Laboratory Identification of E. coli PDF

Laboratory Identification of E. coli Escherichia coli (E. coli) is a bacterium commonly found in the intestines of humans and animals. While most strains are harmless, certain pathogenic strains can cause severe illnesses. by Heba Rashed Learning Outcomes (LOs) On completion of this lecture, t...

Laboratory Identification of E. coli Escherichia coli (E. coli) is a bacterium commonly found in the intestines of humans and animals. While most strains are harmless, certain pathogenic strains can cause severe illnesses. by Heba Rashed Learning Outcomes (LOs) On completion of this lecture, the student will be able to: To know the Enterobacteriaceae To know the lactose fermenter organisms Recognize of laboratory identification of E.coli & klebsiella The family Enterobactericeae includes the following genera 1. Escherichia coli , 2. Klebsiella, 3. Citrobacter 4. Salmonella 5. Shigella 6. Proteus 7. Serratia 8. Enterobacter 9. Morganella 10.Providencia 11.Yersinia Classification of Enterobacteriaceae The family Enterobacteriaceae is divided according to fermentation of lactose into A. Lactose fermenters that ferment lactose producing rose pink colonies on MacConkey’s medium such as E.coli , Klebsiella , Enterobacter, Citrobacter. B. Lactose non-fermenters that do not ferment lactose producing pale yellow colonies on MacConkey’s medium such as Salmonella , Shigella, Proteus. E. coli can only survive in oxygen-rich environments. A. True B. False ANSWER: B ANSWER: B Uses of MacConkey Agar Medium MacConkey agar is selective and differential medium. MacConkey agar is commonly used for the isolation of Gram negative enteric bacteria. It differentiates between lactose-fermenting and non lactose fermenting gram-negative rods by the color of What is E. coli? Gram-Negative BacteriaRod-Shaped Escherichia coli, commonly E. coli is a facultative known as E. coli, is a type anaerobic bacterium that of bacterium found in the can thrive in both oxygen- intestines of humans and rich and oxygen-deprived animals. environments. Commensal Pathogenic Strains Most strains of E. coli Certain strains of E. coli are harmless and part of have evolved virulence the normal intestinal factors that can cause flora, aiding in digestion illness, leading to food and nutrient absorption. poisoning, urinary tract infections, and other  Importance of E. coli Identification Public Health Clinical Diagnosis E. coli identification plays a Accurate identification is crucial role in protecting essential for diagnosing E. public health, especially coli infections, allowing for when associated with appropriate treatment and foodborne outbreaks or preventing complications. water contamination. Epidemiology Identifying E. coli strains helps researchers track outbreaks, identify sources of contamination, and develop effective prevention strategies. E. coli Morphology and Characteristics Rod-shaped Flagella for Capsule for Pili for Adhesion Morphology Movement Protection Pili are thin, hair-like E. coli bacteria are E. coli possesses Some strains of E. coli structures that extend Gram-negative, rod- flagella, which are hair- have a capsule that from the cell surface and shaped, with a length like appendages that surrounds the cell wall, facilitate adherence to of 2-3 micrometers enable them to freely in providing protection host cells, promoting and a width of 0.5-1 liquid environments, such against host defenses and colonization. micrometer. They are as the intestinal tract. environmental stresses. commonly found in the intestines of animals, including humans. Natural host of E.coli is intestinal tract of humans and animals They grow on ordinary medium. It is one of the organism that cause fecal pollution of water Morphology: Gram negative bacilli , motile, non-spore forming Some strain are capsulated Culture characters E.coli are aerobic and facultative anaerobic E.coli isolated from extraintestinal sources produce beta haemolysis on blood agar. Diseases caused by E.coli intestinal diseases ( Diarrhea ) There are 5 types of diarrheagenic E.coli 1-Enterotoxigenic E.coli 2-Enteropathogenic E.coli 3-Enteroinvasive E.coli 4- Enteroaggregative E.coli 5-Enterohaemorrhagic E.coli: A. It causes bloody diarrhea or hemorrhagic colitis. B. It produce verocytotoxigenic toxins C. Antigenically O157:H7 All strains of _Escherichia coli_ are pathogenic and cause severe illnesses in humans. A. True B. False ANSWER: B Specimen Collection and Handling Sample Selection Choose the appropriate specimen based on the suspected infection, such as stool, urine, or blood. Collection Procedures Follow aseptic techniques to avoid contamination and ensure accurate results. Transport Media Use transport media to preserve the viability and integrity of the E. coli bacteria during transport to the laboratory. Labeling and Documentation Label the specimen container with patient information and date/time of collection. Timely Delivery Transport the specimen to the laboratory promptly to minimize any potential bacterial growth or degradation.  Primary Isolation and Culture Media Selective Media Differential Media Selective media inhibit Differential media allow the growth of other for the differentiation of bacteria, allowing E. coli E. coli from other to flourish. MacConkey bacteria based on their agar, for example, metabolic selects for gram-negative characteristics. Eosin bacteria like E. coli. methylene blue (EMB) agar differentiates lactose fermenters from non-fermenters.  Biochemical Identification Tests 1. Indole Production 2. Methyl Red Test The indole test identifies This test determines the bacteria that produce indole ability of bacteria to from tryptophan, a crucial produce acid from glucose amino acid. fermentation, leading to a red color change. 3. Voges-Proskauer Test 4. Citrate Utilization This test identifies bacteria This test determines the that produce acetoin, a ability of bacteria to use neutral product of glucose citrate as a sole carbon fermentation, indicated by a source, leading to a change pink color change. in the medium's color. Interpretation of E. coli Test Results Positive Result Negative Result Indicates the presence of E. coli in the sample. Indicates the absence of E. coli in the sample. Further investigation needed to determine the specific May not rule out the possibility of other bacterial or strain and associated risks. viral infections. Laboratory diagnosis Specimen : diarrhoeal stool sample, urine pus, CSF and blood Samples are examines by 1. Direct smear stained with Gram stain 2. Culture on MacConkey’s medium 3. Lactose fermenting colonies are identified by morphology and biochemical reactions In case of diarrhea isolated E.coli is tested by serotyping using specific O antigen and for toxin production by latex agglutination or ELISA E. coli Pathogenic Strains Enterohemorrhagic Enterotoxigenic E. Enteroinvasive E. coli Enteropathogenic E. E. coli (EHEC) coli (ETEC) (EIEC) coli (EPEC) EHEC strains produce ETEC strains cause EIEC strains invade the EPEC strains attach to the Shiga toxins, causing traveler's diarrhea, intestinal lining, leading intestinal lining, causing bloody diarrhea and characterized by watery to bloody diarrhea and diarrhea, vomiting, and potentially life- diarrhea, abdominal dysentery-like symptoms. dehydration, particularly threatening cramps, and vomiting. in infants. complications, such as hemolytic uremic syndrome (HUS). E. coli-associated Diseases and Infections Gastroenteritis Urinary Tract Respiratory Bacteremia Infections (UTIs) Infections E. coli is a common cause E. coli can spread to the of diarrhea, abdominal E. coli can cause UTIs, In rare cases, E. coli can bloodstream, causing cramps, and fever. This leading to symptoms like cause pneumonia, which bacteremia, a serious infection is often self- pain during urination, is an infection of the condition that requires limiting, but can be frequent urination, and lungs. immediate medical severe in infants, blood in the urine. attention. children, and the elderly. Prevention and Control Measures Sanitation and Safe Food Handling Water Treatment Vaccination Hygiene Cooking meat and Public water systems are No vaccine is currently Proper handwashing poultry to the required to treat water available to protect with soap and water recommended internal to remove pathogens, against E. coli infection. after using the restroom temperature kills E. coli. including E. coli. However, some vaccines and before handling food Cross-contamination can Home water treatment for other diarrheal is essential. Thorough cleaning and be prevented by methods, such as diseases, like rotavirus, disinfection of surfaces, separating raw meat boiling, can be used to can reduce the risk of especially in food from other foods and further reduce the risk complications. preparation areas, help using separate cutting of contamination. reduce contamination. boards and utensils. Clinical Significance and Public Health Impact Human Health Food Safety E. coli infections can lead Contaminated food and to severe complications, water are major sources of especially in vulnerable E. coli outbreaks, populations. impacting food security. Economic Burden Public Health Strategies E. coli outbreaks can result in significant Prevention and control healthcare costs and measures, including economic losses. hygiene practices, are crucial for managing E. coli. Laboratory Diagnosis of Klebsiella Klebsiella is a genus of Gram-negative bacteria that can cause various infections in humans. Accurate diagnosis is crucial for effective treatment. Introduction to Klebsiella Gram-Negative Bacilli Facultative Anaerobes Klebsiella are Gram-negative bacteria, meaning they stain pink Klebsiella can survive with or under a microscope, with a without oxygen, making them characteristic capsule surrounding adaptable to different them. environments. Ubiquitous in Nature Opportunistic Pathogens These bacteria are commonly found in the environment, Klebsiella typically become including soil, water, and the pathogenic when the host's gastrointestinal tracts of humans immune system is compromised or and animals. weakened. Epidemiology and Clinical Significance Klebsiella is a prevalent bacterium found in various environments, including soil, water, and human intestines. It can cause a wide range of infections, especially in individuals with weakened immune systems. Klebsiella is a significant cause of healthcare-associated infections, particularly pneumonia, urinary tract infections, and wound infections. It is also responsible for community-acquired infections, primarily in individuals with underlying health conditions. 10% 20% 50% Hospital Infections Pneumonia Drug Resistance Klebsiella contributes to approximately 10% Klebsiella pneumonia is a common cause of Klebsiella strains have developed resistance of hospital-acquired infections. pneumonia, especially in patients with to multiple antibiotics, making treatment weakened immune systems. challenging. Klebsiella Taxonomy and Identification Genus Klebsiella Species Identification Klebsiella belongs to the family Enterobacteriaceae, a Identification of Klebsiella species relies on a diverse group of Gram-negative bacteria. These combination of phenotypic and genotypic methods. bacteria are commonly found in the environment, Traditional methods include culture characteristics, particularly in soil, water, and the gastrointestinal biochemical tests, and antimicrobial susceptibility tracts of humans and animals. testing. Molecular techniques, such as polymerase The genus Klebsiella includes several species, with chain reaction (PCR) and DNA sequencing, are Klebsiella pneumoniae being the most clinically increasingly used for accurate identification and strain significant. typing. Specimen Collection and Transport Specimen Selection Appropriate specimens are crucial for accurate diagnosis. Common specimens include respiratory secretions, blood, urine, and cerebrospinal fluid. Collection Procedures Sterile collection techniques are essential to minimize contamination and ensure accurate results. Transport Media Specimens should be transported in appropriate media to preserve viability and prevent overgrowth of contaminants. Transport Time Rapid transport is essential to ensure the integrity of specimens and minimize the risk of bacterial overgrowth. Labeling and Documentation Accurate labeling and proper documentation of specimen collection details are vital for laboratory processing and results interpretation. Gram Staining and Microscopic Examination Gram staining is a crucial initial step in the laboratory diagnosis of Klebsiella. This differential staining technique differentiates bacteria based on their cell wall composition, highlighting Klebsiella's characteristic gram-negative morphology. Microscopic examination of gram-stained smears reveals Klebsiella as short, plump, encapsulated, and often arranged in pairs or short chains. This visual identification provides a presumptive diagnosis, prompting further investigations. Culture Characteristics on Selective Media Selective Media Colony Morphology Isolation and Identification Klebsiella species are Klebsiella colonies are typically commonly isolated on selective large, mucoid, and exhibit a Isolation of pure cultures from media like MacConkey agar, distinctive pink-red color on clinical specimens is crucial for which inhibits the growth of MacConkey agar due to lactose accurate identification and most gram-positive bacteria. fermentation. antimicrobial susceptibility testing. Biochemical Identification Tests 1 1. Carbohydrate 2 2. Indole Fermentation Production Tests Klebsiella typically does Klebsiella species exhibit not produce indole, while specific patterns of some related species do. fermentation reactions. 3 3. Citrate 4 4. Urease Test Utilization Test Klebsiella species Klebsiella species utilize produce urease, an citrate as a carbon enzyme that hydrolyzes source. urea. Biochemical reaction Laboratory diagnosis 1. Sugar fermentation Specimen : urine, pus, CSF, blood, with production of acid sputum 1. Direct smear stained and gas with Gram stain 2. Indole negative 2. Culture on MacConkey’s 3. Methyl red negative 4. TSI; A/A medium 5. H2S –ve 3. On blood agar: string test 7. Citrate positive 4. The colonies are identified by 8. Urease positive morphology and biochemical reactions Antimicrobial Susceptibility Testing Antibiotic Susceptibility Testing Minimum Inhibitory Concentration (MIC) This determines the effectiveness of antibiotics The MIC is the lowest concentration of an against isolated Klebsiella strains. The Kirby- antibiotic that inhibits visible bacterial growth. It Bauer disk diffusion method or broth provides a quantitative measure of antibiotic microdilution methods are used to assess sensitivity. antibiotic sensitivity. Antibiotic Resistance Mechanisms Klebsiella can exhibit various resistance mechanisms like beta-lactamases, aminoglycoside modifying enzymes, and efflux pumps. Molecular Techniques for Identification 1 1. Polymerase Chain 2 2. Real-Time PCR Reaction (PCR) Quantitative real-time PCR provides rapid identification PCR amplifies specific DNA and quantification of target sequences for detection and genes. characterization of Klebsiella species. 3 3. Sequencing 4 4. Multiplex PCR DNA sequencing, including Multiplex PCR assays detect whole-genome sequencing, multiple virulence factors provides comprehensive simultaneously, providing a genetic information for comprehensive picture of accurate identification. Klebsiella's virulence potential. Klebsiella Pneumoniae Subspecies Identification Klebsiella pneumoniae subsp. Klebsiella pneumoniae subsp. ozaenae pneumoniae Klebsiella pneumoniae subsp. pneumoniae is the most Klebsiella pneumoniae subsp. ozaenae is less common subspecies, responsible for various infections, commonly encountered, associated with chronic, foul- including pneumonia, urinary tract infections, and smelling nasal infections (ozena). bacteremia. It lacks the typical capsule produced by K. pneumoniae It produces a capsule that contributes to its virulence subsp. pneumoniae, which may explain its different and helps evade the host's immune system. clinical manifestations. Klebsiella Oxytoca Subspecies Identification Biochemical Testing Molecular Methods Antibiotic Susceptibility K. oxytoca can be distinguished from PCR assays targeting specific genes K. oxytoca is often more resistant to K. pneumoniae by its ability to can be used to confirm the antibiotics than K. pneumoniae. produce indole and its sensitivity to identification of K. oxytoca. the antibiotic colistin. Emerging Klebsiella Species and Variants Klebsiella pneumoniae Klebsiella oxytoca Klebsiella aerogenes Klebsiella pneumoniae is a leading Klebsiella oxytoca is commonly It is an opportunistic pathogen, cause of healthcare-associated found in the environment, commonly found in hospital infections. particularly in water. environments, and can cause infections in the respiratory tract, urinary tract, and bloodstream Diseases caused by Klebsiella 1. Klebsiella pneumoniae causing pneumonia 2. Klebsiella aerogenes cause urinary tract infection 3. Klebsiella rhinoscleromatis cause granuloma of mucous membrane of the nose Reporting and Interpretation of Results Colony Morphology and Microscopic Examination Antibiotic Susceptibility Growth Laboratory reports detail Klebsiella The report includes details about The report lists antibiotics tested colony characteristics, including Gram stain results, such as and their associated sensitivity or size, shape, color, and growth morphology, arrangement, and resistance patterns, providing patterns on specific media. presence of capsules. crucial information for treatment decisions.