Microbio Lec 10 - Enterobacteriaceae Gen, Shigella And Salmo

MICROBIOLOGY LECTURE 10 - Enterobacteriaceae, Shigella, Salmonella Notes from Lecture USTMED ’07 Sec C AsM ENTEROBACTERIACEAE

Enterobacteriaceae General Characteristics 1. ubiquitous organisms that are found in the soil, water, decaying matter and the large intestines of animal and insects 2. natural habitat in humans – gastrointestinal tract referred to as “enteric bacilli” or “enterics” 3. constitutes only a minor fraction of the total microbial flora of the gastrointestinal tract 4. includes some of the most important cause of gastrointestinal disease a. salmonellae – typhoid fever b. shigella – bacillary dysentery 5. some are members of the normal commensal flora that can cause opportunistic infections a. Escherichia coli b. Klebsiella pneumonia c. Proteus mirabilis 6. Responsible for the majority of nosocomial (hospitalacquired) infections 7. causes disease a. in the gastrointestinal tract i. Shigella b. Outside the gastrointestinal tract i. Klebsiella ii. Enterobacter iii. Serratia iv. Proteus c. Both inside and outside the gastrointestinal tract i. Escherichia coli ii. Salmonella iii. Yersinia 8. Infection may originate from the: a. Animal reservoir – most Salmonella infections b. Human carrier – Shigella and Salmonella typhi c. Endogenous spread of organisms in susceptible patient – Escherichia coli 9. Possesses complex antigenic structure 10. Produces a variety of toxins and other virulence factors Classification of Enterobacteriaceae is based on: 1. DNA homology 2. Biochemical properties 3. Serologic reactions 4. Susceptibility to genus specific and species specific bacteriophages 5. Antibiotic susceptibility pattern Edwards and Ewing Classification of Enterobacteriaceae Tribe I. Escherichiae

II. Edwardsiellae III. Salmonellae

IV. Klebsiella

V. Proteae

Genus I. Escherichia

Species Eschericia coli

II. Shigella

Shigella dysenteriae Shigella flexneri Shigella boydii Shigella sonnei Edwardsiella tarda Salmonella choleraesuis Salmonella typhi Salmonella enteritidis

I. Edwardsiella I. Salmonella

II. Arizona

Arizona hinshawii Citrobacter freundii

III. Citrobacter I. Klebsiella

Citrobacter diversus Klebsiella pneumoniae Klebsiella ozaenae Klebsiella rhinoscleromatis

II. Enterobacter

Enterobacter Enterobacter Enterobacter Enterobacter

III. Seratia

Serratia marcescens Serratia liquefasciens Serratia rubidaea Proteus vulgaris Proteus mirabilis Proteus morganii Proteus rettgeri

I. Proteus

II. Providencia VI. Yersineae

I. Yersinia

VII. Erwiniae (plant pathogens)

I. Erwinia

cloacae aerogenes hafnia agglomerans

Providencia stuartii Providencia alcalifaciens Yersinia enterocolitica Yersinia pseudotuberculosis Yersinia pestis

Microscopic Morphology 1. small (0.5 by 3.0 um), gram negative non-sporeforming rods 2. motile by means of peritrichous flagella; non-motile – Shigella and Klebsiella 3. well-defined capsule • Klebsiella • Enterobacter • Serratia • Some Escherichia strains slime layer – loose ill defined coating 4. fimbriae or pili – responsible for attachment of the bacterial cells to other bacteria, host cells and bacteriophage 5. cell wall consists of: 20% of the cell wall; responsible for a. murein cellular rigidity b. lipoprotein c. phospholipids d. protein e. lipopolysaccharide – contains the specific polysaccharide side chain i. determines the antigenicity of the various species ii. portion of the cell responsible for the endotoxic activity -

80% of the cell wall is joined to the lipid of the lipoprotein to form a lipid bilayer

Colonial Morphology 1. Similar colonial morphology in blood agar plate a. Moist, smooth, gray colonies b. Some strains are beta hemolytic 2. Eschericia coli – convex, cicular, smooth with distinct edges 3. Enterobacter – similar but somewhat more mucoid 4. Klebsiella – large and very mucoid and tend to coalesce with prolonged incubation 5. Salmonella and Shigella – similar to Escherichia coli but do not ferment lactose 6. Pigmented genera • Serratia – reddish orange • Edwardsiella – yellowish white Enterobacteriaceae on 5% sheep blood agar. Characteristic colonial morphology on 5% sheep blood agar showing large, dull, grey, nonhemolytic colonies. Hemolysis is variable and not characteristic of any one genus.

Klebsiella pneumoniae on MacConkey agar. Rapid lactose fermenting colonies of Klebsiella pneumoniae appears pink, large, glistening, and mucoid. This strain is probably encapsulated and therefore appears mucoid. Although this appearance is associated with Klebsiella pneumoniae. It is not unique for that species. Pigmented Serratia sp. on MacConkey agar. These colonies appear red and should not be confused with the pink color due to lactose fermentation shown in 8.3. Rare strains of Serratia spp. produce pigment, which is seen on all solid media including the blood agar plate.

•

Shigella -

•

citrate positive differs from salmonella – (-) lysine decarboxylation ferments lactose very slowly nonmotile nonlactose fermenter ferments other carbohydrates producing acid but not gas closely related to Escherichia coli share common antigen with one another and with other enteric bacteria

Salmonella motile ferments glucose and mannose without gas but do not ferment lactose or sucrose

-

produces H2S Proteus species on 5% sheep blood agar. Growth appears to spread as a film on the plate from the original colony or streak line, often extending in waves. This characteristic of Proteus spp. is called swarming and suggests that the microorganism is motile by means of flagella.

Biochemical Characteristics 1. facultatively anaerobic or in low oxygen atmosphere ferments carbohydrates 2. with sufficient oxygen – utilize the tricarboxylic acid cycle and the electron transport system for energy production Enterobacteriaceae All are facultative anaerobes All ferment glucose with acid or acid and gas Oxidase negative Reduce nitrates to nitrites

•

Pseudomonadaceae Strict aerobes Does not ferment glucose Oxidase positive Does not reduce nitrates to nitrites

Escherichia 1. positive indole 2. positive lysine decarboxylation 3. positive mannitol fermentation 4. produce gas from glucose 5. urine isolate – identified by: a. hemolysis in blood agar b. typical colonial morphology in EMB – greenish metallic sheen or iridescent sheen c. positive spot indole test

d. e.

positive β-glucoronidase using the substrate 4methylumbelliferyl-β-glucoronide (MUG) negative oxidase test

•

Klebsiella-Enterobacter-Serratia Group 1. Klebsiella a. mucoid growth b. large polysaccharide capsule c. lack of motility d. positive lysine decarboxylase and citrate 2. Enterobacter a. positive tests for motility b. positive citrate c. positive ornithine decarboxylase d. produce gas from glucose e. enterobacter agglomerans – small capsule 3. Serratia produces DNase, lipase and gelatinase all are positive for Voges Proskauer test

•

Proteus-Morganella-Providencia Group deaminate phenylalanine motile grow on KCN medium ferment xylose 1. Proteus swarming on solid media – actively motile by means of peritrichous flagella 2. proteus species and Morganella morganii – urease positive 3. Providencia urease negative

•

4.

Proteus-Providencia – ferment lactose very slowly or not at all

5.

Proteus mirabilis – more susceptible to antimicrobial drugs including penicillin

Citrobacter

Antigenic Structure 1. O-antigens – somatic – heat stable a. species specific polysaccharide which makes up a part of the LPS component of the cell wall b. endotoxic activity expressed by the LPS complex reside in the lipid A molecule 2. K or capsular antigen – heat labile a. a polysaccharide component found in some members of the enteric bacilli b. called Vi antigen in Salmonella c. basis of serologic typing of Klebsiella d. cells possessing K antigen are more pathogenic than those that lack them e. inhibits phagocytosis and the effects of serum antibody 3. H antigens or flagellar antigens – heat labile a. protein found only in motile forms b. motile bacteria may lose their capacity to produce flagella but retain their O antigen specificity Other antigens 4. Enterobacterial common antigen or kunin antigen a. haptenic substance occurring in two aggregative forms o linked to the lipopolysaccharide and is immunogenic o linked to a carrier molecule and nonimmunogenic b. LPS linked ECA rare but found in Escherichia coli 014 c. Role in virulence and pathogenesis not established d. Chemical nature undefined 5. Fimbrial antigens a. believed to be virulence factors because of their adhesive properties b. protein in nature and present in all enteric bacilli

Determinants of Pathogenicity

1.

2.

3.

4.

Endotoxin a. LPS of the cell wall – endotoxin – associated with the bacterial cell and is toxic to animals b. Toxicity of LPS resides in the lipid A molecule c. Effects when injected to animals fever fatal shock leukocyte alterations regression of tumors alterations in host response to infection Sanarelli-Shwartzman reaction Various metabolic changes d. cellular targets are varied e. exact mechanism of action – not clearly delineated f. 30% of enteric bacteremia will develop endotoxic shock chief defect – pooling of blood in the microcirculation – causes cellular hypoxia and metabolic failure due to inadequacy of blood in vital organs Enterotoxins a. toxins that usually affect the small intestine causing a transduction of fluid into the intestinal lumen and subsequent diarrhea o salmonella o shigella o strains of Escherichia coli, Klebsiella pneumoniae o citrobacter freundii o Enterobacter b. enterotoxin producing Escherichia coli – major causes of travelers diarrhea and diarrhea in developing countries c. incidence of disease cause by enterotoxin producing Citrobacter, Klebsiella and Enterobacter species – unknown d. role of enterotoxins in salmonellosis and shigellosis – unclear; tissue penetration important in pathology Shiga toxins and Shigalike toxins (verotoxins) a. Shiga toxin – interferes with protein synthesis of mammalian cells; role in shigellos is unclear b. Certain E. coli strains – produce similar toxins called verotoxins because of their action of Vero (African green monkey) tissue culture cells; important causes of hemolytic diarrhea and hemolytic uremic syndrome Colonization factors a. capsule of Klebsiella pneumoniae – to prevent phagocytosis b. Vi antigen of Salmonella typhi – prevents intracellular destruction of the bacterial cell c. Fimbriae such as the CFA of human isolates – necessary for the attachment of the organism to target tissues d. O antigen – may bind the organism to certain tissue receptor sites

Laboratory Diagnosis Specimens – sputum, tissue, pus, body fluids, rectal swabs or feces 1.

Culture

a. b.

c. d.

cefsulodin-irgasan-novobiocin (CIN) – selective for Yersinia isolation and differentiation of lactose fermenters from nonlactose fermenters Eosin Methylene Blue agar MacConkey agar Gram-negative broth (GN broth) – enrichment broth used to enhance isolation of enteric pathogens Isolation and differentiation of Salmonella and SHigella; inhibition of normal flora coliforms Hektoen enteric agar

e.

2.

Clinical Infection Types of infection 1. leading cause of bacteremia and urinary tract infections 2. can invade any body site and can cause wound infections, pneumonia, meningitis and various gastrointestinal disorders 3. opportunistic infections occur outside of the intestine and require an alteration of the host by some mechanical, physiologic or infectious process before they can cause disease 4. true enteric pathogens – Salmonella, Shigella and Yersinia 5. potential danger – secondary bacteremia and endotoxic shock Sites of infections with members of the enterobacteriaceae

Salmonella Shigella agar Xylose-lysine-desoxycholate (XLD) enrichment broth used to enhance Salmonella and Shigella selenite broth tetrathionate broth

recovery

of

Biochemical Tests a. Triple sugar iron Agar – to distinguish morphologically similar bacteria of Enterobacteriacea all of which ferment glucose to an acid end product b. IMVIC reactions – for the identification of lactosefermenting members of Enterobacteriaceae. Escherichia, Enterobacter and Klebsiella. i. Indol test ii. Methyl red test iii. Voges Proskaeur test iv. Citrate utilization test c. Urease test – for the identification of: i. Rapid urease producers – Proteus and Morganella ii. Weak urease producers – Klebsiella and some enterobacter d. deaminase reactions – to identify organisms producing the enzyme deaminase such as Proteus, Providencia and Morganella e. decarboxylase reactions – to determine the production of decarboxylases by bacteria f. motility – determines the motility of bacteria through semisolid media i. Nonmotile – Shigella and Klebsiella

ii.

g.

3.

Yersinia enterocolitica – nonmotile at 37oC but motile at 22oC ONPG reaction i. Determines the presence of late or slow lactose fermenting strains o useful in detecting late-lactose fermenting strains of Escherichia coli o distinguishes Citrobacter species and arizonae subspecies from similar Salmonella subspecies o useful in the speciation of shigella

Serologic grouping of the O, H and K antigens to characterize certain enteric isolates a. Salmonella and Shigella – for complete identification b. Other organisms – for epidemiologic purposes

Rapid, presumptive identification of gram negative bacteria Lactose fermented rapidly Escherichia coli metallic sheen on differential media; motile, flat, nonviscous colonies Enterobacter aerogenes raised colonies, no metallic sheen; often motile; more viscous growth Klebsiella pneumniae very viscous, mucoid growth; nonmotile Lactose fermented slowly Edwardshiella Serratia Citrobacter Arizona Providencia Erwinia Lactose not fermented Shigella species nonmotile; no gas from dextrose Salmonella species motile; acid and usually gas from dextrose Proteus species swarming on agar, urea rapidly hydrolyzed (smell of ammonia)

SHIGELLA

1. 2. 3.

Gram negative nonsporeformers none are motile, hence they do not contain flagellar antigens some resemble anaerogenic type of coliform bacilli and typhoid bacilli in that they ferment carbohydrates with the production of acid without gas

4.

Differ from one immunologically

5.

Facultative anaerobes; optimum temperature for growth is 37oC Grow upon the ordinary nutrient (beef extract medium) relatively resistant to the bacteriostatic action dyes can grow in EMB, MacConkey, SS agar, deoxycholate agar Only Shigella sonnei is a slow lactose fermenter

6. 7. 8. 9.

another

biochemically

and

Properties that distinguish Shigella from most Salmonellae 1. lack of motility 2. failure to produce gas during fermentation except for some biotypes of Shigella flexneri 6 and rare strains of Shigella boydii 13 3. lack of lysine decarboxylase 4. possess specific polysaccharide O antigens but no H antigens 5. much less invasion rarely causing bacteremia 6. much narrower distribution in nature inhabiting only the intestinal tracts of primates

Treatment 1. factors in the difficult treatment of enterobacterial infections • underlying disease of patient • emergence of resistant strains 2. The appropriate antibiotic must be chosen by: • careful evaluation of the isolate’s susceptibility pattern • condition of the host • site of infection 3. Treatment of shock – centers on the cardiovascular system • restoration of the intravascular volume • digitalization • administration of isoproterenol • steroids, pressor amines and norepinephrine

Classification is based on fermentation of mannitol I. Nonfermenting group – serogroup A – Shigella dysenteriae II. Mannitol fermenters – serogroup B – Shigella flexneri serogroup C – Shigella boydii serogroup D – Shigella sonnei • Group A – Shigella dysenteriae 1. Shigella dysenteriae type I – Shiga bacillus o produces an endotoxic LPS which is responsible for O antigenicity of the bacillus o unique in its production of an exotoxin, a neurotoxin, which affects the central nervous system o Similar to diphtheria toxin in potency and mechanism of action o Produces an enterotoxin which closely resembles cholera and coliform enterotoxins in its mechanism of action 2. Shigella dysenteriae type 2 – Schmitz bacillus o encountered in institutional and other outbreaks of dysentery • Group B – Shigella flexneri – Flexners bacillus, Hiss and Russell’s bacillus 1. Worldwide in distribution 2. most commonly found of the dysentery bacilli making up more than half of isolates • Group C – Shigella boydii – Newcastle Manchester bacillus similar in pathogenicity to Shigella flexneri but differs immunologically • Group D – Shigella sonnei – Duval’s bacillus 1. most common species in the US – 72% of isolates 2. slow lactose fermenter, producing acid in one week to 10 days Pathogenesis

1. 2. 3. 4. 5.

natural habitat of dysentery bacilli – large intestine of humans – BACILLARY DYSENTERY infection limited to gastrointestinal tract blood stream invasion is rare highly communicable infective dose – less that 10 microorganisms Bacilli penetrate epithelial cells of the colonic mucosa

Multiplication of bacilli

Microabscesses develop in the lamina propria

Necrosis of mucosa due to action of enterotoxin

ulcerations

Formation of diphtheritic or pseudomembrane consisting of fibrin, WBC, debris, tissues and bacteria

all the consequences of such a mistake

Epidemiology 1. occurs in children from 6 months to 10 years of age 2. endemic disease in adults is frequently due to contact with infected children 3. epidemic outbreaks of disease are associated with day care centers, nurseries and custodial institutions 4. transmitted by the fecal oral route, primarily by contaminated hands and less commonly in water or food – food, fingers, flies, feces 5. spreads rapidly in communities where sanitary standards and the level of personal hygiene are low Laboratory Diagnosis Specimens 1. fresh stool, mucus flecks, rectal swab – culture 2. serum – serology 1. Culture a. EMB or MacConkey – colorless colonies b. Salmonella-Shigella agar – colorless colonies without black centers c. Hektoen enteric agar – green colonies without black centers

Granulation and scar formation

Cllinical Manifestations 1. incubation period – 1 to 4 days 2. begins with fever, abdominal cramping and pain, and diarrhea 3. Two stages of the infection a. First stage – involves a watery diarrhea, which may last up to 3 days b. Dysenteric phase – characterized by frequent stools, with red blood cells, white blood cells, and mucus present o the bacteria have invaded the epithelial lining of the intestine, causing severe inflammation DIFFERENTIAL DIAGNOSIS BETWEEN AMEBIC DYSENTERY AND TISSUEINVASIVE BACTERIAL GASTRO-ENTERITIS Amebic Dysentery Tissue-invasive bacterial Colitis (Shegella dysentery) Incubation period Variable (usually 2- Short (24-72 hours) Onset Fever Toxemia Dehydration Hepatomegaly

Colonic mucosal changes

4 weeks or longer) Insidious Usually absent, unless complicated Usually absent Unusual Common (if complicated or disseminated) Gross findings: segmented: ulcers with undermined borders and normal intervening mucosa; lack of inflammation Microscopic findings – trophozoites in flask-shaped ulcers; wet mount shows motile amoeba containing RBC

Stool appearance Fecal leucocytes (methylene blue staining) Red cells E. histolytica

Indirect hemogglutination test (IHA)

Bloody mucoid semiformed Uncommon or few Numerous and in clumps Hematophagous trophozoites present Positive in invasive amebiasis (85-95% of cases)

Acute Common, moderately high Often present usual uncommon Diffuse erythema with loss of vascular pattern; mucopurulence, mild friability, occasional ophthoid ulcers

Edema, capillary congestion, focal hemorrhages, cyst, hyperplasia, goblet depletion, mononuclear polymorphonuclear leukocyte infiltrate; loss of epithelial cells with microulcerations Bloody mucoid less formed Usually abundant with macrophages Numerous, discrete absent negative

* Confusion in differentiating amebic cysts from fecal leucocytes and amebic cysts from

fecal leucocytes and amebic trophozoites from large motile macrophages may occur with

2. Biochemical tests

a. b. c. d. e. f. g. h.

TSIA – acid butt, alkaline slant, no gas, no H2S MR positive Citrate – negative ODC – negative ADH – negative Deaminase (phenylalanine) – negative Urease – negative Carbohydrate fermentation o sucrose – negative o salicin – negative o adonitol – negative o dulcitol – negative o D-mannose – positive

3. Slide agglutination by specific Shigella antisera Reaction Dysenteriae Flexneri Boydii Fermentation of: Lactose Mannitol + + ODC ONPG -

Sonnei + + +

Treatment 1. supportive a. prompt replacement of fluids and electrolytes b. opiates should be avoided 2. specific a. potent specific antitoxin against Shigella dysenteriae b. antimicrobials i. Ampicillin or the analogue amoxicillin ii. Chloramphenicol iii. Tetracycline iv. Norfloxacin or Ciprofloxacin v. Trimethoprim sulfamethoxazole Prevention and Control 1. mass chemoprophylaxis 2. eliminate organism from reservoir hosts a. sanitary control of water, food and milk; sewage disposal and fly control b. isolation of patients and disinfection of excreta c. detection of subclinical cases particularly in food handlers SALMONELLAE

1. 2. 3.

4.

Gram negative, nonencapsulated, nonsporulating rods All species are actively motile by means of peritrichous flagella except Salmonella pullorum and Salmonella gallinarum. stain readily with the usual dyes such as methylene blue and carbol fuchsin have simple nutritional requirements growing readily on the usual nutrient media

5.

optimum temperature is 37 C but growth occurs at a reasonable rate at room temperature

6.

facultative anaerobes growing equally well under either aerobic or anaerobic conditions

7.

characterized biochemically by failure to ferment lactose or salicin and inability to liquefy gelatin or produce indole

8.

ferments sugar with gas but there are anaerogenic strains of Salmonella enteritidis, Salmonella typhimurium and Salmonella paratyphi C

Kauffman White Classification scheme – based on agglutination tests with absorbed antisera, permitting identification of different O and H antigens in an unknown organism. Salmonella Nomenclature Salmonella subgroup 1 2 3a 3b 4 5 6

Former Genus Salmonella Salmonella Arizona Arizona Salmonella Salmonella Salmonella

Subspecies Choleraesuis Salamae Arizonae Diarizonae Houtenae Bongor idnica

Antigenic Structure 1. somatic O antigen – LPS a. associated with the cell substance b. antibodies are mostly IgM 2. flagellar or H antigen a. associated with the flagella b. antibodies are mostly IgG 3. Vi antigen a. surface antigen of the K class b. occurs in Salmonella typhi, Salmonella paratyphi A and B, and some strains of Citrobacter c. blocks agglutination by antiserum against the O antigen d. virulence antigen – presence associated with virulence and antibody; it is protective 4. Additional K antigen – R antigen

Enteric Fevers 1. Paratyphoid fever a. relatively mild course with sudden chills but otherwise similar to a mild typhoid fever b. most often milk borne and transmitted by carriers c. predominant in younger age groups d. causative agents  Salmonella paratyphi A – sewage contaminated food  Salmonella paratyphi B (Salmonella schottmulleri) – often in carriers  Salmonella paratyphi C (salmonella hirschfeldii) – tends to produce endocarditis 2. Typhoid fever a. Salmonella typhi – a strict parasite of man b. Not only an intestinal infection but a general invasion particularly the lymphatic system

Host factors that contribute to the resistance to Salmonella infection 1. Gastric acidity 2. normal intestinal microbial flora 3. local intestinal immunity Three Clinical Entities 1. Salmonella gastroenteritis or Salmonella food poisoning 2. enteric fevers 3. septicemia Salmonella Gastroenteritis or Salmonella food poisoning 1. Most common manifestation of Salmonella infection

2. 3. 4. 5. 6. 7. 8.

8-48 hours after the ingestion ---------à nausea, headache, vomiting and profuse diarrhea with few leukocytes in the stools but rarely blood Low grade fever Recovery within 2-3 days Blood stream invasion quite rare an infection not an intoxication because no toxin is involved Large numbers of bacilli cause irritation of mucosa Causative agents a. Salmonella typhimurium b. Salmonella enteritidis(Gartners baciluus) c. Salmonella choleraesuis

Clinical Manifestations 1. First week a. gradual onset with increasing remittent fever b. dull, continuous headache c. anorexia, malaise d. nonproductive cough e. epistaxis f. vague abdominal discomfort g. constipation 2. Second week

a. b. c. d. e.

temperature sustained at 40oC appears acutely ill and weak dull, lethargic, expressionless face mental state normal to mental confusion to delirium abdominal discomfort and distention

f.

diarrhea more common with blood - Physical Findings 1) spleen enlarged and soft 2) maculopapular lesions on anterior chest and upper abdomen 3) rose spots 2-3 mm. in diameter, blanch on pressure lasting 2-4 days 4) slow pulse rate or bradycardia inspite of high temperature

3. 4.

3rd and 4th week – recovery when fever subsides; death when untreated Complications a. intestinal hemorrhage b. perforation and peritonitis c. splenic rupture

5.

Typhoid carriers a. more in females than males between 50-59 years b. maybe convalescent carriers or chronic carriers c. bacilli in the gall bladder or in the urinary bladder d. best treated with massive ampicillin and cholecystectomy

black sheen or with dotted black or greenish gray 2.

Biochemical identification a. TSI: KI

b. c. d. e. f. g. h. i. j.

Septicemia 1. may occur as a sequelae of enteric fever or rarely from gastroenteritis or may have no intestinal focus 2. characterized by rapid rise with spiking temperature 3. Signs and symptoms refer to the area involved a. pneumonia b. meningitis c. conjunctivitis d. sinusitis e. suppurative arthritis f. pyelonephritis 3.

H2S: positive Motility: positive MR: positive VP: negative Indole: negative LDC: positive Urease: negative Deaminase (phenylalanine): negative Fermentation of: o sucrose – negative o myoinositol - positive o sucrose – positive

Serology a. slide agglutination with specific antisera – clumping b. Widal Test – tube dilution agglutination o Patients serum + known antigen o Serum obtained twice 7-10 days apart

o

Significant reaction: rising titer or high titer (1:160) 2nd-3rd week

Interpretation: o if predominantly O – active or acute infection o if predominantly H – past infection or vaccination o If predominantly V – carrier state What makes Salmonellae pathogenic? 1. rarely, formation of exotoxins. But all contain endotoxins 2. ECA or kunin antigen – inhibits phagocytosis and decreases their susceptibility to bactericidal action of serum 3. fimbriae – mediate attachment of bacteria to intestinal mucosa, but have no pathogenic function extraintestinally 4. tendency to plasmid-mediated multiple drug resistance 5. tendency to a carrier state especially in highly nutritive environment of gall bladder 6. Past infection and vaccination confer only partial or temporary active immunity 7. Enterotoxin similar to Escherichia is produced by Salmonella typhimurium, causative agent of food poisoning. Epidemiology Sources of infection – food and drink contaminated with salmonellae a. water b. milk and other dairy products (ice cream, cheese, custard) c. shellfish d. dried or frozen eggs e. meats and meat products f. recreational drugs g. animal dyes h. household pets Laboratory Diagnosis Specimens

• •

• •

Blood – culture + 1st week Bone marrow – culture Urine – culture + after 2nd week Stool – culture

o o

Typhoid fever + 2nd week or 3rd week Enterocolitis + 1st week

•

Serology

1.

Culture a. enrichment cultures – selenite F broth or tetrathionate broth b. differential media o MacConkey – colorless colonies, nonlactose fermenter c. selective o Salmonella Shigella agar – colorless colonies, sometimes with black colonies due to H2S production (black center with clear periphery) o Hektoen enteric agar – green with black centers (lactose negative; H2S positive) o Bismuth sulfite agar – rapid detection of Salmonella typhi producing jet black colonies,

c.

Typhidot – the 60 minute test o Dot EIA for the rapid detection of specific IgM and IgG antibodies

Result 1. IgM positive only 2. IgM and IgG positive 3. IgG positive 4. IgG and IgG

Clinical Interpretation Acute typhoid fever Acute typhoid fever (in the middle stage of infection) Previous infection (not due to typhoid); or relapse or reinfection Probably not typhoid

Treatment 1. Enterocolits a. fluid and electrolyte replacement b. antibiotic treatment not needed o may prolong excretion of the organisms o increase the frequency of carrier state o select mutants resistant to the antibiotic 2. Enteric fevers and bacteremias a. antimicrobial treatment o ceftriaxone or ciproflaxin – treatment of choice o chloramphenicol o trimethoprim-sulfamethoxazole o ampicillin b. carriers o ampicillin alone o cholecystectomy combined with drug treatment Prevention and Control 1. Through cooking of infected poultry, meats and eggs 2. carriers should not be allowed to work as food handlers 3. sanitary measures to prevent contamination of food and water 4. immunization a. two injections of acetone killed bacterials suspensions of Salmonella typhi followed by booster injection b. oral administration of a live avirulent mutant strain of Salmonella tyhi

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