PHT313 Lecture 1 2nd Term 1436-1437

PHT313 Lecture 1 2nd Term 1436-1437

PHT313 Lecture 2 2nd Term 1436-1437 Dr. Hesham Radwan Objectives By the end of this lecture the student must be: A) Identify the genus of Enterobacteriaceae B) describe the chemical tests for Family C) Differentiate between different genuses D) List and match the symptoms, diagnosis

and treatment for different sps. E) Recognize the differences between lactose fermenters and lactose non-fermenters The Gram-negative cell wall is composed of a thin, inner layer of peptidoglycan and an outer membrane consisting of molecules of phospholipids, lipopolysaccharides (LPS), lipoproteins and sutface proteins. The lipopolysaccharide consists of lipid A and O polysaccharide. Enterobacteriaceae 1. Coliforms (lactose fermenters) Normal inhabitants of GIT of human and animals Source of noscomial infections

Opportunistic or cause secondary infections of wounds, urinary and respiratory tracts and the circulatory system e.g. E. coli, Klebsiella E. coli used as biological indicator in water pollution 2. True pathogens (Lactose non-fermenters) Salmonella spp., Shigella spp., Yersinia spp. Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC) 4 General characteristics of most Enterobacteriaceae

Gram negative rods Non-spore forming Grow in simple media Ferment glucose and produce acid Have peritrichous flagella and are motile Possess a capsule Media for isolation Selective differential media for enteric pathogens MacConkey agar EMB agar SS agar Selective by incorporation of dyes and bile salts Differential by incorporation of lactose and/or Fe+3 Fe+3 is incorporated to detect H2S Classified as lactose fermenters & non-lactose fermenters 6

The Enterotube II contains 12 different agars enabling the performance of a total of 15 biochemical tests as well as an enclosed inoculating wire. I- Escherichia coli General characteristics: Gram-negative Motile rods Non-spore forming,Facultative anaerobic, Oxidase -ve Ferment glucose and lactose Normal flora of intestine Opportunistic pathogens E. coli may be pathogenic inside or outside Intestine Some strains (Pathogenic) cause various forms of gastroenteritis 8 EMB

Gram Stain MacConkey agar Citrate Indole Methyl Red +ve and VP -ve Virulence factors 1. 2. Adhesions (Colonization factors)

Pili or fimbriae & nonfimbrial factors Host defense Capsule OMPs are involved in helping the organism to invade by helping in attachment and in initiating endocytosis 3. Exotoxin production (Enterotoxin) Heat-Labile (LT) & Heat-Stable Toxin (ST) (ETEC) Shiga-like toxin (Verotoxin) (EHEC) 4. Endotoxin (Pyrogen) Lipid A of LPS causes fever and endotoxic shock 10 Diseases caused by E. coli A. Intestinal: Diarrhea (Toxin and/or adhesion) A. Enterotoxegenic E. coli (ETEC) Acquired by B. Enteropathogenic E. coli (EPEC)

ingestion of C. Enteroinvasive E. coli (EIEC) contaminated D. Enterohemorrhagic E. coli (EHEC) food and water E. Enteroaggregative E. coli (EAEC) B. Extraintestinal: 1. Urinary Tract Infections [UTI] (Pili) 2. Neonatal meningitis (K1 antigen) 3. Sepsis (commonly in debilitated hospitalized patients) Endotoxic shock Due to lipid A (Pyrogen) Fever and sudden hypotension 11 Summary of E. coli gastroenteritis Symptoms Watery

diarrhea, cramps, nausea, low grade fever dysentery-like diarrhea, severe inflammation, fever Watery diarrhea With mucous without blood or pus With fever & vomiting Hemorrhagic colitis with sever abdominal cramps, watery diarrhea followed by blood, no fever HUS

Watery diarrhea Vomiting Abdominal pain Without inflammation or fever Diseases Traveler's diarrhea Infant diarrhea Dysentery Invasion Non invasive Pathogenesis Site

M.O. LT + STcAMP + cGMP Small ETEC Fluid + electrolyte loss intestine Invasive nonfimbrial adhesin (NFA): Large EIEC OMP intestine Infantile diarrhea Poorly invasive

NFA: intimin EPEC adherence factor Some reports of shiga-like toxin Cytotoxic shiga-like toxin (verotoxin) Bloody Poorly diarrhoea and invasive haemolytic uraemic syndrome Aute and Non persistent

invasive diarrhoea in children and adults Small EPEC intestine Large EHEC intestine Production of enterotoxin Small EAEC that similar to ETEC intestine 12

Urinary Tract Infection (UTI) E. coli is the most common organism causing UTI Community acquired 90% Hospital acquired (50%) UTI is the disease of female (Short urethra) Fecal E. coli acquires pili to colonize mucosa of UT Travel up urethra & infect balder (Cystitis)& sometimes move further up to infect kidney (pyelonephritis) Symptoms:urinary frequency, dysuria, hematuria, 13 Diagnosis of UTI Specimen MSU (Mid-Stream Urine) Culture On MacConkey agar

Gram negative, Lactose colonies) Viable count 100,000 (105) cfu/ml urine IMViC + + - - fermented (Pink 14 Neonatal meningitis E. coli is the second most common cause S. galactaiae (Group B) is the first Occur during the first month of life Lab diagnosis Specimen: CSF

Culture: Pink colonies on MacConkey agar (LF) 15 II- Salmonella General characteristics: Gram-negative rods belonging to Enterobacteriaceae Do not ferment lactose and H2S positive Salmonellae live in the intestinal tracts of animals Not

considered part of normal intestinal flora in human Always PATHOGENIC to human 16 Classification of Salmonella A. Kaufman-White- Le Minor Classification Based on O and H antigen serotyping 64 O and 114 H variants identified Classified into 9 groups (A-I) according to O antigen Each group can be classified into subgroups according to H Ag

Salmonella can be detected by its group O antigen and then by its type specific H antigen >2500 known serovars 17 Classification of Salmonella B. US CDC (Center for Disease Control) 1. S. enterica Subdivided into 6 subspecies enterica, salamae, arizonae, diarizonae, indica, houtanae Of these six subspecies, only subspecies enterica is associated with disease in warm-blooded animals S. enterica subsp. enterica serovar Typhi or S. Typhi S. enterica subsp. enterica ser.Typhimurium or S. Typhimurium

S. enterica subsp. enterica ser. Enteritidis or S. Enteritidis 2. Salmonella bongori (Subspecies V) 18 Species of Salmonella Two important members of Salmonella causing diseases: A. Salmonella causing enteric fever Salmonella Typhi or Salmonella Paratyphi A,B, and C These organisms penetrate intestinal mucosa Detected in blood, urine and stool B. Salmonella causing food poisoning Salmonella Enteritidis and Salmonella Typhimurium These organisms do not penetrate intestinal mucosa Detected in stool only

19 Enteric Fever Typhoid Enteric fevers are severe systemic forms of salmonellosis. Caused by S. typhi whereas a milder form Paratyphoid caused by S. paratyphi A, B or C Mode of Transmission Via fecal-oral route through fecally-contaminated food or water from either chronic carrier or case A few individuals continue to harbour Salmonella in their gall-bladders and intermittently excrete organisms in their faces Temporary excretors: Patients who excrete bacteria for year Incubation Period 10-14 day during which bacteria multiplies in Peyers patches Pass to blood via lymphatics resulting in bacteriaemia in 1st week

In 2nd week M.O. passes to different organs including peyers patches causing ulcers, gall bladder, liver, kidney & rarely menings 20 Symptoms of Enteric Fever Symptoms begin after an incubation period of 2 weeks Enteric fevers may be preceded by gastroenteritis, which usually resolves before the onset of systemic disease The symptoms of enteric fevers are nonspecific and include Fever, headache, delirium (sustained fever), malaise and tender abdomen Complications include intestinal hemorrhage and perforation 21 Laboratory diagnosis

A. Direct diagnosis: 1. Specimen: Blood during 1st week, urine during 2nd week and stool during 3rd week 2. Isolation of microorganism: I. From blood using blood culture Five to 10 ml of blood is taken during the 1st week of infection, Add to 50-100 ml sterile nutrient broth and incubate at 370C for 24 hrs. Subculture is done on MacConkey's agar which shows colorless colonies in positive case 23 Laboratory diagnosis II. From stool By culture on enrichment medium such as selenite F or tetrathionate broth which inhibits the growth of coliform and allow the growth of Salmonella and Shigella

Subculture on MacConkey, SS or DCA agar(Deoxycholate Citrate Agar) On MacConkey's agar they give colorless colonies On SS agar they give colorless colonies with black edges due to H2S production 3. Biochemical Reactions: The suspected colonies were subjected to biochemical reactions Oxidase negative, not ferment lactose and sucrose, H2S positive 24 Laboratory diagnosis A. Indirect: Serological diagnosis (Widal Test):

In the 2nd week of the disease, antibodies against Salmonella are present in the patient's serum and can be detected serologically by Widal test (agglutination test) Widal test is positive and valid during 2nd week Serial dilutions of patient's serum are added to an equal volume of common O and specific H antigens Agglutination of O- antigen and one only of the H-antigens at a titer 1/80 or above is diagnostic 25

Salmonella causing food poisoning The causative agent : S. typhimurium & S. enteritidis Mode of infection: Consumption of contaminated food Food as cakes, pastries and various milk and egg dishes

Cattle, sheep, hens, ducks and turkeys are often infected and the organism may contaminate meat and meat products Infective dose 100,000 bacteria Gastric juice is the an important host defense and decreased acidity is a predisposing factor Incubation period is 12-24 hrs and recovery within 4-7 days It is self limiting disease Manifestations include Nausea, vomiting, and abdominal discomfort, non-bloody diarrhea and slight fever 26 I- Shigella General Characteristics Gram negative rods Non motile Non spore-forming

Non capsulated Oxidase negative Ferment glucose with acid only Non lactose fermentating organism H S negative 2 27

Shigella species Disease: Bacillary dysentery (Shigellosis) S. sonnei is the most common, followed by S. flexneri Mode of Transmission Oral-fecal transmission <200 bacilli are needed for infection in health individuals Incubation periods It varies between 1-3 days Symptoms Ranges from asymptomatic to severe bacillary dysentery Watery diarrhea changing to dysentery with frequent small stools with blood, pus and mucus Fever, tenesmus and abdominal cramps

28 Stages of shigellosis A. Early stage: 1. Ingestion of contaminated food or water 2. Noninvasive colonization and cell multiplication 3. Production of the enterotoxin in the small intestine 4. Watery diarrhea attributed to enterotoxic activity of Shiga toxin (similar to LT of ETEC) 5. Fever attributed to neurotoxic activity of toxin B. Second stage: 1. Adherence to and tissue invasion of large intestine 2. Typical symptoms of dysentery 3. Cytotoxic activity of Shiga toxin increases severity 29 Laboratory identification Specimen:

Stool (mucous bloody part of stool) or rectal swap Stool culture Specimen is inoculated in Selenite broth at 370C for 24 h Then subculture on MacConkey or SS On MacConkey agar: they give colorless colonies On SS agar: they give colorless colonies Gram stain Gram negative bacilli, NON MOTILE Biochemical reactions Oxidase negative, ferment glucose, non ferment lactose and H2S NEGATIVE 30 COMMENSAL ENTEROBACTERIAE Opportunistic pathogens and are common cause of nosocomial infections 1. Klebsiella Lactose Fermnters

2. Enterobacter 3. Serratia 4. Proteus 5. Morganella Non-Lactose Fermnters 6. Providencia 31 Klebsiella-Enterobacter-Serratia group General characteristics: These organisms are very similar All are motile except Klebsiella MR negative; VP positive, Lactose Fermenter Simmons citrate positive H2S negative Some weakly urease positive Phenylalanine deaminase negative These organisms belonging to Enterobacteriaceae

Frequently in large intestine but also found in soil and water Usually opportunistic pathogens Wide variety of infections: primarily pneumonia, wound and UTI 32 Klebsiella pneumoniae Virulence factors 1. Polysaccharide capsule Protects against phagocytosis and antibiotics Makes the colonies moist and mucoid 2. Adhesions Clinical significance Major cause of nosocomial infections Nosocomial Pneumonia Important respiratory tract pathogen outside hospitals 3% of bacterial pneumonia Bloody sputum (50%) (Thick, Jelly and Red Sputum)

Septicemia, Meningitis and UTI 33 Proteus-Providencia-Morganella These organisms belonging to Enterobacteriaceae All are normal intestinal flora but also found in soil and water Opportunistic pathogens Non-lactose fermenters Phenylalanine deaminase and urease positive Urease positive after 2-6 hrs (urea NH3+ CO2) All are highly motile Proteus sp. swarming on blood agar Swarming characterized by expanding waves (rings) of organisms over the surface of blood agar 34 Proteus species

P. mirabilis and P. vulgaris are widely recognized human pathogens Isolated from urine, wounds and ear and bacteremic infections They found in colon and able to colonize urethra especially in female 1. P. mirabilis causes urinary tract infections (UTI): Proteus sp. produces urease which alkalinizes urine precipitation of calcium and magnesium salts stone formation renal epithelium damage 2. P. vulgaris causes nosocomial infections (pneumonia, bacteremia) and UTIs 35 Laboratory diagnosis

Specimen: Urine or Stool. Culture: On MacConkey agar non lactose fermenters colorless colonies On SS agar non lactose fermenters colorless colonies with black center

On ordinary media, such as nutrient agar, blood agar, show swarming (successive waves on the surface) due to high motility of Proteus The Weil-Felix test which is an antigen antibody reaction Biochemical reactions: Urease , phenyldeaminase and H2S positive 36

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