Enterobacteriaceae Introduction 2024 PDF

Enterobacteriales Prof Dr Rıza Durmaz 2022-YBU Order: Enterobacteriales  Enterobacteriales Enterobacteriales should possibly be divided into seven or more separate families. Enterobacteriaceae, Erwiniaceae Pectobacteriaceae Yersiniaceae Hafniaceae...

Enterobacteriales Prof Dr Rıza Durmaz 2022-YBU Order: Enterobacteriales  Enterobacteriales Enterobacteriales should possibly be divided into seven or more separate families. Enterobacteriaceae, Erwiniaceae Pectobacteriaceae Yersiniaceae Hafniaceae Morganellaceae Budviciaceae *Enterobacteriaceae is one of the most taxonomically diverse bacterial families.  Enterobacteriales  Small gram-negative rods (2-5 by 0.5 microns)  Most motile with peritrichous flagella Shigella and Klebsiella are nonmotile  They share a common antigen (enterobacterial common antigen)  All members can grow rapidly on a variety of nonselective (e.g., blood agar) and selective (e.g., MacConkey agar) media.  Oxidase-negative facultative anaerobes  Reduce nitrate  Ferment glucose and other carbohydrates Catalase, glucose, nitrate +ve; oxidase -ve. 6 Proteus species move very actively by means of peritrichous flagella, resulting in "swarming" on solid medium. Some strains of E. coli produce hemolysis on blood plates. Proteus spp.  Enterobacteriales cont.. The ability to ferment lactose has been used to differentiate; Lactose fermenters (LF): Escherichia, Klebsiella, Enterobacter, Citrobacter, and Serratia… No Lactose fermenters Salmonella, Shigella, and Yersinia spp. LF Late Lactose fermenters Shigella sonnei, Proteus spp LNF Many genera (50 genera) Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Enterobacter, Yersinia, etc. Some strains opportunistic pathogens Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis…. Some strains true pathogens Salmonella, Shigella, Yersinia, some strains of E. coli  Antigenic Structure Capsule (most Klebsiella, some Enter obacter and Escherichia strains) lipopolysaccharide (LPS): somatic O polysaccharides H proteins K antigens Fimbriae--adherens Pili (sex or conjugative pili).genetic transfer The epidemiologic (serologic) classification is based on three major groups of antigens:  Somatic O polysaccharides,  K antigens in the capsule (type-specific polysaccharides), and  H proteins in the bacterial flagella Antigenic Structure of Enterobacteriales S. Typhi O antigen side chain (Fimbriae)  Transmitted by the fecal-oral route Primary pathogens Opportunistic pathogens Organisms capable of causing Organisms that can only cause disease under certain conditions or in certain disease in anyone hosts Shigella E. coli Providencia Salmonella Klebsiella Morganella pneumoniae Yersinia Enterobacter Proteus Serratia Enterobacteriales Common Virulence Factors Associated with Enterobacteriales cont.. Sequestration of growth factors: Siderophores, enterobactin, aerobactin Iron is an important growth factor required by bacteria, but it is bound in heme proteins (e.g., hemoglobin, myoglobin) or in iron- chelating proteins(e.g., transferrin, lactoferrin). The bacteria counteract the binding by producing their own competitive siderophores or iron-chelating compounds (e.g., enterobactin, aerobactin) Resistance to serum killing: ◦ The capsule can protect the organsim from serum killing as well as other factors that prevent the binding of complement components to the bacteria Antimicrobial resistance: ESBL and Carbapenemase production  Endotoxin Many of the systemic manifestations of gram-negative bacterial infections are initiated by endotoxin— ◦ activation of complement, ◦ release of cytokines, ◦ leukocytosis, ◦ thrombocytopenia, ◦ disseminated intravascular coagulation, ◦ fever, ◦ decreased peripheral circulation, ◦ shock, and ◦ death  Other virulance factors.. Exotoxins: ETEC produce one or more enterotoxins. Cytotoxins: Shiga toxin produced by S. dysenteriae Medically Important Enterobacteriales Citrobacter species Enterobacter spp. Escherichia spp. Klebsiella spp. Morganella spp. Proteus spp. Salmonella spp. Serratia spp. Incidence of Enterobacteriales associated with bacteremia Shigella spp. Yersinia spp. Origins and sites of infections with Enterobacteriales ◦ Infections with the Enterobacteriales can originate from; ◦ Animal reservoir (e.g., most Salmonella species, Yersinia species), ◦ Human carrier (e.g., Shigella species, Salmonella serotype Typhi), or ◦ Endogenous spread of organisms (e.g., spread of E. coli from the intestine to the peritoneal cavity following perforation of the intestine) Escherichia coli a) The most common gram-negative rods isolated from patients with sepsis b) Responsible for causing more than 80% of all community-acquired UTIs as well as many hospital-acquired infections, and c) A prominent cause of gastroenteritis. Most infections (with the exception of neonatal meningitis and gastroenteritis) are endogenous. ESCHERICHIA COLI Specialized Virulence Factors Associated with Escherichia coli  coli (ETEC) Infections are primarily acquired through consumption of fecally contaminated food or water. Cuase Traveler’s diarrhea and infant diarrhea Person-to-person spread does not occur. Secretory diarrhea develops after a 1- to 2-day incubation period and persists for an average of 3 to 5 days The symptoms: ◦ watery, nonbloody diarrhea and ◦ abdominal cramps; ◦ less commonly nausea and vomiting  ETEC.. ETEC produce two classes of enterotoxins: ◦ heat-labile toxins (LT-I, LT-II) ◦ increase cyclic adenosine monophosphate (cAMP) levels, resulting in enhanced secretion of chloride and a decreased absorption of sodium and chloride ◦ These changes are manifested in a watery diarrhea ◦ heat-stable toxins (STa and STb) ◦ Increase in cyclic guanosine monophosphate (cGMP) and subsequent hyper secretion of fluids. Genes for LT-I and STa are present on a transferable plasmid, ADP-ribosylation Enhance chloride secretion Decrease sodium and chloride absorption  Enteropathogenic E. coli (EPEC) EPEC is major cause of infant diarrhea in developing countries. Person-to-person spread occurs, so the infectious dose is likely to be low Disease is characterized by watery diarrhea and vomiting, nonblody stools Not invasive. Do not produce enterotoxins. Enteropathogenic E. coli (EPEC) Enteropathogenic E. coli (EPEC) Enteroaggregative E. coli (EAEC) EAEC FORM STACKED BRICK LIKE FORMATION. 34 Shiga toxin–producing E. coli (STEC) (Enterohemorrhagic E. coli (EHEC), Verotoxıgenic E. coli, VITEC) ◦ STEC are the most common strains producing disease in developed countries. ◦ STEC disease is most common in the warm months, and the highest incidence is in children younger than 5 years. ◦ Most infections are attributed to the consumption of ◦ undercooked ground beef or other meat products, ◦ water, ◦ unpasteurized milk or ◦ fruit juices (e.g., cider made from apples contaminated with feces from cattle), ◦ uncooked vegetables such as spinach, and fruits. ◦ The ingestion of fewer than 100 bacteria can produce disease, ◦ Person-to-person spread occurs.  STEC cont.. ◦ The most common strain of STEC is serotype O157:H7, although disease has been associated with other serotypes, including E. coliO104:H4, which was responsible for a 2011 outbreak in Germany ◦ these strains have acquired Shiga toxin ◦ Disease caused by STEC ranges from mild, uncomplicated diarrhea to hemorrhagic colitis with severe abdominal pain and bloody diarrhea ◦ Hemolytic uremic syndrome (HUS), a disorder characterized by ◦ acute renal failure, ◦ thrombocytopenia, and ◦ microangiopathic hemolytic anemia, ◦ HUS is a complication in 5% to 10% of infected children younger than 10 years.  Shiga toxins Cytotoxic Shiga toxins (Stx1, Stx2) disrupt protein synthesis  Enteroinvasive E. coli (EIEC) EIEC strains are rare in both developed and developing countries. The strains are closely related by phenotypic and pathogenic properties to Shigella. The bacteria are able to invade and destroy the colonic epithelium, producing watery diarrhea..  Pathogenic strains are primarily associated with a few restricted O serotypes: O124, O143, and O164 A series of genes on a plasmid mediate bacterial invasion (pInv genes) into the colonic epithelium. ◦ The bacteria then lyse the phagocytic vacuole and replicate in the cell cytoplasm.  DAEC strains DAEC strains are defined based on the presence of a diffuse adherence pattern (DA) on HeLa and HEp-2 epithelial cells Afa/Dr DAEC strains are associated with acute diarrhoea in children, especially in those 6 months and older, with persistent diarrhoea It was found that the relative risk of diarrhoea associated with DAEC increases with the child's age from 18 months to 5 years Its pathogenicity has not been identified in adults  Extraintestinal Infections of E. coli Urinary Tract Infection: ◦ E. coli is the most common cause of urinary tract infection (Cystitis, pyelonephritis..). ◦ Uropathogenic E. coli produces ◦ adhesins (primarily P pili, AAF/I, AAF/III, and Dr) that bind to cells lining the bladder and upper urinary tract ◦ hemolysin HlyA that lyses erythrocytes and other cell types Neonatal Meningitis: E. coli and group B streptococci cause the majority of central nervous system infections in infants younger than 1 month. ◦ Approximately 75% of the E. coli strains possess the K1 capsular antigen. Septicemia: ◦ May be originated from UT or GIS ◦ Mortality is high E. coli UTI URINARY TRACT INFECTIONS. 44 Ascending urinary tract infection. 46  Other opportunistic Enterobacteriales Klebsiella K. pneumoniae (FRIEDLANDER’S BACILLUS ) and K. oxytoca are the most commonly isolated. community-acquired primary lobar pneumonia (frequently involves necrotic destruction of alveolar space), infections of wound, soft tissue, and urinary tract. Risk factors for pneumonia: alcoholism; compromised pulmonary function. K. rhinoscleromatis causes rhinoscleroma Rhinoscleroma is a slowly progressive, chronic granulomatous infectious disease. In 95% to 100% of cases nose is the most affected part  K. granulomatis K. granulomatis may cause granuloma inguinale (donovanosis), a granulomatous disease affecting the genitalia and inguinal area, a sexually transmitted disease K. ozaenae causes ozena (chronic atrophic rhinitis). Ozena is a chronic disease of the nasal cavity characterised by atrophy of the mucosa and bone caused by Klebsiella ozaenae.  KLEBSIELLA Gram negative bacilli with capsule Mucoid lactose fermenting colonies on MacConkey’s agar KLEBSIELLA - MICROSCOPY. 52 KLEBSIELLA - on CULTURE. 53 Proteus Most common isolates: P. mirabilis. Cause urinary tract infections and bacteremia. Produce urease, making the urine of the patients alkaline, promoting stone formation by precipitating Mg and Ca. Enterobacterales such as Enterobacter spp., Serratia marcescens, Citrobacter freundii, Providencia spp., and Morganella morganii (ESCPM group) are increasingly identified as causative agents of nosocomial infections, frequently including bloodstream, urinary tract, gastrointestinal, respiratory, and skin and soft tissue infections These genera, particularly Enterobacter, are resistant to multiple antibiotics. multiresistant ESCPM infections carries the risk of promoting carbapenem resistance. 55  Escherichia coli and other opportunistic Enterobacteriales Laboratory diagnosis Smears: the Enterobacteriales pathogens resemble each other. The presence of large capsules is suggestive Klebsiella. Culture: blood agar and selective differential media (e.g., MacConkey agar), the latter is useful for preliminary identification. Biochemical test systems can be used for identification of Enterobacteriales members. Serologic tests are used for determining the clinical significance of an isolate and for epidemiologic purpose.  Diagnosis of E. coli strains responsible for gastroenteritis Diagnosis of E. coli strains responsible for gastroenteritis cont.. EAEC: Characteristic adherence to HEp-2 cells; DNA probe and amplification assays developed for conserved plasmid EPEC: Characteristic adherence to HEp-2 or HeLa cells; probes and amplification assays ETEC: commercial immunoassays for detecting ST in clinical specimens and cultures; PCR assays used with clinical specimens EIEC: Sereny (guinea pig keratoconjunctivitis) test; plaque assay in HeLa cells; probes and amplification assays EMB plate MacConkey agar BIOCHEMICAL TEST & REACTIONS They provide additional information for the identification of the bacterium. The tests include: ◦Triple sugar iron agar (TSI) ◦Indole test ◦Citrate utilization ◦Urease test TRIPLE SUGAR IRON AGAR (TSI) It is a composite media used to study different properties of a bacterium – sugar fermentation, gas production and H2S production. In addition to peptone, yeast extract & agar, it contains ◦ 3 sugars – Glucose, Lactose, Sucrose. ◦ The Iron salt – Ferric citrate indicates H2S production. ◦ Phenol red is the indicator. It is an orange red medium with a slant and a butt. pH of the medium – 7.4 TSI REACTIONS: Yellow – Acid Pink - Alkaline Yellow slant / Yellow butt (A/A) – Lactose fermenters (LF). Pink slant / Yellow butt (K/A) – Non lactose fermenters (NLF). Pink slant / no colour change (K/K) – Non fermenters Black colour – H2S production. Gas bubbles or crack in the medium – gas production. Lactose Fermenter (LF) – E.coli, Klebsiella Non-Lactose Fermenter – Salmonella, Shigella H2S - Proteus INDOLE TEST Used to detect indole production by the organism. They produce indole from tryptophan present in peptone water. After overnight incubation, a few drops of indole reagent (Kovac’s reagent) is added. Positive test is indicated by a pink ring. ◦ Positive indole test – pink ring ◦ Negative indole test - yellow ring Indole positive – E.coli Indole negative – Klebsiella, Salmonella. CITRATE UTILIZATION To detect the ability of certain bacteria to utilize citrate as the sole source of carbon. ◦ Contains Sodium citrate and bromothymol blue as the indicator. If citrate is utilized, alkali is produced which turns the medium to blue. ◦Citrate positive – blue colour ◦Citrate negative – green colour Positive – Klebsiella Negative – E.coli UREASE TEST This test is used to detect organisms that produce urease. Urease produced by the organisms split urea into ammonia and CO2. ◦Urease positive – pink colour ◦Urease negative – yellow colour Positive – Proteus, Klebsiella Negative – E.coli, Salmonella Triple sugar iron agar (TSI)  References Murray et al. Medical Microbiology, 2020 Sherris Medical Microbiology 2018 68 References Jawets…Medical Microbiology 2010 http://www.eplantscience.com/index/medicinal_microbiolo gy/spiral_bacteria.php http://www.life.umd.edu/classroom/bsci424/Lectures/ LectureSummaryList.htm