic environment favourable for the tetanus pacillus. The extent of surgical treatment may vary from simple cleansing to radical excision, depending (on the type of the wound, © Antibiotle ‘prophylaxis aims at destroying or inhibiting tetanus bacilli and pyogenic bacteria in wounds so thatthe production of toxin is prevented In experimentally infected animals, tetanus can bg Prevented by antibiotics administered within four hours after infection but not after eight hours. Thi emphasises the need for prompt administration OF ‘antibiotics. Long-acting pencilin injection the drug of choice. An alternative is erythromycin puma bad. fr five days. Antibiotics ae tobe saricd tion Passive immunisation i by injection, of tetanus anti- ‘oxin, Antitetanusérum (ATS) from hyperimmune =e orci BacTERioLoGy ae lly usa oan after receiving any tetanus-prons but also in prolonging the incubs reducing the mor the disease. However, equine ATS carried tw gig advantages implicit in the use of any heterologa serum: “immune elimination” and hypersensitnge The half-life of ATS in human beings is normaly about seven days but in persons previously injestey with horse serum, its eliminated much more quick because it combines with pre-existing antibodies Prior sensitisation also leads to hypersensing, reactions which may range from mild local reacting to serum sickness, and even fal anaphylanis ne ‘ble therefor, obligatory that ates for hypersensin oti invariably be made before administration of te Passive immunity without risk of hypersensiticy ‘Treatm can be obtained by the use of human antcuns a immunoglobulin (T1G). This is effective in smal det 250 ui) a nn eres aie) a Fase ss ze mention | om human volunteers, its availability is limited, zt Passive immunisation is an emergency procedure tobe used only once. Itis better to eliminate the use ofATS altogether, tetanus being controlled by acne immunisation, with human TIG being reserved for emergency use in the non-immune, Active immui iThe tetanus toxoid is given alone or with the diph theria toxoid and the pertussis vaccine as the “triple Yaceine’, in which the pertussis vaccine acts as an adjuvant also,fxd at monthly intervals Its important to use ih his pe of fod pening Cluinn ee fey morbed toxoid as the immune respons isolated by soning. C.botulinum was fist adsorbe ponse to plain ed by van Ermengem (1896) fi immunisation should be used whenever passive Widely distributed saprophyte occuing in ec ca iqmanisaion is called for. vegetubles, hay, slage, sninal manute euleceaaiet rable 275 shows the recommended integrated rae tanus following injury Morphology prophylaxis of It is a Gram-positive bacillus about 5 fe bacillus about 5 1 yim in size, on hon-capsulated, motile by peritrichate flagella, produc ninal, oval, bulging spores. anspatients should be treated in hospitals, preferably jrapaial units. Isolation is necessary to protect them fom noise and light which may provoke convulsions Treament consists of ensuring quiet, controlling It isa strict anaerobe. Optimum temperature is 35°C maintaining airway by tracheostomy with butsomestrains may grow even at 1-5°C. Good growth asmitent positive pressure respiration and attention occurs on ordinary media, Surface colonies are large to feding, Human TIG 10,000 IU suitably diluted irregular and semitransparent, witha fimbriate border soy be given by slow IV infusion, followed, if needed, Biochemical reactions vary indifferent types. Spores fy another 5,000 TU later. Even though TIG may not ate produced consistently when grown in alkaline glu- cose gelatin media at 20-25 produced at higher temperatures. spasms neutralise the toxin already bound to the nervous tis sug, it can inactivate the unbound toxin and any further toxin that may be produced. Antibacterial therapy with peniilin or metronidazole should be started at once Resistanee and continued for a week or more, ATS used to be given Spores are heat and radiation resistant, surviving intravenously in massive doses as part of the treatment. several hours at 100°C and for up to 10 minutes at Twle2733. Tetanus prophylaxis in the wounded atareof ound Papeete ean (wound toilet performed within six hours) Toxoid « 1° Contaminated (soil or other foreign or ‘ert material presen Toxoidss* ——Tonald = 116 4 . antibiotics antibiotics Infected, Toxoid « 1° Toxoid « 1 TIG Toxoid * 3 TIG anions anions auibitcs "mune - Patient as had full cours of tre nections of trod . aly immune Patt hos hed we jection of 0, ; Bee Hovimmune anes es had one ore necson of tron munsaonsatsi500tMow See have elapsed after active immunisation or the last “The toroid needs to be given only if three years or more "epbined immunisation consists of adi SSE) Cultural characteristics Toxoid * 1 ‘They are not usually Toxold » 3 Toxoid » 31161C. Spores of the non-proted Classificati Eight types of C bot (A, B, Cl, C2, D, E, Fand G) based on the immune logical difference in the toxins produced by them. The toxins produced by the different types are identical in their pharmacological activity but are neutralised only the homologous antiserum. An exception is the C2 Fe ees caren iy whee oh ers are neurotoxins Texin: C.botulinum produces a powerful exotoxin that is responsible for its pathogenicity. The toxin differs from other exotoxins in that i not released during the life ofthe organism. 11s produced intracel. lularly and appears in the medism only on cell death and autolysis, Its believed tobe synthesised initially as 4 non-toxic protoxin or progenitor toxin. Trypsin and other proteolytic enzymes activate the progenitor toxin to produce active toxin, The toxin has been isolated as a pure crystalline pro. {ein which is probably the most toxic substance known Ithas an MW of 70,000 and the lethal dose for mice is .000,000.055 mg. The lethal dose for human beings is probably 1-2 ug. It is a neurotoxin and acts slowly taking several hours to kill The toxin is relatively stable, being inactivated only alter 3040 minutes at 80°C and 10 minutes at 100°C Food suspected to be contaminated withthe botulinum toxin can be rendered completely safe by pressure cooking or boiling for 20 minutes. It resists digestion and is absorbed through the small intestines in active form. It acts by blocking the production or release of acetylcholine at the synapses and neuromuscular junc- tions. Onset is marked by diplopia, dysphagia and dys arthria due to cranial nerve involvement. A symmetric descending paralysis isthe characteristic pattern, end- ing in death by respiratory paralysis. I used of toxins: A small quantity of C.botulinum Type A toxin injected into a muscle selec tively weakens it by blocking the release of acetylcho- fine atthe neuromuscular junction. Muscles so injected ‘trophy but recover in 24 months as new terminal axon sprouts form and restore transmission. Intramuscular injection of the toxin, first used to treat strabismus, is now recognised as a safe and effective symptomatic therapy for many neuromuscular diseases. um toxin can be tOxOIdeM neutralised by its antitoxin and is & 6008 a toxins produced by the different types of Cat appear to be identical, except for immunclogieal T ppears to be determi ferences. Toxin production apped by the presence of bacteriophages, at least im Types and D. Pathogeni botulinums non-invasive and virtually non-infeetioy, Its pathogenicity is due to the action of is toxin, the manifestations of which are collectively called bots, BOTULISM Food-borne botulism is caused by the ingestion of preformed toxin. The types of the bacillus ang the nature of the food responsible vary in differen, regions. Human disease is usually caused by types a y rarely F. Types C and D are usualy associated with outbreaks in cattle and wild fowl Type G has been associated with sudden death in a few Patients, The source of botulism is usually preserved food—meat and meat products in Europe, canned vegetables in America and fish in Japan. Type E is associated with fish and other seafood, Protecytc varieties of C.botulinum can digest food, which then | appears spoiled. The cans are often inflated and show bubbles on opening. Non-proteolytic varieties lave food unchanged. ‘Symptoms usually begin 12-36 hours after ingestion of food. No vomiting or diarrhoea is present. Coma ot delirium may supervene. Death is due to respirator failure and occurs 1-7 days after onset, Case fatality varies at 25-70 per cent. Wound botulism is a very rare condition result ing from wound infection with C.botulinum. Toxin Produced at the site of infection and is absorbed. The symptoms are those of food-borne botulism excert {or the gastrointestinal components which are absent Type A has been responsible for most of the cases studied. Infant botulism was recognised as a clinical ent in 1976, This isa toxico-infection. C.botulinum spores are ingested in food, get established in the gut and there produce the toxin, Cases oceur in infants below six months. Older children and adults are not suscept ble, The manifestations are constipation, poor feeding lethargy, weakness, pooled oral secretions, weak of see iaaieeniaalia capi ernie as gerry + remem aiele ai aryES DR eis stoma a in snstrable in blood. Management consis eerie nage ot indicated. Degrees of se It pss fatal disease. Some cates of ery i eath syndrome have been found to be ia is. Hone) has benineriniated oa rough, which the bacillus enters atoratory diagnosis reas ray be confirmed by demonstration ofthe ose the toxin in food oF feces. Gram-positive fil may be demonstrated in smears made pod. Cibotulium may be isolated from the spe patient's feces. The food is macerated in 2 ne and the ftrate inoculated into mice or se igsinrapritonealy- Control animals protected se gant attoxin remain healthy. Typing is done foe protection with type-specific anttoxin. The 2 Fnay occasionally be demonstrable in the patient's pada nthe liver postmortem. aropective diagnosis may be made by detection afanitorn in the patient's serum but it may not be enn pom th seen inal cases. Control As most cases of botulism follow consumption of inadequately canned or preserved food, control can be icheed by proper canning and preservation. When an cubreak occurs, a prophylactic dose of antitoxin shold be given intramuscularly to all who consumed the article of food. Ave immunisation has been shown to be e! Ufimnunisation is needed, as in laboratory workers cexgsed to the risk, two injections of aluminium sul- hae adsorbed toxoid may be given at an interval of ten ‘ets, followed by a booster a year later. Antitoxin may ‘etre for treatment. Polyvalent antiserum to types A: fective. 265 Band E maybe administered is made. Supportive therapy with manteranes of respiration is of ‘of equal or gre ‘eater importance CC TTY EC cys aie Cificite was fs e Sac w fist inlted in 1935 fom the fess taal Srey aug eT ae Gram-positive ba ata iheaitine eae Gramps bain wih a proncunesd tends subterminal I is nonchemolts safaris and weakly proteolytic, Iwas not considered pathogen i 1977 when fas found tobe responsible fran eee colitis. aor oe 4 difficile causes acute colts with bloody diarehea and pseudomembranous els Cdifiles the most common cause of nosocomial diarthea. The disease usually follows from th use of broad-spectrum antibi tics (clindamycin) to which the orgeism is resistant. “Two high-molecular-weight exotoxin, A and B, are involved inthe pathogenesis ofthe condition. C fice is an opportunistic organism that rarely causes disease land does so only when normal flora is lst. There appears tobe lite protective immunity following infec: tion. The disease is prevented by restricting the use of antibiotics associated with C.dfcle outbreaks; for treatment, the antibiotic causing the disease should be discontinued and the disease treated with vancomycin or metronidazole. Diagnosis can be made by demonstrating the toxin in the feces of patients by its characteristic effect on Hep-2 and human diptoid cell cultures or by ELISA. ‘The toxin is specifically neutralised by the C.sordelli antitoxin. C.difficile can also be grown from the feces of patient. Cdifficile strains are usvally resistant to most ant ‘otics. Treatment is with metronidazole, the drug of Choice, Vancomycin and bacitracin are also useful cileTE & Classification ANAE ‘ANAEROBIC GRAM-POSITIVE BACILL ‘ANAEROBIC GRAM-NEGATIVE Bi ‘ANAEROBIC INFECTIONS Laboratory diagnosis Treatment INTRODUCTION Anaerobic bacteria outnumber aerobie bacteria in many habitats, including most sites of the human body, especially the gastrointestinal tract. Even in such seemingly aerobic locations as the mouth and skin, ‘anaerobic bacteria are ten to thirty times mote frequent than aerobes. In the human intestines, they outnumber ‘aerobic bacteria a thousandfold. The number of anaer: ‘bes present has been estimated to be 10*-10'/ml in the small intestine, 10'/ml in saliva and 10!/g in the colon Clinical Case A 65-year-old dlabetic woman devel ‘oped seizures and presented to neurosurgery with 3 provisional diagnosis of space occupying lesion. On further investigation, itwas diagnosed a brain abscess. The patient underwent an operation and the pus wos sentto the laboratory in a sealed val filed with carbon dioxide. Microscopic examination showed the presence | of Gram-positive cocci and a few fine Gram-negative baci. Culture from Robertson's cooked meat medium | was positive for peptostreptococci and Bacteroides rn ails The patient responded to treatment with amoxici, Uin/sutbactam and metronidazole Anaerobic bacteria differ wide anaerobiosis required for their fail to grow if the atmospher ty in the degree of growth. Some species f contains as little as See ste tect cc eee cesion sad stedy of ina aie Classification Depending on DNA base composition and analy fatty acid end products of metabolism, medical tant anggrobes may be broadly classified as flan Coe 7 Gram positive _ Peptostreptococcus Peptococcus B. Gram negative ——~veilfonella —"Closirid 2, Non-sporing A. Gram positive cEubacterium Propionibacterium “Tactobacillus Mobiles — Bifidobacterium Actinomyces B. Gram negative Bacteroides Prevotellla ~~ Porphyromonas Fusobacterium Leptotrichia| IIL Spirochetes ___ Treponema —Borretia — Besides the medically important species listed there are several anaerobes that occur in soil an! and which may be of industrial and agricultural i tance (for example, methanobacteria, butyrvbi®i a TST earns of cocci. They can be divided into the Gram-positive ai Caters =o eae Gram postive: These had been classified into th genera Peptosireptococcus. They are cocci of small sia (02°25 yan) Mange ease ean well under 10% CO, in an aerobic atmosph They are normal inhabitants ofthe vagina, intestine and mouth, They may cause several clinical infections such as puerperal sepsis and other genital infections, wound infections, gangrenous appendicitis, urinary tract infections, osteomyelitis and abscesses in thi brain, lungs and other internal of i fernal organs. They are often seen in large numbers in pus from suppurative lesions, so a Gram-stained smear may be helpful in diagnosis. ine are usually mixed, the cocci being present along with clostridia or anaerobic Gram-negative bacilli. Peptostreptococcus anaerobius is most often responsible for puerperal sepsis and Pst.magnus for abscesses. Pst.asaccharolyticus, Pst.tetradius and Ps, prevoti are some other species commonly present in clinical specimens. Gram negative: Veillonellae are Gram-negative cocci of varying sizes occurring as diplococci, short chains ‘or groups. They are normal inhabitants of the mouth, and intestinal and genital tracts. Veillonella parvula has been reported from clinical specimens but its patho- genic role is uncertain. All anaerobic cocci are generally sensitive to penicil- lin, chloramphenicol and metronidazole, and resistant to streptomycin and gentamicin. ANAEROBIC GRAM-POSITIVE BACILLI This group contains many genera, of which the medi cally relevant are Eubacterium, Propionibacterium, Lactobacillus, Mobiluncus and Bifidobacterium. Other genera in this group, Actinomyces and Arachnia, are dealt with elsewhere. S Members of the genus Eubacterium are strictly anaerobig and grow very slowly. They are part of the normal mouth and intestinal flora. Some species (Ebracky, E.timidum, Enodatum) are commonly "-E:lentum is commonly isolated seen in periodor from non-oral clinical specimens. ypicalintheadulvagna (Doderen’sbasl) te yenerally non-pathogenic, though L ha beon asoitd wih onshoplnonary into dobacterium isa pleomorphic rod that shows true tecil that ay, ope a ee ae Mimuliris and M. curtis have been isolated fromthe ‘vagina in bacterial vaginosis, along with Gardnerella buginalis. Bacterial vaginosis is a polymicrobia infection characterised by a thin malodorous vaginal discharge. Its “rotten fish’ smells accentuated when itis mixed with a drop of KOH solution, The ‘aginal pH is more than 4:5. Clue cels (epithelial calls whose surface is covered by adherent bacll are seen in stained or unstained films, PNT sea Medically important anaerobic Gram-negative bacilli belong to the family Bacteroidaceae and are classified into the genera Bacteroides, Porphyromonas, Prevotella Fusobacterium and Leptotrichia. Bacteroides: This genus comprises the most com- mon anaerobes isolated from cfinical specimens. They are non-sporing, non-motile, strict anaerobes, usually very pleomorphic, appearing as slender rods, branching forms or coccobacili, seen singly, in pairs or in short chains, They grow well on media such as brain heart infusion agar in an anaerobic atmosphere containing 10% CO,. They possess capsular polysaccharides which appear to be virulence factors, and antibodies to them can be detected in patients. They are normal inhabitants of the intestinal, respiratory and female genital tracts, The Bacteroides species is susceptible ee metronidazole and usually to clindamycin and chloramphenicol. Bumelaninogenicus is susceptible to penicilin, but B fragilis isnot susceptible t pencil fragilis is the most frequent of the non-spot: ing anaerobes isolated from clinical specimens, Itis ing Anvecovered {rom blood, pleural and peritoneal fluids, CSF, brain abscesses, wounds and urogenital infections.Partill_ BACTERIOLOGY Porphyromonas Bacteroides, the asacchare Previously classified und ytic pigmented been separated as the genus P. ing Pgingivalis responsible for periodontal disease Pendedomtalis—eausing dental root canal infection: and other species. Prevotella: Previously the moderately saccharolytic species inhibited by 20% bile have been placed in the genus Prew Pmelaninogenica, Pbuccalis, Pdenticol Pmelaninogenica is easy of the black or colonies (Fig. 28.1). The colour is not due to the melanin pig. ment as was once thought but to a hemin derivative It has been isolated from various infections inc lung or liver abscess, mastoiditis, inte and lesions of the mouth and gums Pmelaninogenica sified under Bacteroides and others, brown colour of the Huding stinal lesions Cultures of and even dressings. from infected with the bacillus produce a ch fluorescence when exposed to ult Fusobacterium: This contains long, thin or spindle shaped bacilli with pointed ends. Fnucleatum ia inhabitant of the mouth and is found in orl and pleuropulmonary sepsis. Enecrop wide range of exotoxins and has bee liver abscess and other abdominal inte and less often in humans Leptotrichia: This contains the single species, Lbuccalis which was formerly known as Vincent's fusiform bacillus or Fusobacterium fusiforme. They are long, straight or slightly curved rods, often with Pointed ends. They are part of the normal and are seen in acute necrotising lesions in t ‘wounds aracteristic red violet light anormal infection phorum produces responsible for ‘ctions in animals oral flora he mouth, Fig 28.1 Pmela ninogenica obic blood aga angina, whieh 1 pharymgeg hich peel Spina Ea iaae Anaerobic infections are usually endogenous and ay caused by tissue invasion by bacteria normaly reside on the respective body surfaces. Anaerobic bactery are normally present on the skin, mouth, nasopharyne and upp (Table 28.1) Fespiratory tract, intestines and vaging ® Anacrobie infections generally follow some py cipitating factor such as trauma, tissue necreng impaired circulation, hematoma formation or th presence of foreign bodies * Diabetes, malnutrition, malignaney oF prolonged treatment with aminoglycoside antibiotics may se 4s predisposing factors, © They are typically pol ierobial, more than one robe being responsible besides aerobic bacteria (Case) While the infection is usually localised, general ds semination may occur by bacteremia, * Some anaerobic infection, Pus produced by anaerobes is ch putrid, with there may B fragilis aractersicaly pervasive, nauseating odour. However be exceptions; infections solely due ts may be free of this smell © Pronounced cellulitis is a common festire anaerobic wound infections * Toxemia and fev er are not marked, Tible 28.2 lists the common sites and types of anaerobic infections and the bacteria responsible. Laboratory diagnosis 1. Specimen: As anaerobes form part of the normal flora of the skin and mucous surfaces, their isolation ‘rom specimens has to be interpreted cautiously. The inere Presence of an anaerobe does not prove its caustl role. Specimens should be collected in such a mannet tneayctoid resident flora. For example, the sputum is Unsatisfactory for culture from a suspected ease Of ab scess; only material collected by aspiration would be acceptable, a aohse238 994°. Eee