BACTERIA LECTURE NOTES FINAL EXAM - PDF

 LEARNING OBJECTIVES 1. Characterize the significant Gram-negative enterics; 2. Explain the pathophysiology of diseases; 3. Distinguish features of the enteric rods for accurate laboratory diagnosis; 4. Recognize their medical implications to public health General Character...

 LEARNING OBJECTIVES 1. Characterize the significant Gram-negative enterics; 2. Explain the pathophysiology of diseases; 3. Distinguish features of the enteric rods for accurate laboratory diagnosis; 4. Recognize their medical implications to public health General Characteristics Often referred as ENTERICS - Consists of a large number of diverse organisms (Frequently encountered in the lab) Normal flora of the GI tract of animals and humans Gram negative bacilli or coccobacilli Associated in infections almost throughout the body. Ubiquitous they are everywhere General Characteristics All Enterobacteriaceae are: does not require oxygen, but can survive oxygen § Facultative anaerobe, non-spore-former § Glucose fermenters AND FRUCTOSE NOT ALL ARE FERMENTERS OF LACTOSE § Cytochrome oxidase negative EXCEPT : Plesiomonas § All reduce nitrates to nitrites except: Erwinia, Pantoea agglomerans, Photorhabdus, and Xenorhabdus they have NITRATE REDUCTASE General Characteristics All members are motile except: § Klebsiella NM @370C & RT § Shigella § Yersinia Generally are NM @370C, but M at RT motile at room temp Motile members have peritrichous flagella except: § Tatumella ptyseos which has polar flagella General Characteristics All members grow luxuriantly on CHOC and BAP as moist, smooth, gray, shiny, entire, convex and opaque colonies. Klebsiella and sometimes Enterobacter produce mucoid colonies Beta-hemolysis is seen in some strains. Enterobacteriaceae on SBA Mucoid Colonies of Klebsiella spp., on SBA All are catalase positive except for one group of Shigella species and Xenorhabdus.  General Characteristics EMB- E.coli- blue-black colonies with metallic greenish sheen - Enterobacter- pink colonies - non-lactose fermenter - translucent and colorless/light purple Selective and Differential Media § Primary culture media used in the presumptive identification of enteric organisms in the laboratory. § EMB, MAC, XLD, HEA, SSA MacConkey Agar Eosin Methylene Blue Agar Note: 1. For the culture media, especially for the primary isolation of Enterobacteriaceae, Please read Appendix A of Mahon, C. R., Lehman, D. C., & Manuselis, G. (2018). Textbook of diagnostic microbiology-e-book. Elsevier Health Sciences. 2. A huge percentage of the post quiz for Enterobacteriaceae will cover the principle, procedure of inoculation, incubation requirement, and interpretation of these culture media and also in their correlation with the results of the biochemical tests for enteric bacteria. Xylose Lysine Salmonella-Shigella Agar Hektoen-Enteric Agar Deoxycholate Agar -isolate Salmonella, Shigella - has sodium deoxycholate - inhibit gram+ cocci, gram- rods MAC - LF - pink or red colonies - has sucrose, lactose, xylose - NLF - colorless/transparent colonies - phenol red as pH indicator - sodium thiosulfate -sulfur source Serotyping (serogrouping) of Enterobacteriaceae Serotyping – a serologic test that determines Note: 1. Salmonella/Escherichia/Shigella species may look the same under species/subtypes of bacteria based on the the microscope, but they can be separated into different many serotypes according to the variations of the antigens found on their antigens present on its surface. determined by the agglutination of bacteria in a specific antisera surface. 2. Serotyping is determined by the agglutination of bacteria with specific antisera to identify variants of O and H antigens. Antigens – substances (proteins) that react with specific antibodies Antibodies (anti-sera) – proteins that react to antigens. Bacteria with Antibodies specific Agglutination of the antigens of to the bacterial bacteria interest antigens to be Antigenic variations – refers to the variations detected and serotyped in the antigenic structures of antigens. Antigenic Structures used for Identification Through Serotyping heat labile H antigen – flagellar antigen § An antigen found in the flagellum of motile members § Protein in nature § Heat labile § Used in the serotyping between species of Salmonella and other motile bacteria. Antigenic Structures used for Identification Through Serotyping O antigen – somatic antigen § Heat stable, located in the cell wall § Lipopolysaccharide of the cell wall composing the endotoxin portion § over 160 types of O antigen for E.coli and specific types are associated with diseases. § Shigella : 4 serotypes based on the O antigen: serotypes A, B, C, and D § 60 types of O antigens exists for Salmonella Antigenic Structures used for I.D K antigen – capsular antigen/envelope antigen § Heat labile polysaccharide found in encapsulated strains. § Covers the O antigen (must be removed to detect the O antigen!!) § To remove the K antigen – the organism must be boiled for 30 minutes. § Klebsiella, Salmonella, and E.coli have K antigens. § Vi antigen for Salmonella is categorized as K antigens. Serogroups of Escherichia coli based on its O and H antigens Route of Infections Infections are associated with lapses in personal hygiene via: § Fecal-oral route – Most Common! § Poor sanitation in impoverished countries § Colonization of the skin and respiratory tract of hospitalized patients (nosocomial) Common types of infections include: § UTI, Gastroenteritis, Septicemia Factors Affecting Virulence The organisms ability to cause disease is influenced by their ability to: § Adhere to host cells LPO, pili, fimbrae § Colonize body surfaces LPO, pili, fimbrae § Release certain toxins (endotoxins) § Invade deeper tissues dysentery (diarrhea with blood) § Ability to transfer plasmids carrying antibiotic resistance genes conjugation mediated by the pili Opportunistic Pathogens 2 Broad Categories of Primary Pathogens Enterobacteriaceae Escherichia coli (Colon bacillus) Previously considered as a harmless member of the colon biota § First described by Theodore Escherich in 1885 § Associated with wide range of clinical syndromes § Primary marker of fecal contamination in water § Closely related to Shigella in terms of DNA homology but can be easily differentiated through lactose fermentation: o E.coli is lactose fermenter o Shigella is non-lactose fermenter Escherichia coli (Colon bacillus) Characteristics § Motile, possess an adhesive fimbriae and sex pili § Has O, H and K antigens Escherichia coli (Colon bacillus) General laboratory findings Fermenter of: Glucose, lactose, trehalose and xylose indole (+) MR (+) H2S, DNAse, Urease, PAD, citrate (-) Escherichia coli (Colon bacillus) Enterotoxigenic E.coli (ETEC) Associated with diarrhea of infants (major cause!) and adults in tropical and subtropical areas Most common cause of TRAVELLER’S DIARRHEA/TOURIST’S, DIARRHEA/ “MONTEZUMA’S REVENGE” Acquired via ingestion of contaminated food and water Escherichia coli (Colon bacillus) Enterotoxigenic E.coli (ETEC) MAJOR CONTRIBUTING FACTORS Poor hygiene Reduced availability of sources of potable water Inadequate sanitation Escherichia coli (Colon bacillus) Enterotoxigenic E.coli (ETEC) 106 – 1010 (1,000,000–10,000,000,000) organisms is the infective dose of organisms in immunocompetent individuals. STOMACH ACIDITY § Serves as a protective mechanism against colonization and disease initiation § Patients suffering from achlorhydria – higher risk compared to normal persons. Escherichia coli (Colon bacillus) Enterotoxigenic E.coli (ETEC) Pathophysiology Colonization of ETEC in the small intestine mediated by fimbriae Releases toxins in the small intestine § Heat – Labile (LT) toxin –Similar in action to cholera toxin –Has A and B portion § Heat – stable (ST) toxin Escherichia coli (Colon bacillus) Enterotoxigenic E.coli (ETEC) Pathophysiology LT toxin (heat labile) – consists of 2 subunits, one A subunit and five B subunit. B subunit binds to the GM1 ganglioside receptor, the same receptor used by cholera toxin to bind on the epithelial cells in the small intestine. The whole toxin is endocytosed by the epithelial cells of the intestine Escherichia coli (Colon bacillus) Enterotoxigenic E.coli (ETEC) Pathophysiology The vesicle that contain the whole toxin transports the toxin to the golgi apparatus In the golgi apparatus, the A subunit dissociates from the B subunit and it moves across the membrane of the vacuole and interact with a membrane protein (Gs) that regulates adenylate cyclase Adenylate cyclase enzyme catalyzes the transformation of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). The effect of this interaction is an increase in cyclic adenosine monophosphate (cAMP) level Escherichia coli (Colon bacillus) Enterotoxigenic E.coli (ETEC) Pathophysiology The increased levels of cAMP stimulate the intestinal cells to hyper secret fluids and electrolytes into the lumen of the intestine. This results in watery diarrhea (similar to cholera). Escherichia coli (Colon bacillus) Enterotoxigenic E.coli (ETEC) Pathophysiology Heat – stable (ST) toxin § Binds to the transmembrane guanylate peptide cyclase receptor found on the surface of the intestine. § Binding results in an increased accumulation of cyclic guanosine monophsophate (cGMP) which also causes hyper-secretion of fluid and electrolytes. § This results in watery diarrhea Escherichia coli (Colon bacillus) Enterotoxigenic E.coli (ETEC) Diagnosis watery diarrhea, abdominal cramps, and nausea Isolation of lactose fermenting organisms on selective and differential media Biochemical tests Multiplex PCR Lactose fermenting colonies as PRESUMPTIVE IDENTIFICATION for Escherichia coli Note: The presence of colonies on EMB that exhibit a “green metallic sheen” does not definitively indicate that presence of Escherichia coli since some Citrobacter and Enterobacter species are also able to produce the same type of colony characteristic on EMB. Escherichia coli (Colon bacillus) Enteroinvasive E.coli (EIEC) Associated with dysentery (watery diarrhea with blood, leukocytes, and mucus) similar to Shigella spp. Occur in adults and children Direct person-to-person transmission via fecal oral route Clinical infection characterized by: fever, severe abdominal cramps, malaise, and watery diarrhea.  Escherichia coli (Colon bacillus) Enteroinvasive E.coli (EIEC) Pathophysiology § Plasmid mediated bacterial invasion (pInv genes) - mediate the colonization of EIEC into the colonic epithelium § EIEC lyses phagocytic vacuole and replicate into the cell cytoplasm § Movement within the cytoplasm and into the adjacent cells is regulated by formation of actin filaments This destroys colonic epithelial cells and results in inflammation that could progress to colonic ulceration. Escherichia coli (Colon bacillus) Enteroinvasive E.coli (EIEC) Fecal – oral route Manifestation: § Fever, severe abdominal cramps, malaise and watery diarrhea (with blood) § Clinical symptoms similar to Shigella spp. (Must be differentiated from Shigella species) Virulence test: SERENY TEST Escherichia coli (Colon bacillus) Enteroinvasive E.coli (EIEC) Similarities of EIEC with Shigella Differentiation is done thru: § Both are gram negative, facultative anaerobic and non- spore-forming bacilli § Their infective dose § Non-motile EIEC – has higher infective dose (106) § Non-lactose fermenters § Negative for lysine decarboxylation Shigella – as few as 10 organisms § Cross reactions between O antigens § Acetate or mucate utilization as carbon source – EIEC is positive Escherichia coli (Colon bacillus) Enteropathogenic E.coli (EPEC) Non-toxigenic and non-invasive strain that causes diarrhea Pathophysiology is based on its adhesive properties Major cause of infant diarrhea in impoverished countries Characterized by watery diarrhea with large amounts of mucus but no blood! Escherichia coli (Colon bacillus) Enteropathogenic E.coli (EPEC) Pathophysiology Infection is initiated by EPEC attachment to the epithelial cells of the small intestine with subsequent effacement (destruction) of the microvillus (attachment/effacement). Bundle-forming pili (BFP)- mediates the initial aggregation of EPEC that leads to the formation of micro-colonies on the epithelial cell surface. Escherichia coli (Colon bacillus) Enteropathogenic E.coli (EPEC) Pathophysiology Attachment is followed by the active secretion of proteins into the host epithelial cells through the type III secretion of EPEC § Translocated intimin receptor (Tir) – inserted into the host’s epithelial cell membrane and functions as a receptor for an outer membrane bacterial adhesin, intimin. § Binding of intimin to Tir – results in polymerization of actin and accumulation of cytoskeletal elements beneath the attached bacteria, loss of cell surface integrity, and death. Escherichia coli (Colon bacillus) Enteropathogenic E.coli (EPEC) Pathophysiology Attachment is followed by the active secretion of proteins into the host epithelial cells through the type III secretion of EPEC § Escherichia coliI secreted protein F (EspF) – causes loss of intestinal barrier function through disruption of tight junctions and induces host cell death through apoptosis. Escherichia coli (Colon bacillus) Enterohemorrhagic E.coli (EHEC) O157: H7 Causes hemorrhagic diarrhea and colitis associated with hemorrhagic diarrhea, colitis, and hemolytic uremic syndrome (HUS) watery diarrhea that progresses to bloody diarrhea with abdominal cramps, low grade fever or an absence of fever. Stool contains no leukocytes – distinguishes it with dysentery caused by Shigella or EIEC infections Risk factors –consumptionof processed meats such as undercooked hamburgers served at fast food restaurants, unpasteurized dairy products and apple cider, bean sprouts, and spinach. Escherichia coli (Colon bacillus) Enterohemorrhagic E.coli (EHEC) Pathophysiology: Hemorrhagic diarrhea and colitis EHEC O157:H7 (a.k.a STEC) produces two toxins: verotoxin 1 and verotoxin 2 § Verotoxin 1 –phage encoded toxin identical to the shiga toxin (Stx) produced by Shigella dysenteriae type 1. It produces damage to Vero Cells (African Green Monkey kidney cells). § Biologically similar to, but immunologically different from both Stx and verotoxin 1 Note: Verotoxin 1 can react and be neutralized by antibodies against Stx indicating antigenic similarities, while verotoxin 2 does not, indicating the lack of antigenic similarities. Escherichia coli (Colon bacillus) Enterohemorrhagic E.coli (EHEC) Pathophysiology: Hemorrhagic diarrhea and colitis Verotoxins and Shiga toxins are similar in quaternary structure to the heat-labile toxin of ETEC; they are composed of a pentamer of receptor-binding B subunits that are noncovalently bound to a single enzymatic A subunit. B subunit of the toxin - binds to the specific glycolipid called Globotriaosylceramide (Gb3) expressed on host cells particularly on the intestinal villi and renal endothelial cells. Note: High concentrations of Gb3 are found on intestinal villi and renal endothelial cells Escherichia coli (Colon bacillus) Enterohemorrhagic E.coli (EHEC) Pathophysiology: Hemorrhagic diarrhea and colitis A subunit of the toxin – is internalized and cleaved into two molecules. The A1 fragment binds to 28S rRNa and causes cessation of protein synthesis. This results in the damage of the intestinal cells leading to colitis and hemorrhagic diarrhea. Escherichia coli (Colon bacillus) Enterohemorrhagic E.coli (EHEC) Pathophysiology: Hemolytic Uremic Syndrome (HUS) Endothelial cells appear to be particularly important target cells for Shiga toxins. The toxin is transported retrograde in membrane-bound vesicles to the endoplasmic reticulum. The A subunit attaches to the 28S rRNA leading to cessation of protein synthesis and death of the cell.  Escherichia coli (Colon bacillus) Enterohemorrhagic E.coli (EHEC) Pathophysiology: Hemolytic Uremic Syndrome (HUS) ST toxin binding t o glomerular endothelium inactivates a metalloproteinase called von Willebrand factor-cleaving protease (VWFCP). Once the VWFCP is disabled, multimers of von Willebrand Factor (vWF) form and initiate platelet activation and cause micro thrombi formation. The arterioles and capillaries of the body become obstructed by the resulting complexes of activated platelets which have adhered to endothelium via large multimeric vWF Escherichia coli (Colon bacillus) Enterohemorrhagic E.coli (EHEC) Pathophysiology: Hemolytic Uremic Syndrome (HUS) The growing thrombi lodged in smaller vessels destroy red blood cells (RBCs) as they squeeze through the narrowed blood vessels, forming schistocytes, or fragments of sheared RBCs This mechanism, known as microangiopathic hemolysis. The consumption of platelets as they adhere to the thrombi lodged in the small vessels can lead to severe thrombocytopenia Kidney Failure – Due to reduced blood flow leading to Ischemia and also due to the direct damage caused by the toxin to the renal endothelial cells Thus the triad symptoms of HUS – thrombocytopenia, microangiopathic hemolytic anemia, and kidney failure. Escherichia coli (Colon bacillus) Enterohemorrhagic E.coli (EHEC) Laboratory Diagnosis Stool culture with serotyping Isolation using MAC using sorbitol instead of lactose Demonstration of a four-fold increase or greater in Shiga- toxin neutralizing antibody titer in patients’ sera with HUS Escherichia coli (Colon bacillus) Enterohemorrhagic E.coli (EHEC) Laboratory Diagnosis MUG test (4-methylumbelliferyl-β-D-glucoronide) Beta-glucuronidase 4-Methylumbelliferyl-β-D Glucuronide 4-methylumbelliferone Fuoresces blue under long- wave UV light (366 nm) EHEC O157:H7 rarely produces the enzyme, whereas 92% of other E.coli strains do produce it. Note: Sorbitol-negative and MUG-negative colonies are subcultured for serotyping using anti serum for the detection of E.coli O157:H7 Escherichia coli (Colon bacillus) Enteroaggregative E.coli (EAEC) Implicated in persistent watery diarrhea in infants in developing countries and in travelers to these countries Associated with chronic diarrhea and growth retardation in children Characterized by their auto-agglutination in a “stacked-brick”arrangement”. Escherichia coli (Colon bacillus) Enteroaggregative E.coli (EAEC) Pathophysiology The “stacked-brick” arrangement is mediated by aggregative adherence fimbriae I (AAF1) adhesin that are similar to the bundle-forming pilus (BFP) of EPEC. After adherence of EAEC to the surface of the intestine, mucus secretion is stimulated leading to the formation of a thick biofilm. Thick biofilm – protects the aggregated bacteria from antibiotics and phagocytic cells. Escherichia coli (Colon bacillus) Enteroaggregative E.coli (EAEC) Pathophysiology Two toxins are also produced: § Enteroaggregative heat stable toxin –antigenically related to the heat-stable toxin of ETEC § Plasmid encoded toxin Both toxins induce fluid secretion Watery diarrhea with no wbcs and rbcs present  Escherichia coli (Colon bacillus) Uropathogenic E.coli Most common cause of UTI, mostly occurring among females Originate from the large intestine as resident biota, contaminate the urethra, and ascend into the bladder and may migrate to the kidney or prostate Pili – primary virulence factor which uropathogenic strain used to adhere to epithelial cells and not be washed off with urine flow Cytolysins – characterized as a hemolysin that can lyse white blood cells and inhibit phagocytosis and chemotaxis Male Urethra (20cm [8inches]) Female Urethra (4-5cm [1.5 – 2 Aerobactin – allows uropathogenic strains to chelate iron inches]) Escherichia coli (Colon bacillus) Extra-intestinal Infections Meningitis – caused by E.coli (and also group B streptococci!) § Acquired in the birth canal during delivery § May also result if contamination of the amniotic fluid. Septicemia- originates from infections in the urinary tract or gastrointestinal tract Encapsulated strains of E.coli (possess K1 Ag) – mostly associated with neonatal meningitis and septicemia Escherichia coli (Colon bacillus) Extra-intestinal Infections Bacteremia – E.coli is a clinically significant isolate in blood cultures from adults. Bacteremia in adults may result from a urogenital tract infection or from GI source. Escherichia coli (Colon bacillus) Other Escherichia species E. hermannii E. vulneris § Formerly called E.coli atypical or § Isolated from humans with infected enteric group II wounds § Yellow pigmented colonies § Half of the strains produced yellow- § Isolated from CSF, wounds and blood pigmented colonies E. albertii § Associated with diarrheal disease in children Escherichia coli (Colon bacillus) Laboratory Diagnosis MacConkey dry pink colonies due to Eosin methylene blue colonies with XLD dry yellow colonies lactose fermentation greenish metallic sheen Escherichia coli Note: E.coli that produces CO2 gas as additional by product of General Biochemical Reaction CHO fermentation are called aerogenic. Anaerogenic E.coli – a type of E.coli that does not produce gas during fermentation. They are also lactose negative, and non- motile and previously been known as the “alkalescens- dispar” group. Note: Exception to these biochemical reactions is the EIEC strain which share similar biochemical reactions to Shigella species Kblebsiella General Characteristics Genus consists of several species: K. pneumoniae subsp. pnuemoniae, K. oxytoca, K. planticola, K. terrigena , K. pnuemoniae subsp. ozaenae and K. rhinoscleromatis, K.ornithimlytica, and K.terrigena Absence of motility – distinguishes Klebsiella spp., from other members of the family Enterobacteriaceae Usually found in the G.I tract; free living in soil, water and plants All are possess a polysaccharide capsule (encapsulated) Kblebsiella General Characteristics Capable of utilizing citrate as carbon source Do not produce indole (except K. oyxtoca!) and can grow in KCN broth They possess O and K antigens Unable to produce H2S Urease (+) and ornithine decarboxylase (-) All are lactose fermenter except K. ozaenae and K. rhinoscleromatis Further ferments glucose via the VP pathway Kblebsiella pneumoniae (Friedlander’s bacilli) Most common isolated species Disease Association Has a polysaccharide capsule – DISTINCT § Community – Acquired Pneumonia FEATURE (CAP), nosocomial § Confers protection against § Opportunistic infections among phagocytosis and antimicrobial newborns, elderly patients, and absorption. seriously ill patients. § Responsible for the moist, MUCOID, colonies § Wound infections, UTI and bacteraemia § Also tests positive for Neufeld Quellung test (capsular swelling) Kblebsiella pneumoniae (Friedlander’s bacilli) Carbapenemase – producing K.pneumoniae is an important cause of ventilator-associated pnuemonia. § Carbapenemase – these are versatile Beta- lactamases that can hydrolyze penicillins, cephalosporins, monobactams, and carbapenems § Renders many beta lactam drugs inactive Kblebsiella pneumoniae (Friedlander’s bacilli) Laboratory Diagnosis Gram stain – Gram negative bacilli Culture - large, moist, mucoid colonies on EMB, MacConkey and XLD Kblebsiella pneumoniae (Friedlander’s bacilli) Laboratory Diagnosis Growth in KCN using Moeller KCN Broth Base § Supplemented with potassium cyanide, is useful in differentiating organisms based on their ability to grow in the presence of cyanide. § Used for the differentiation of the members of Enterobacteriaceae on the basis of potassium cyanide tolerance. § Test is positive if there is marked turbidity in the tube. In negative tests, there is no growth. § Bacteria that tolerate KCN – Klebsiella, Citrobacter, Proteus § Bacteria that are inhibited by KCN – Salmonella, Shigella, Escherichia Kblebsiella oxytoca Causative agent of antibiotic-associated hemorrhagic colitis (AAHC). § Observed during therapy with amoxicillin-clavulanate, amoxicillin, penicillins, or ampicillin. § Sudden onset of bloody diarrhea a after 2 to 7 days of treatment with the oral antibiotics (mentioned in the previous bullet). § Antibiotic therapy disrupts the normal gut microbiota and promotes the overgrowth of Klebsiella oxytoca with subsequent production of cytotoxin called tilivallin that causes mitotic arrest of host cells and induces apoptosis. § AAHC resolves spontaneously within 2-3 days when the offending antibiotics are discontinued. Kblebsiella granulomatis Formerly called Donovania granulomatis then Calymmatobacterium granulomatis before being reclassified as Klebsiella granulomatis § Causes granuloma inguinale – a granulomatous disease affecting the genitalia and inguinal area. § Disease is called donovanosis § Transmitted through unprotected sexual intercourse § After prolonged incubation of weeks to months, subcutaneous nodules appear on the genitalia or in the genital area. Kblebsiella granulomatis Donovan bodies are rod-shaped, oval organisms that can be seen in the cytoplasm of mononuclear phagocytes or histiocytes in tissue samples from patients with granuloma inguinale. They appear deep purple when stained with Wright's stain. Illustration of a cell showing donovan bodies with bipolar Actual macrophage showing densities giving the bacteria the donovan bodies appearance of a closed “safety pin” Closed safety as reference for the description of donovan bodies Kblebsiella rhinoscleromatis Causes Rhinoscleroma a chronic granulomatous disease. It commonly affects the nasal cavity and nasopharynx, but it can also involve the larynx, trachea, bronchi, middle ear, and orbit. Acquired via inhalation of respiratory droplets containing the causative agents. Kblebsiella rhinoscleromatis Diagnosis of rhinoscleromatis depends on the identification of the pathognomonic Mickulicz cells (MCs) which is most prominent during granulomatous phase. Mickulicz cells Mickulicz cells (MCs)- foamy macrophages with numerous cytoplasmic vacuoles containing viable and nonviable Klebsiella bacilli Kblebsiella ozanae Causative agent of chronic atrophic rhinitis called ozena. Ozena is a disease of the nose in which the bony ridges and mucous membranes of the nose waste away Transmitted via person-to-person contact. Differential Biochemical Reactions for Klebsiella spp. Enterobacter General Characteristics Inhabits soil and water and, to a lesser extent, the large bowels of man and animals Often confused with Klebsiella in terms of growth in MAC Also share biochemical reactions with Klebsiella spp: § Citrate positive § Grows in KCN broth § Methyl Red test negative Enterobacter General Characteristics Growth on EMB – “fish eye” colonies Difference Klebsiella spp: § Positive for ornithine decarboxylase (Klebsiella are negative) § Positive for motility (Klebsiella are non-motile) Enterobacter on EMB – “FISH EYE” colonies Enterobacter Disease Association Enterobacter cloacae and Enterobacter aerogenes § Two common isolates from the genus. § Isolated from wounds, urine, blood, and CSF Pantoea (Enterobacter) agglomerans – gained notoriety with a nationwide (U.S) outbreak of septicemia resulting from contaminated IV fluids § LOA negative. A.K.A “triple decarboxylases” negative § Pantoea agglomerans HG XIII – produce a yellow pigment, a plant pathogen. Enterobacter Disease Association Enterobacter gergoviae – found in respiratory samples and is rarely isolated from blood cultures. Cronobacter (Enterobacter) sakazakii – produces a yellow pigment and has been documented as a pathogen in neonates causing meningitis and bacteremia often coming from powdered infant formula. § Isolated from cultures taken from brain abscesses, respiratory, and wound infections. Enterobacter hormaechei –isolated from human sources such as blood, wound, and sputum. Enterobacter asburiae – similar biochemically to Enterobacter cloacae and has been isolated from blood, urine, feces, sputum, and wounds. Enterobacter cancerogenus - formerly Enterobacter taylorae has been associated with osteomyelitis after traumatic wounds. Enterobacter Differential Characteristics Serratia General Characteristic Comprise of species that are opportunistic pathogens associated with outbreaks in health care settings. § Serratia marcescens § Serratia rubidea Pigment production at room temperature. “Prodigiosin” – pink-to-red in color § Serratia plymuthica § Serratia liquifaciens § Serratia odorifera – emits a dirty, musty odor resembling “rotten potatoes” § Serratia ficaria Note: All Serratia species except for Serratia fonticola are late lactose fermenters, positive for § Serratia fonticola ONPG. All are DNAse, Lipase, and Gelatinase positive (differential for other members of the § Serratia entomophila family Enterobacteriaceae. Serratia species are known for their resistance to a wide range of antimicrobials. All are sucrose fermenters Serratia Disease Association Serratia marcescens – the most clinically significant species § Frequently found in hospital-acquired infections of the urinary tract § Bacteremic outbreaks in nurseries and cardiac surgery and burn units. § Contamination of antiseptic solution used for joint injections has result in an epidemic of septic arthritis. Serratia odorifera biogroups § S.odorifera biogroup 1 – isolated predominantly from the respiratory tract and is positive for the fermentation of sucrose and raffinose, and is positive for ornithine decarboxylation § S.odorifera biogroup 2 – isolated from blood and CSF and is negative for the fermentation of sucrose and raffinose, and negative for ornithine decarboxylation. o 50% of isolates are indole positive. Serratia marcescens Laboratory Diagnosis Biochemical reactions TSI: A/A or K/A, H2S negative IMVIC: - - + + Urease: Negative Lipase: Positive DNAse: Positive Gelatinase: Positive MaConKey: Clear colonies (Lactose negative) Hafnia General Characteristics Delayed positive citrate reaction – MAJOR Hafnia alvei (Enterobacter alvei) CHARACTERISTICS OF Hafnia Resembles Enterobacter but differentiated by Clinical significance is still questionable its inability to ferment lactose, sucrose, despite being recovered from a number of sorbitol, and raffinose human sources, including stools and wounds. Differentiated from Serratia because it is Infrequent pathogen, but has been isolated in DNAse and Lipase (-) cases of bacteremia, respiratory tract infections, and gastroenteritis. Differential Reactions of Pantoea, Hafnia, and Serratia Tribe Proteeae General Characteristics Comprised of the genera: Proteus, Providencia, and Morganella (PPM) Widely distributed in the environment, are normal intestinal flora, and are recognized as opportunistic pathogens. Distinguished from other members of the Enterobacteriaceae by their ability to deaminate phenylalanine deaminase (PAD +) None of the members of this tribe ferment lactose Tribe Proteeae Genus Proteus Proteus mirabilis and Proteus vulgaris are widely recognized human pathogens Isolated from urine, wounds, ear, and blood Responsible for 3% of UTI in the U.S § Ascend the urinary tract causing infections both in the lower and upper tracts § Can infect proximal kidney tubules and can cause acute glomerulonephritis particularly in patients with urinary tract defects or catheterization § Urease activity of Proteus – lead to struvite Struvite Crystals – formed after the urine turns alkaline as a result of the conversion of kidney stones urea to ammonia by the bacterium’s urease enzyme. As urine pH increases, calcium and magnesium phosphates begin to precipitate out of solution, leading to the formation of struvite calculi (kidney stones) Tribe Proteeae Genus Proteus Proteus mirabilis and Proteus vulgaris easily identified by their swarming colonies on culture media. Swarming – results form a tightly regulated cycle of differentiation from standard vegetative cells (swimmers) to hyper-flagellated, elongated, polypoid cells (swarmers) capable of coordinated movement. Swarming ability – thought to play a role in the ascending nature of Proteus-associated UTI Swarmers – produce a distinct “burnt chocolate” odor Tribe Proteeae Genus Proteus Diene’s Phenomenon When different Proteus species swarm towards each other, a line of inhibited growth results where strains meet. This was first observed in 1946 by Dienes and is referred to as the Dienes phenomenon. This line of inhibited growth results from the production of and sensitivity to different types of bacteriocins, namely, proticines, produced by different strains of Proteus species. Tribe Proteeae Genus Proteus Differential Characteristics of Proteus Species Tribe Proteeae Genus Proteus Nice to Know Information Proteus antigens are used in the diagnosis of rickettsial disease Proteus antigens are cross-reactive with Rickettsia antigens Proteus antigens are used in the Weil-Felix test – an agglutination test to differentiate rickettsial disease. Note: Weil-Felix test is extensively Antigens: OXK, OX19, OX2 discussed in Immunology and Serology. You will learn more of this in your I.S class. Just wait J Tribe Proteeae Genus Providencia Providencia species are lactose-negative, deaminase (PAD) positive, H2S-negative, and motile Characterized by a pungent odor Ferment mannose, and do not swarm on SBA Five species: § Providencia alcalifaciens § Providencia heimbachae § Providencia rettgeri – the only urease positive § Providencia rustigianni § Providencia stuartii Tribe Proteeae Genus Providencia Disease Association Providencia species have been found in specimens from the urine, throat, stool, and blood and from wounds. P.rettgeri and P.stuartii have been associated with human infections. Associated with nosocomial infections of the urinary tract and the skin Providencia-associated UTI – involved patients with predisposing urological problems Providencia-associated skin infections – are often found in burn patients P.stuartii – associated with UTI in those with indwelling catheters Tribe Proteeae Genus Morganella Morganella morganii – only species within the genus Morganella Biochemical characteristics – NLF, negative reactions for citrate, H2S, and LDC. Urease and deaminase (PAD) positive. Opportunistic pathogen that causes gastrointestinal disease, urinary tract, and wound infections. Infections are more frequently seen in immunocompromised patients and those undergoing prolonged antibiotic therapy. More serious infections include septicemia and abscesses Tribe Proteeae Differential Characteristics Citrobacter General Characteristics Considered as inhabitants of the GIT tract and are associated with hospital-acquired infections, most frequently UTI. Three species most often isolated from diarrheal stool cultures § Citrobacter koseri § Citrobacter braakii § Citrobacter freundii – associated with infectious diseases in hospital settings such as UTIs, pneumonias, and intra-abdominal abscess. It is also associated with endocarditis in IV drug abusers. Citrobacter General Characteristics Motile, biochemically resembles Salmonella 80% Produce H2S (black colonies in HEA, SSA, TSI) 50% Fails to ferment lactose (colorless colonies on lactose containing media) Differentiated with Salmonella through: § Urea hydrolysis – 70% of C.freundii (+); Salmonella are negative § LDC - all species of Citrobacter are negative Edwardsiella General Characteristics Composed of three species: E. tarda; E. hoshinae; E. ictaluri All of them are negative for urea § Isolated from cold blooded animals hydrolysis including reptiles, freshwater and All of them are positive for: aquarium fish, frogs, and turtles. § Lysine decarboxylase § H2S, and indole E.tarda – the only recognized pathogen. All of them do not grow on Simmon’s § Opportunistic citrate § Isolated in GI infections Plesiomonas General Characteristics Formerly under the family Vibrionaceae Oxidase-positive (take note that Enterobacteriaceae are oxidase negative!), glucose-fermenting, facultatively anaerobic, gram-negative bacilli Phylogenetically closely related to Enterobacteriaceae, but it does not have the ability to produce gas from glucose Only oxidase-positive member of the family Enterobacteriaceae Plesiomonas General Characteristics Plesiomonas shigelloides – the only species in this genus. Does not form a capsule and are motile by monotrichous or two to five lophotrichous flagella (in contrast to other motile members of Note: Three major clinical types of Enterobacteriaceae that have peritrichous flagella) gastroenteritis caused by Plesiomonas: 1. Common watery or secretory diarrhea Plesiomonas and Shigella share biochemical and antigenic features 2. Subacute or chronic diseases that lasts from 14 days to 2-3 months 3. A more invasive, dysenteric form that Plesiomonas shigelloides– appear to have much lower virulence resembles colitis compared to Shigella spp. It has emerged as a potential cause of enteric disease in humans, most often after consumption of undercooked seafood or untreated water. References Mahon, C. R., Lehman, D. C., & Manuselis, G. (2018). Textbook of diagnostic microbiology-e-book. Elsevier Health Sciences. Delost, M. D. (2020). Introduction to diagnostic microbiology for the laboratory sciences. Jones & Bartlett Learning. Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2020). Medical microbiology E-book. Elsevier Health Sciences. Opportunistic pathogens 2 Broad Categories of Primary Pathogens Opportunistic Pathogen - generally harmless but cause harm if patients becomes immunocompromised Enterobacteriaceae Primary pathogens - cause infection virtually to all individuals regardless of age and immune status  IN MAC: Shigella Shigella - colorless colonies (non-lactose fermenters Escherichia - pink colonies (lactose fermenters) General Characteristics Closely related to Escherichia – both belong to the same tribe, Escherichieae can be differentiated based on colonies formed in MAC Shigella – not members of the GI microbiota, and are considered as “primary pathogens” All species causes dysentery (bloody diarrhea with mucus, blood, and leukocytes) Biochemical characteristics: § Non-lactose fermenters § Non-motile no flagella § Anaerogenic, except Shigella flexneri cannot produce CO2 as byproduct § Do not hyrdrolyse urea § Do not produce H2S Biochemically “inert” mostly negative results in biochem tests § Do not decarboxylate lysine Note: On selective and differential media used to § Do not use acetate or mucate as carbon source selectively isolate intestinal pathogens, Shigella appear as clear, non-lactose fermenting colonies. Shigella General Characteristics Shigella sonnei – unique member of the genus § Ability to ferment lactose slowly (LLF) producing pink colonies on MAC agar only after 48 hours of incubation Differentiate it with other members of the genus § ONPG positive has B-galactosidase (only in late lactose fermenters) Shigella species – fragile and susceptible to Note: Shigella are susceptible to the acid pH of various effects of physical and chemical the stool. Feces suspected of containing Shigella agents: spp., must be immediately inoculated to primary § Disinfectants culture media to increase recovery of the organisms. § High concentrations of acids and bile Shigella General Characteristics All Shigella species – possess O antigens, and certain strains possess K antigens K antigens (capsular) if present, interfere with the detection of the O antigen during serologic grouping. § May be removed by boiling the bacterial suspension to remove heat labile K antigen and to expose the heat stable O antigen. Non motile, do not have H antigen Shigella Serotypes based on their O antigen Subgroup A – S. dysenteriae (Dysentery bacillus) § Serotypes 1-10 § Ex. S.dysenteriae (shiga toxin) Subgroup B – S. flexneri (Strong’s bacillus) Subgroup C – S. boydii (S. ambigua) Subgroup D – S. sonnei LLF) Shigella Mode of Transmission Humans and other large primates are the only known reservoir! Mode of transmission: direct person-to-person contact, fecal-oral-route with carriers as the source. May also be transmitted by flies, fingers, and food and water contaminated by infected persons Personal hygiene plays a major role in the transmission Shigella Disease Association Shigella species cause all types of bacillary dysentery – painful type of diarrhea characterized by watery feces w/ mucus, blood streaks & pus cells Infective dose: as low as 100 bacilli are needed to initiate the disease in some healthy individuals. Predominant isolates: § S.sonnei – predominant in the U.S and other industrialized countries § S.flexneri – affects mostly young children. Leading isolate of gastroenteritis among men who have sex with men. HIV infections increases this risk. § S.dysenteriae type 1 and S.boydii are the most common isolates in developing countries. S.dysenteriae type 1 remains the most virulence of all the species with a significantly high morbidity and mortality rate. Shigella Pathophysiology Shigella – attach to and invade the M cells located in Peyer patches. The organism secretes four invasion-plasmid antigen (Ipa) proteins into epithelial cells and macrophages that induce membrane ruffling on the target cell, leading to engulfment of bacteria. These proteins include: § IpaA § IpaB § IpaC § IpaD  Shigella Pathophysiology Shigella – lyses the phagocytic vacuole and replicate in the cytoplasm. The rearrangement of actin filaments in the host cells propel the bacteria through the cytoplasm through the adjacent cells where cell- to-cell passage occurs. § Cell-to-cell passage protects Shigella from immune-mediated clearance. § They survive phagocytosis by inducing apoptosis. Apoptotic phagocytes release IL1B that acts as a chemoattractant for PMNs into the infected tissues. This destabilizes the integrity of the intestinal wall and allows bacteria to reach the deeper epithelial cells. PMNs - Polymorphonuclear neutrophils Shigella Pathophysiology Shigella dysenteriae – produces shiga toxins, similar to the verotoxins produced by EHEC. The B subunit binds to the host cell glycolipid Gb3 receptor and facilitate the transfer of the A subunit into the cell. The A subunit cleaves the 28S rRNA in the 60S ribosomal subunit, resulting in the disruption of protein synthesis resulting in cell damage and death. Note: High concentrations of Gb3 are found on intestinal villi and renal endothelial cells. This results in hemorrhagic diarrhea and hemolytic uremic syndrome (HUS) Shigella Laboratory Diagnosis Selenite broth – enrichment medium for the isolation of Salmonella spp., and Shigella species from heavily contaminated specimens such as stool. Primary culture media: § EMB eosin methylene blue agar § MAC MacConkey Agar § XLD Xylose-Lysine deoxycholate § SSA Salmonella-shigella agar § HEA Hektoen Enteric agar Salmonella General Characteristics Many Salmonella serotypes are typically found in cold-blooded animals as well as in rodents and in birds, which serve as their natural hosts. Gram-negative, facultative anaerobic bacilli. On selective and differential media – Salmonellae produce clear, colorless, non-lactose fermenting colonies. § Colonies with black centers are seen in media with sodium thiosulfate such as Note for the following exceptions: HEA, SSA, and XLD S.pullorum and S.gallinarum – Non-motile § Black precipitates indicate H2S production. All are NLF –except for Salmonella Arizona -LLF All are aerogenic – except Salmonella Typhi All are H2S positive –except Salmonella Paratyphi  Salmonella Antigenic Structures and Virulence Factors O Ag: Stable to boiling and acid alcohol Surface Ag’s - invasiveness (enables them to traverse the intestinal mucosa); H Ag: More stable endotoxin, enterotoxins, intracellular § O and H are the primary antigenic structures capability Vi Ag: Virulence Ag (K antigen); occurs in Salmonella Fimbriae – for adhesion serotype Typhi and a few strains of Salmonella serotype Cholerasius § Plays a role in preventing phagocytosis Salmonella Classification Formerly, there are only 3 known species: § S.enteritidis; S.cholerasius; S.typhi § S.enteritidis – also known as the Gardner’s bacillus; S.typhi formerly S.typhosa is also known as the Eberth’s bacillus Genetic studies have shown that the three previously named species are genetically related and that there are only 2 designated species; § S.enterica (Type species of the genus); and with 6 subspecies. § S,bongori (rare isolate) – initially isolated from a lizard and is usually isolated from cold-blooded animals and the environment. Salmonella Classification Note: Proper way of writing/naming. Type species is: Salmonella enterica. In writing/naming subspecies it should be: Salmonella enterica subspecies enterica The formerly known three species have been placed as seroytpe under the level of Salmonella enterica subspecies enterica. They must be written/named this way: Salmonella enterica subspecies enterica serotype Typhi Salmonella enterica subspecies enterica serotype Cholerasius Salmonella enterica subspecies entericia serotype Paratyphi Salmonella Disease Association and Pathophysiology In humans, salmonellosis occurs occurs in many forms: § Acute gastroenteritis or food poisoning characterized by vomiting and diarrhea § Typhoid fever, the most severe form of enteric fever, caused by Salmonella serotype Typhi, and enteric fevers caused by other serotypes such as Salmonella serotype Paratyhpi and Salmonella serotype Cholerasius. Mode of transmission – ingestion of the organism in food, milk, and water contaminated with human or animal excreta. § Except for Salmonella Typhi and Salmonella Paratyphi, Salmonella infect animals that serve as their reservoir § Salmonella Typhi and Salmonella Paratyphi have no known animal reservoirs and infections only occurs in humans. Carriers are often the source of infection. Salmonella Disease Association and Pathophysiology A. Gastroenteritis Source of infection have been attributed to poultry, milk, eggs, and egg products, as well as to handling pets. Insufficiently cooked eggs and domestic fowl, such as chicken, turkey, and duck and common sources of infection. Outbreaks associated with consumptions of foodstuffs such as peanut butter, cantaloupe, puffed rice and wheat cereals, corn-and vegetable-coated snacks, vegan cheese, alfalfa sprouts, cucumber, and raw tomatoes. Cross –contamination through cooking utensils also occur Salmonella Disease Association and Pathophysiology B. Enteric Fever Also known as typhoid fever – a febrile disease that results from the ingestion of food contaminated with organisms originating from infected individuals or asymptomatic carriers. Salmonella Typhi – does not have an animal reservoir and infection only occurs between humans. Other causes – Salmonella Paratyphi A, B,and C and Salmonella Cholerasius Salmonella Disease Association and Pathophysiology B. Enteric Fever 1st Week of infection When organisms are ingested – they are resistant to gastric acid Upon reaching the proximal end of the small intestine, they invade and penetrate the intestinal mucosa – patients suffer constipation instead of diarrhea Organisms gain entrance to the lymphatic system and are sustained in the mesenteric lymph nodes. They eventually reach the blood stream and spread to the liver, spleen, and bone marrow, where they are immediately engulfed by mononuclear phagocytes. Organisms multiply intracellularly inside phagocytes and are released in the blood stream for the second time. This results to febrile episode and the organism can be easily isolated from blood Salmonella Disease Association and Pathophysiology B. Enteric Fever 2nd and 3rd Weeks of Infection Patient generally sustained fever with prolonged bacteremia The organism invade the gall bladder and the Peyer’s patches of the bowel. They also reach the intestinal tract via the biliary tract. “Rose-spots” - blanching rose-colored papules around the umbilical region also appear during the second week of fever. Involvement of the biliary system initiates GI symptoms as the organisms reinfect the intestinal tract. The organism now exists in large number in the bowel and may be isolated from the stool Salmonella Disease Association and Pathophysiology B. Enteric Fever Asymptomatic Carriers For asymptomatic carriers, the gallbladder becomes the foci of long- term carriage of the organism From the gallbladder of carriers, the organism is occasionally/intermittently shed in the feces which then result in the spread of infection via fecal-oral route. They become important sources of infections for susceptible persons. Salmonella Specimen Collection and Laboratory Diagnosis Blood – 1st and 2nd week Urine/Stool– 3rd and subsequent weeks Stool – can be used indefinitely as clinical specimen NOTE: Culture is the best diagnostic procedure to identify Salmonella Salmonella Specimen Collection and Laboratory Diagnosis Gram stain – Gram negative bacilli Culture: § EMB/MAC – colorless § SSA – colorless-black center (H2S+) § NA – maple leaf colonies w/irregular margins § XLD – slightly pink to white opaque colonies S. Typhi: “Metallic colonies with a black ring” on BSA BSA Salmonella Specimen Collection and Laboratory Diagnosis Widal Test Serologic test that detects the presence of antibodies in the person’s serum against O and H antigens of Salmonella Typhi. Agglutination test. Performed on a slide (qualitative) and in a tube (quantitative) Rapid Slide Method for Widal Test Salmonella Specimen Collection and Laboratory Diagnosis Widal Test Serologic test that detects the presence of antibodies in the person’s serum against O and H antigens of Salmonella Typhi. Agglutination test. Performed on a slide (qualitative) and in a tube (quantitative) Tube Method to Determine Antibody Titer to Salmonella Antigens The titer is the highest dilution that gives a visible agglutination Salmonella Specimen Collection and Laboratory Diagnosis Widal Test Interpretation: Titer of 1:100 or more for O agglutinins and 1:200 or more for H agglutinin should be considered clinically significant as these indicate active infection. Tube Method to Determine Antibody Titer to Salmonella Antigens The titer is the highest dilution that gives a visible agglutination Yersinia General Characteristics Three human pathogens: § Yersinia pestis – causative agent of plague § Yersinia pseudotuberculosis Implicated in sporadic cases of gastroenteritis, mesenteric lymphadenitis especially § Yersinia enterocolitica in children, and generalized septicemic infections in immunocompromised hosts. Yersinia enterocolitica – has been a cause of diarrhea outbreaks in numerous communities and it has been seen to mimic appendicitis. MAC – colonies are small, pinpoint colonies; Non-motile at 37C, but motile at 25C, except Yersinia pestis. Optimal growth is at 25—30C TSI – Acid/orange (A/K) in 24 hours due to weak acid reaction in the slant with no change in the butt – suspected of Yersinia Yersinia pestis The causative agent of plague – a primary disease in rodents that are spread to humans by bites of fleas. Xenopsylla cheopis (oriental rat flea) – flea vector (most common and effective vector of Yersinia pestis) § Parasite of the rodent Rattus rattus – a black rat, as the main rodent host of Yersinia pestis Yersinia pestis Yersinia pestis (Plague bacillus) Microaerophilic, gram negative, short, plump bacillus Shows marked bipolar staining when stained with “Wayson Stain”- cells having a characteristic “Safety Pin” appearance Yersinia pestis In culture, the organism prefers a temperature between 25C to 30C, though it can still grow at 37C. Non-motile both at 25C and at 37C Catalase positive Oxidase negative Yersinia pestis Plague – occurs in three distinct forms: Bubonic plague – most common form of the disease, results from the bite of a flea, or by direct inoculation of an open skin would by plague-infected material. Buboes, lymph nodes filled with inflammatory cells that can ulcerate, can be several in diameters in centimeters. Yersinia pestis Plague – occurs in three distinct forms: Secondary septicemic plague – results from the hematogenous dissemination of the organism, following the damaged of the lymph nodes in patients with bubonic plague. § Primary septicemic plague – occurs in patients without buboes as primary infections. Symptoms – similar to bubonic plague, but then progressively worsen as the bacterial endotoxins set off an immunologic cascade of events, ultimately leading to multiple organ failure, respiratory distress, and DIC, noted by petechiae and gangrene in the extremities (fingers, toes, and noes). Yersinia pestis Plague – occurs in three distinct forms: Secondary pneumonic plague – results form the dissemination of the organism to other organ systems such as the lungs, brain, liver, and spleen, with days- near and hemoptysis, plague meningitis, and hepatic and splenic abscesses. Individuals are extremely infectious and should be isolated and respiratory precautions should be followed to reduce the spread of the disease. If Yersinia pestis is to be weaponized – its optimal release would be via aerosols, and most patients would develop primary pneumonic plague. Yersinia enterocolitica Found in a wide variety of animals, including domestic swine, cats, and dogs. Mode of transmission – contact with household pets. Transmission via pigs has been greatly emphasized in Europe Human infections – most often occurs after the ingestion of contaminated food, often pork, and vacuum packed deli meat, lamb, chicken, and possibly milk and water. The ability of the organism to survive in cold temperatures – is a potential risk of transmitting infections via consumption of refrigerated foods (refrigeration is ineffective as a measure to control the infection) In transfusion medicine, reports of the transmission of Yersinia enterocolitica from contaminated packed red blood cell components have been recorded. Yersinia enterocolitica Clinical manifestations: § Acute gastroenteritis § Appendicitis – like syndrome § Less frequent manifestations such as septicemia, arthritis, and erythema nodosum. Erythema Nodosum Yersinia enterocolitica Resembles other Yersinia species Gram-negative coccobacilli, with bipolar staining Optimal growth at 25C-30C Clearly motile at 25C, but non-motile at 35C. Cold-enrichment – used to increase the recovery in fecal samples. Yersinia enterocolitica Cold enrichment of fecal specimens for Yersinia enterocolitica a. Fecal material is inoculated into isotonic saline and kept at 4C for 1- 3 weeks with weekly subculture to selective agar for Yersinia. b. Subculture can be performed using Cefsulodin –Irgasan-Novobiocin (CIN) – selective for Yersinia that contains cefsulodin, irgasan, novobiocin, bile salts, and crystal violet as inhibitory agents c. CIN – better than MAC in inhibiting normal colon microbiota and provides better opportunity for the recovery of Yersinia enterocolitica in feces. “Bull’s Eye” colonies of Yersinia enterocolitica on CIN medium. Results from the fermentation of mannitol and d. YSA – Yersinia-selective agar, a modified version of CIN that contains production of acids that cause the pH indicator neutral to turn red at the center of the colony and precipitate bile. mannitol as differential agent. Yersinia pseudotuberculosis Similar to Yersinia pestis for being a disease primarily of rodents, particularly guinea pigs. Birds are natural reservoirs. Disease is characterized by caseous swelling called “pseudo- tubercles” Human infections are rare, and are associated with close contact with infected animals, or their fecal material, or ingestion of contaminated drink or foodstuff. Morphology – resembles Yersinia pestis Differences from Yersinia pestis – motility at 18C – 22C, urease production, and ability to ferment rhamnose. Yersinia Differential Characteristics Laboratory Diagnosis of Enterobacteriaceae Specimen collection, transport, and Direct Microscopic Examination processing A. Generally gram-stain is not valuable since Enterobacteriaceae are indistinguishable A.Isolated from wide variety of clinical B. Gram stain – must be performed on specimens specimens from sterile sites such as CSF, B.Transport media to ensure isolation other body fluids or exudates. of opportunistic and fastidious C.Stool - gram stain is not helpful in the I.D, but Enterobacteriaceae must be used may reveal the presence of white blood such as: Cary-Blair, Amies, or cells that may help in determining Stuarts whether the GI disease is toxin mediated or an invasive process. Laboratory Diagnosis of Enterobacteriaceae Direct Microscopic Examination Culture A. Generally gram-stain is not valuable since A.Enterobacteriaceae are facultative Enterobacteriaceae are indistinguishable anaerobe and grow at optimal B. Gram stain – must be performed on temperature of 35C-37C specimens from sterile sites such as CSF, B.Some species require lower temperatures other body fluids or exudates. C.Stool - gram stain is not helpful in the I.D, but (1C – 5C for Serratia and Yersinia) may reveal the presence of white blood C.Routine primary media include: SBA, cells that may help in determining CHOC, and selective media such as whether the GI disease is toxin mediated MAC or an invasive process. Laboratory Diagnosis of Enterobacteriaceae Screening for Stool Cut-lures for Pathogens A.Fecal specimens should be routinely screened for Salmonella, Shigella, and EHEC B.Protocol must be in place for Yersinia (such as cold enrichment) C.Salmonella should be suspected if a lactose-negative, H2S positive colonies are isolated from stool culture. D.Inoculate stool samples on highly selective media such as HE, XLD in addition to MAC and SMAC E.Enrichment broth (selenite, tetrathionate broth, GN broth) – may be used to enhance recovery especially of Salmonella and Shigella Brief Run Through of the Algorithm for the Identification of Enterobacteriaceae Note: 1. Oxidase test is a presumptive Colony Morphology and Determination of test that differentiates Pseudomonas spp. and Vibrio its reaction with lactose (LF, NLF, LLF) spp. which are oxidase positive from Enterobacteriaceae which are oxidase negative. 2. A gram-negative bacilli primary isolated from MAC that tested negative for oxidase is presumptively identified as The colony examined for its morphology and Enterobacteriaceae. reaction to lactose is tested for Oxidase test. 3. An exception to rule #2 is Based on the ability of bacteria to produce Plesiomonas spp which is an Cytochrome C Oxidase that oxidizes the oxidase positive member of the oxidase reagent in the presence of family Enterobacteriaceae atmospheric oxygen to produce indophenols which are purple or dark-blue end products. Brief Run Through of the Algorithm for the Identification of Enterobacteriaceae Triple Sugar Iron test MR and VP (IMViC) test CHO utilization patterns A gram-negative, oxidase ONPG test for LLF negative isolate on MAC will be Lysine Iron Agar test further identified based on a Action towards amino acids (deamination/decarboxylation) series of biochemical tests. LOA Reaction using Moeller Decarboxylase test Phenylalanine deaminase test H2S production Other characteristics Motility, Indole production from tryptophan Citrate utilization, gelatin hydrolysis, urea hydrolysis Nitrate reduction Schematic Diagram for the Identification of Enterobacteriaceae References Mahon, C. R., Lehman, D. C., & Manuselis, G. (2018). Textbook of diagnostic microbiology-e-book. Elsevier Health Sciences. Delost, M. D. (2020). Introduction to diagnostic microbiology for the laboratory sciences. Jones & Bartlett Learning. Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2020). Medical microbiology E-book. Elsevier Health Sciences. https://labpedia.net  Non-fermentative Gram-Negative Bacilli  LEARNING OBJECTIVES At the end of this unit, the student should be able to: 1. Characterize the significant species of NF Gram negative bacilli, their general properties, and virulence factor; 2. Describe and explain the clinical significance and pathogenesis of NFGNB; 3. Distinguish features of NFGNB for accurate laboratory diagnosis and determine possible treatments. TOPIC Non-fermentative or Oxidative GNB üOxidative metabolism üMolecular oxygen as final hydrogen acceptor üForms very weak acids Other GNB Non-saccharolytic or non-oxidizers üDo not utilize carbohydrate (with or w/o O2) üOther compounds as source of energy  Gram-Negative Bacilli Oxidase Test Oxidase positive Oxidase Negative O/F O/F O+/F+ O+/F- O+/F+ Enterobacteriaceae Pseudomonadaceae Vibrionaceae  CHARACTERISTICS OF NON-FERMENTERS Non-spore formers Obligate aerobes TSI: ______________ Point of colonization: mucus membrane & skin of humans Entry to sterile sites of the body: trauma, burns, or wounds Opportunistic pathogens  IDENTIFICATION OF NON-FERMENTERS üOxidase positive GNB üGrow better in SBA than on MacConkey agar (+/-) üTSI – K/K Tests: qFlagellar morphology q Nitrate reduction qModified indole test q Acetamide qGrowth on MacConkey q Urease qMotility q Decarboxylase reactions qO-F CHO utilization patterns  Oxidation-Fermentation (O-F) Test HUGH & LEIFSON PURPOSE: § To determine whether an organism can utilize CHO oxidatively or a fermentatively. § High conc’n of CHO (1%) and a low conc’n of peptone or protein (0.2%). § __:__ peptone to CHO concentration COMPOSITION: § OF Basal media § CHO: Glucose, Maltose, Lactose, Mannitol, & Xylose § Bromthymol blue = indicator  Oxidative-Fermentation Reaction Alcaligenes faecalis Pseudomonas Enterobacteriaceae OPEN tube CLOSED tube RESULT 1. Green / Blue Green Green / Blue Green Non-saccharolytic 2. Yellow Green / Blue Green ? Oxidative 3. ? Yellow ? Yellow Fermentative  Nitrate Reduction Test Nitrate Reductase Further reduction Nitrogen gas Nitrate broth Nitrite (NO2) N2 (NO3) Sulfanilic acid α-naphthylamine If no red color Red diazonium salt (RED COLOR) =Add _______ zinc dust = Positive for Nitrate reduction NO RED color à Positive for NO3 reduction W/ RED color à Negative for NO3 reduction  Nitrate Reduction Test: Procedure POSITIVE Red color NB 10 drops Sulfanilic acid Incubate at 10 drops a-naphthylamine 37oC for 24 hrs or upto 48 hrs NEGATIVE NEGATIVE Add zinc dust No red color POSITIVE  FAMILY Pseudomonadaceae (NFGNB) rRNA Homology Group & Genus and Species subgroup I. Fluorescent Group Pseudomonas aeruginosa (pyoverdin & pyocyanin) P. fluorescens (pyoverdin) P. putida (pyoverdin) Non-fluorescent Group P. stutzeri (Soil denitrifiers) P. mendocina II. Pseudomallei Group Burkholderia pseudomallei B. mallei Resistant to polymyxin B and B. cepacia colistin *Ralstonia pickettii III. *Comamonas species *Acidovorax species IV. *Brevundimonas species V Stenotrophomonas maltophilia  Pseudomonas aeruginosa (PAE) Not a major normal flora BUT… most frequently isolated N-F Found in environment Can survive in harsh environment Major human pathogen in the group most important opportunistic pathogen Invasive and toxigenic  Pseudomonas aeruginosa (PAE) Obligate aerobe; motile Pigments: ________, __________, ________, _________, & ________ Fruity odor or corn tortilla-like odor (______________) Grow at RT°, 35-37°C and at 42°C  Pseudomonas aeruginosa: LABORATORY TESTS § Skin lesions, pus, urine, blood, spinal fluid, sputum, etc § Smears: Gram (-) rods § Culture: ú BAP à large, rough, dull grayish, feathered edged/ serrated and spreading colonies & mostly are beta hemolytic; Mucoid (capsular alginate) ú Mac & EMB à NLF § Biochemical test: § TSI: K/K § Oxidase test (+) § Oxidative Fermentation (G,F, X but not M,S,L) § Nitrate reduction test: (+)  LAB TESTS REACTIONS of PAE 1. MacConkey NLF 2. TSI K/K 3. Pyocyanin & Pyoverdin Positive 4. O-F Medium glucose, fructose and xylose (+); maltose, sucrose and lactose (negative) 5. Oxidase Positive 6. LOA (Lysine, Ornithine, Arginine) Negative, Negative, Positive 7. Acetamide Utilization Positive 8. Cetrimide test Positive 9. Nitrate Reduction Positive 10. Citrate Positive 11. Urease Variable 12. Indole Negative 13. Growth at 42 C Positive 14. Motility Motile with one flagella 15. Kanamycin Resistant 16. Carbenicillin Susceptible  Infections Associated with PAE Nosocomial or HAI: Severe wound infections in burn patients UTI Pneumonia (ventilator- associated) Septicemia Chronic lung infections in CF patients (mucoid strain) Meningitis Ecthyma gangrenosum  Infections Associated with PAE Community Acquired: keratitis corneal ulcers in contact lens wearers Swimmer’s ear Folliculitis (“hot tub folliculitis”)  Pseudomonas aeruginosa: VIRULENCE FACTORS Attachment Tissue Invasion ______________ fimbriae Elastase ______________ polar flagella Protease Collagenase Lipopolysaccharide endotoxin Hemolysin effect & Exotoxin Leukocidin Alginate capsule Pyocyanin Pyoverdin Exotoxin A Type III secretion system Exotoxin S,T,U,Y Pseudomonas aeruginosa: TREATMENT Single drug therapy is not recommended. Example: Penicillin based drug + aminoglycoside Beta lactam drugs Carbapenems Fluoroquinolones Do AST  P. fluorescens & P. putida Fluorescent pseudomonads Can grow at 4ºC associated w/ Nosocomial infections (transfusion associated sepsis) rarely associated with opportunistic infxns. P. fluorescens Contaminant Rare cause of UTI & wound infxns in humans P. putida Isolated cases of septicemia, UTI, & pneumonia  P. fluorescens & P. putida Laboratory Characteristics Motile Cetrimide & Acetamide: (-) Growth at 35ºC but not at 42ºC Arginine: (+) Oxidase (+) Resistant to Carbenicillin OF medium: oxidize Glucose Susceptible to Kanamycin Pyocyanin: (-) Pyoverdin: (+) P. fluorescens P. putida Protein Metabolism ? Proteolytic Non-proteolytic Gelatin Hydrolysis Positive ? negative Non-Fluorescent Group: P. stutzeri & P. mendocina “Soil denitrifiers” à uses NH4 (source of _____) nitrogen and acetate (source of _____) carbon Rare cause of eye infections, endocarditis, septic arthritis, postsurgical wound infxn, UTI, septicemia, and pneumonia Motile Grows at 42ºC P. stutzeri Colonies: dry and wrinkled w/ buff or light brown color ADH ( -? ) & starch hydrolysis (? ) P. medocina Colonies: smooth, buttery, flat, unwrinkled on BAP ADH (?+) and starch hydrolysis (-)  Burkholderia 1950: Walter H. Burkholder previously part of Pseudomonas Ubiquitous GNB; obligate aerobes Motile (EXCEPT: B. __________) mallei Pathogenic Species: B. mallei à agent of glanders dse B. pseudomallei à agent of melioidosis B. cepacia complex B. cepacia à pulmonary infection (CF) Burkholderia mallei Glander’s disease Rare human transmission direct contact via skin abrasions, inhalation, & mm of eyes & nose Lab Diagnosis: 1. Non-motile 2. Resistant to polymyxin B 3. weakly oxidase positive 4. Fails to grow at 42ºC 5. Smooth, cream to white on BAP Burkholderia pseudomallei agent of Melioidosis or Whitmore’s disease üAggressive granulomatous pulmonary disease à lead to septicemia LAB DIAGNOSIS: SBAP: wrinkled, cream-tan colonies Ashdown medium àdeep pink colonies with “________” earthy odor üCrystal violet & gentamycin=__________________ Ashdown medium becomes selective media ü4% glycerol üNeutral red -pink color by the colonies Oxidase (+), motile (lophotrichous flagella Arginine (+) OF medium: oxidizes glucose, maltose, lactose, & cellobiose  Burkholderia cepacia § Cause of _______ foot rot in humans § Opportunistic & nosocomial LAB DIAGNOSIS: § Motile w/ polar flagella § Yellow serrated colonies § Weakly oxidase (+), Arginine (-) & lysine (+) § OF : oxidizes glucose, maltose, lactose & mannitol TREATMENT: § sulfamethoxazole, chloramphenicol, or 3rd gen cepahlosporins Stenotrophomonas (Xanthomonas) maltophilia Colonize: respiratory, urinary, genitourinary tracts Nosocomial Infection: pneumonia, septicemia, UTI, & meningitis Lab Diagnosis: vMotile vLarge, smooth, glistening colonies with a yellow to tan pigment on TSA vNo pyocyanin & pyoverdin; _________-like Ammonia odor vLavander-green to light purple pigment on SBA; Grow at 42 C vOF medium: weak oxidizer of glucose, strong (maltose) v Oxidase (_____); negative Dnase (______); positive Nitrate not reduced Treatment: vSusceptible to trimethoprim-sulfamethoxazole  Lab Diagnosis PAE P. fluorescens P. mendocina P. putida P. stutzeri B. cepacia B. pseudomallei S. maltophilia Oxidase + + + + + Weak + + - Motility + + + + + + + + Pyoverdin + + - + - - - - Oxidation of: Glucose + + + + + + + Weak + Maltose - V - V + + + Strong + Lactose - - - - - + + + Mannitol + + - - V + + - Arginine + + + + - + + - Lysine - - - - - + - Slow + NO3 to No2 + V + - + V + V NO3 to N V - + - + - + - DNase - - - - - - - + Polymyxin S S S S S S R R R  Acinetobacter Nosocomial & opportunistic pathogen Infection: UTI, RTI, wound infxn, bacteremia, & meningitis “Mima” à mimic appearance of Neisseria (Do Oxidase Test) 2 most common human pathogen: üA. baumannii üA. lwoffii LAB DIAGNOSIS: Obligate aerobes; Oxidase (-); catalase (+); nonmotile coccobacilli Grows on EMB, Mac & SBA Resistant to penicillin  Differentiation of Acinetobacter species A. baumannii A. lwoffii Oxidase Negative Negative Growth at 42ºC Positive Negative Lysine decarboxylase Negative Negative CHO utilization Glucose Ferments rapidly Asaccharolytic Sucrose Negative Negative Lactose assimilation Rapid assimilation Negative Fluorescence-Lactose Positive Negative Denitrification (FLN) Acid  Alcaligenes Tiny GNB A. faecalis A. xylosoxidans Motile Oxidase Positive Positive A. faecalis Motility + (Peritrichous) + (Peritrichous) Odor: ______________ fruity odor - apples or pears MacConkey agar Growth Growth Opportunistic infxn OXIDATION Lab Diagnosis: Glucose Negative Positive Oxidase & catalase (+) Xylose Negative Positive Grow on MacConkey NO3 to NO2 Negative Positive SBA: Flat, dull colonies w/ irregular margin NO3 to N Negative Variable NO2 to N Positive Negative Growth in 6.5% NaCl Positive Negative Vibrio, Campylobacter, Helicobacter, Aeromonas & Plesiomonas  LEARNING OBJECTIVES At the end of this unit, the student should be able to: 1. Characterize the significant organisms under Vibrio, Campylobacter, Helicobacter, Aeromonas, & Plesiomonas. 2. Describe and explain the clinical significance and pathogenesis; 3. Distinguish features of the organisms under this topic for accurate laboratory diagnosis.  VIBRIOS Natural habitants of sea water (halophilic) Except: _____________________ v. cholerae and V. mimicus MOT: drinking contaminated water or seafood Isolated from GIT; blood and wound infections Motile, GNB comma-shaped, or curved rods Prolonged culture: straight rods Facultative anaerobes or aerobic Oxidase (+) EXCEPT: __________ fermentative VIBRIOS Culture Media Alkaline Peptone water Incubate 5 – 8 hrs at 35ºC à subculture to TCBS Thiosulfate citrate bile salts sucrose (TCBS) agar sucrose, oxgall, sodium cholate, bromothymol blue, thymol blue Sucrose Positive Sucrose Negative pH (8.6) V. cholerae V. parahaemolyticus Color:____________ V. alginolyticus V. vulnificus V. fluvialis V. mimicus V. furnissii  Vibrio cholerae Cause cholera MOT: Ingestion of contaminated seafoods & drinking contaminated water halotolerant Ferment sucrose Antigenic Structure & Biologic Classification flagellar (H) antigen Somatic (O) Ag O1 strain (epidemic & pandemic) & non-O1 (cholera like dse) V. cholerae O1 (3 serogroups): Ogawa, Inaba, Hikojima Vibrio cholerae Antigenic Structure & Biologic Classification Epidemic strainà categorized into 2 biotypes: Classical & El Tor Classic El Tor RBC Hemolysis Nonhemolytic Beta Hemolytic VP Negative Positive Polymyxin B (50 U) Susceptible Resistant Agglutination of chicken RBC Negative Positive Clinical Significance Cholera (First 6 Pandemics = Cholera 1817; 1829; 1846; 1863; 1881, 1899) Seventh Pandemic 1961 V. cholerae O139 is very similar to V. cholerae O1 El Tor biotype O1 Ag Polysaccharide Capsule V. cholerae O139 - + V. cholerae O1 + - Vibrio cholerae PATHOLOGY & PATHOGENESIS pathogenic only for humans Normal gastric acidity ID = > 1010 or more V. cholerae in water = < 102 – 104 organism in food PATHOGENESIS: Severe diarrhea: choleragen (potent enterotoxin) à bowel mucosa à outpouring of water & electrolytes (rice-watery stool) à severe dehydration à severe muscle cramping and anuria à death V. cholerae produces heat labile enterotoxin (A & B subunits) A subunit causes Î level of cAMP Diarrhea = dehydration, shock, acidosis, and death.  Vibrio cholerae DIAGNOSIS: ¡ SPECIMEN: stool; rectal swab ¡ SMEAR: GNB; comma shaped ¡ CULTURE: § peptone agar; BAP w/ pH 9, § TCBS – smooth, yellow colonies § STRING TEST (0.5% ________________) § à viscous string § SEROTYPING: § Serogroups O1 and O139 à cholera epidemics § Biochemical reaction patterns Vibrio cholerae TREATMENT: Water And Electrolyte Replacement Oral Tetracycline Note: Some V. cholerae acquired resistance already (Transmissible Plasmids) Perform AST Differentiation of Clinically Significant VIBRIO Organism Halo- Sucrose ADH Cello- VP Clinical Significance philic Ferm biose V. cholerae - + Epidemic & Pandemic Cholera “Asiatic cholera” V. parahaemolyticus + - - - Gastroenteritis (ingestion of contaminated food) V. vulnificus + - - + Septicemia & wound infxn (marine envi) V. mimicus - - - Gastroenteritis & ear infx (marine environment) V. anginolyticus + + - - + Wound & ear infxn (marine environment) V. fluvialis + + + - Gastroenteritis & diarrhea (marine environment) V. furnissii + + + - Gastroenteritis & diarhhea (marine environment) Vibrio parahaemolyticus à Kanagawa phenomenon àKanagawa toxin, a thermostable hemolysin à _____________ agar  Aeromonas Found in both fresh and seawater gastroenteritis, cellulitis & wound infection Lab Diagnosis: Mac, EMB, SSA, CIN medium fermentation of glucose & indole production Motile: monotrichous A. hydrophilia Heat-labile enterotoxin & heat stable cytotoxic enterotoxin Produce protease, lipase, and nuclease enzymes  Plesiomonas Motile, straight, round GNB Now categorized as enteric bacteria P. shigelloides Infection: gastroenteritis Mild with no blood or mucus in stool MOT: ingestion of contaminated food Lab Diagnosis: BAP, NLF on MacConkey & EMB Oxidase & Indole (+)  CAMPYLOBACTER Campylobacter jejuni Campylobacter coli Motile, microaerophilic and capnophilic Catalase & oxidase (+) Do not oxidize & ferment CHO GNB with comma, S, or “_______” shapes Disease: Gastroenteritis & diarrhea Guillain-Barré syndrome Guillain-Barré syndrome à an autoimmune disorder MOT: Ingestion of contaminated materials, direct resulting from cross-reactivity contact of Campylobacter antibodies with the nerve ganglia Campylobacter PATHOGENESIS Virulence Factor: Cytotoxin, cytotoxic factor and enterotoxin Organism multiplies in the SI invade the epithelium inflammation appearance of blood cells in stool Sometimes bloodstream is invaded & cause endocarditis, septic arthritis and meningitis  Campylobacter LABORATORY DIAGNOSIS SPECIMEN: STOOL SMEAR: “gull-wing” shaped rods CULTURE: Campylobacter blood agar (with Vancomycin, polymyxin B, __________, _______ & Amphotericin B) Skirrow’s medium (Vancomycin, polymyxin B, trimethoprim) Incubated at reduced atmospheric O2 (5% O2) w/ 10% CO2, 85% N Optimal Temperature for growth: 42 to 43°C Gray to pink; yellow to gray colonies, nonhemolytic and slightly mucoid  HELICOBACTER Previously: Campylobacter pylori Characteristics: Small, curved GNB sometimes U form microaerophilic Requirement: Oxygen (5% O2) w/ 10% CO2, 85% N @ 35-37C can’t grow at 42C motile Strong producer of __________ Oxidase and catalase (+); nitrate (-) Helicobacter pylori PATHOGENESIS Helicobacter pylori CLINICAL SYNDROMES Chronic gastritis Duodenal ulcer Gastric biopsy for histologic examination  Helicobacter pylori LABORATORY DIAGNOSIS MICROSCOPY: Special silver stain or Modified Giemsa GOLD STANDARD: Histological staining & culture of biopsy from stomach or duodenum Tissue (mashed) à plated on SBAP = incubate at 37ºC in microaerophilic & increased humidity environment Colonies: small, gray, translucent, slightly beta hemolytic BIOCHEM: strong & very rapid urea production– strongly positive Helicobacter pylori DIAGNOSIS Serum = determination of serum IgG antibodies Stool antigen tests = ELISA Carbon urea breath test = detects urease Endoscopy àto provide sample for testing for: biopsy microbial culture Helicobacter pylori CHEMOTHERAPY Triple therapy ____________ + bismuth subsalicylate or bismuth subcitrate + amoxycillin or tetracycline 14 days Acid suppressing agent given 4-6 wks Proton pump inhibitor Differentiation of Campylobacter & Helicobacter species C. jejuni subsp. C. coli C. fetus subsp. fetus Helicobacter pylori jejuni Catalase + + + + Nitrate + + + - Urease - - - ++ Hippurate + - - - hydrolysis Growth at 25C - - + V Growth at 37C + + + + Growth 42C + + - V Susceptible to: Nalidixic acid S S V R Cephalothin R R S S  Finals Week