CLINICAL BACTERIOLOGY FINALS BACTELEC PDF

CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS LESSON 1: FAMILY ENTEROBACTERIACEAE GENERAL CHARACTERISTICS SEROTYPING (SEROGROUPING) OF ENTEROBACTERIACEA ROUTE OF...

CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS LESSON 1: FAMILY ENTEROBACTERIACEAE GENERAL CHARACTERISTICS SEROTYPING (SEROGROUPING) OF ENTEROBACTERIACEA ROUTE OF INFECTIONS Often referred as ENTERICS - Consists of Serotyping - a serologic test that determines species/subtypes of bacteria based Infections are associated with a large number of diverse organisms on the antigens present on its surface. lapses in personal hygiene via: (Frequently encountered in the lab) Normal flora of the Gl tract of animals and Antigens - substances (proteins) that react with specific antibodies ○ Fecal-oral route — humans Most Common! Gram negative bacilli or coccobacilli Antibodies (anti-sera) - proteins that react to antigens. ○ Poor sanitation in Associated in infections almost throughout impoverished countries the body. Antigenic variations - refers to the variations in the antigenic structures of ○ Colonization of the skin Ubiquitous antigens. and respiratory tract of hospitalized patients All Enterobacteriaceae are: Note: (nosocomial) ○ Facultative anaerobe, non-spore-former 1. Salmonella/Escherichia/Shigella species may look the same under the Common types of infections ○ Glucose fermenters microscope, but they can be separated into different many serotypes include: ○ Cytochrome oxidase negative according to the variations of the antigens found on their surface. UTI, Gastroenteritis, Septicemia ○ All reduce nitrates to nitrites except: 2. Serotyping is determined by the agglutination of bacteria with specific Erwinia, Pantoea agglomerans, antisera to identify variants of O and H antigens. FACTORS AFFECTING Photorhabdus, and Xenorhabdus VIRULENCE All members are motile except: The organisms ability to cause disease is influenced by their ○ Klebsiella and Shigella - NM @ 37 degree ability to: celcius & RT ○ Yersinia-Generally are NM @37°C, but M ○ Adhere to host cells at RT ○ Colonize body surfaces ANTIGENIC STRUCTURES FOR ID ○ Release certain toxins Motile members have peritrichous flagella (endotoxins) H antigen - flagellar antigen except: ○ Invade deeper tissues Tatumella ptyseos which has polar flagella ○ Ability to transfer ○ An antigen found in the flagellum of motile members CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS All members grow luxuriantly on CHOC and BAP ○ Protein in nature plasmids carrying as moist, smooth, gray, shiny, entire, convex and ○ Heat labile antibiotic resistance opaque colonies. ○ Used in the serotyping between species of Salmonella and other genes motile bacteria. Klebsiella and sometimes Enterobacter produce mucoid colonies 2 BROAD CATEGORIES Beta-hemolysis is seen in some strains. OPPORTUNISTIC PATHOGENS PRIMARY PATHOGENS All are catalase positive except for one group of Shigella species and Xenorhabdus. Selective and Differential Media ○ Primary culture media used in the O antigen - somatic antigen presumptive identification of enteric organisms in the laboratory. ○ Heat stable, located in the cell wall ○ EMB, MAC, XLD, HEA, SSA ○ Lipopolysaccharide of the cell wall composing the endotoxin portion ○ There are over 160 types of 0 antigen for E.coli and specific types are Note: associated with diseases. 1. For the culture media, especially for the primary ○ Shigella have been grouped into four serotypes based on the O antigen: isolation of Enterobacteriacee, Please read serotypes A, B, C, and D Appendix A of Mahon, C. R., Lehman, D. ○ 60 types of 0 antigens exists for Salmonella C., & Manuselis, G. (2018). Textbook of diagnostic microbiology-e-book. Elsevier Health Sciences. K antigen - capsular antigen/envelope antigen 2. A huge percentage of the post quiz for Enterobacteriacee will cover the principle, ○ Heat labile polysaccharide found in encapsulated strains. procedure of inoculation, incubation requirement, ○ Covers the O antigen (must be removed to detect the O antigen!!) and interpretation of these culture media and also ○ To remove the K antigen - the organism must be boiled for 30 minutes. in their correlation with the results of the ○ Klebsiella, Salmonella, and E.coli have K antigens. biochemical tests for enteric bacteria. ○ Vi antigen for Salmonella is categorized as K antigens.  CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS 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: ○ E.coli is lactose fermenter ○ Shigella is non-lactose fermenter Characteristics ○ Motile, possess an adhesive fimbriae and sex pili ○ Has O, H and K antigens General laboratory findings Fermenter of: Glucose, lactose, trehalose and xylose Can produce indole from tryptophan Further ferments glucose in the MR pathway Negative for H2S, DNAse, Urease and PAD Cannot utilize citrate as source of carbon ENTEROTOXIGENIC E.COLI ENTEROINVASIVE ENTEROPATHOGENIC ENTEROHEMORRHAGIC E.COLI (EHEC) ENTEROAGGREGATIVE E.COLI (ETEC) E.COLI (EIEC) E.COLI (EPEC) (EAEC) -Associated with diarrhea of infants Associated with Non-toxigenic and Causes hemorrhagic diarrhea and colitis Implicated in persistent watery (major cause!) and adults in tropical and dysentery (watery non-invasive strain diarrhea in infants in developing subtropical areas diarrhea with blood, that causes diarrhea EHEC strain 0157: H7-associated with hemorrhagic countries and in travelers to -Most common cause of diarrheal leukocytes, and Pathophysiology is diarrhea, colitis, and hemolytic uremic syndrome (HUS) these countries disease commonly referred as mucus) similar to based on its adhesive On of the few bacteria TRAVELLER'S DIARRHEA/TOURIST'S, Shigella spp. properties Classic symptoms - watery diarrhea that progresses to associated with chronic diarrhea DIARRHEA/ "MUNTEZUMA'S Major cause of infant bloody diarrhea with abdominal cramps, low grade and growth retardation in REVENGE" Occur in adults and diarrhea in fever or an absence of fever. children -Acquired via ingestion of contaminated children impoverished Characterized by their food and water countries Stool contains no leukocytes - distinguishes it with auto-agglutination in a  CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS Direct person-to-person Characterized by dysentery caused by Shigella or ElEC infections "stacked-brick"arrangement" - Major contributing factors transmission via fecal watery diarrhea with Poor hygiene oral route large amounts of Risk factors - consumption Pathophysiology Reduced availability of sources mucus but no blood! of processed meats such as undercooked hamburgers of potable water Clinical infection served at fast food restaurants, unpasteurized dairy The "stacked-brick" arrangement Inadequate sanitation characterized by: fever, PATHOPHYSIOLOGY products and apple cider, bean sprouts, and spinach. is mediated by aggregative sever abdominal adherence fimbriae | (AAF1) 106 - 1010 (1000000 - 10000000000) cramps, malaise, and Infection is initiated by EPEC attachment to A. Pathophysiology: Hemorrhagic diarrhea and adhesin that are similar to the organisms is the infective dose of watery diarrhea. the epithelial cells of colitis bundle-forming pilus (BFP) of organisms in immunocompetent the small intestine with EPEC. individuals. Pathophysiology subsequent ЕНЕС 0157:7 (a.k.a STEC) produces two toxins: After adherence of EAEC to the Stomach acidity ○ Plasmid effacement verotoxin 1 and verotoxin 2 surface of the intestine, mucus mediated (destruction) of the secretion is stimulated leading to ○ Serves as a protective bacterial microvillus ○ Verotoxin 1 -phage encoded toxin identical to the formation of a thick biofilm. mechanism against colonization invasion (plnv (attachment/effacem the shiga toxin (Stx) produced by Shigella Thick biofilm - protects the and disease initiation genes) ent). dysenteriae type 1. It produces damage to aggregated bacteria from ○ Patients suffering from mediate the Vero Cells (African Green Monkey kidney antibiotics and phagocytic cells. achlorhydria - higher risk colonization of Bundle-forming pili cells). compared to normal persons. EIC into the (BFP)- mediates the ○ Biologically similar to, but immunologically Two toxins are also produced: colonic initial aggregation of similar from both Pathophysiology epithelium EPEC that leads to the Stx and verotoxin 1 ○ Enteroaggregative ○ EIEC lyses formation of heat stable toxin Colonization of ETEC in the small phagocytic micro-colonies on the Note: Verotoxin 1 can react and be neutralized by -antigenically related intestine mediated by fimbriae vacuole and epithelial cell surface. antibodies against St indicating antigenic similarities, to the heat-stable replicate into while verotoxin 2 does not, indicating the lack of toxin of ETEC Releases toxins in the small intestine the cell Attachment is followed by the active secretion antigenic similarities. ○ Plasmid encoded toxin cytoplasm of proteins into the Both toxins induce 1. Heat — Labile (LT) toxin ○ Movement Verotoxins and Shiga toxins are similar in quaternary host epithelial cells fluid secretion Similar in action to cholera toxin within the through the type Ill structure to the heat-labile toxin of ETEC; they are Has A and B portion cytoplasm and secretion of composed of a pentamer of receptor-binding B Watery diarrhea with no white 2. Heat - stable (ST) toxin into the ЕРЕС subunits that are noncovalently bound to a single blood cells and red blood cells adjacent cells enzymatic A subunit. present A. LT toxin (heat labile) is regulated by - consists of 2 subunits, one A subunit formation of ○ Translocated and five B subunit. actin intimin B subunit of the toxin - binds to the specific glycolipid receptor (Tir) called Globotriaosylceramide Gb3) expressed on host  CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS filaments - inserted into cells particularly on the intestinal villi and renal B subunit binds to the GM, ganglioside ○ This destroys the host's endothelial cells. receptor, the same receptor used by colonic epithelial cell Note: High concentrations of Gb3 are found on cholera toxin to bind on the epithelial epithelium cells membrane intestinal villi and renal endothelial cells. cells in the small intestine. and results in and functions inflammation as a receptor A subunit of the toxin - is internalized and cleaved into The whole toxin is endocytosed by the that could for an outer two molecules. epithelial cells of the intestine progress to membrane The A1 fragment binds to 28S rRNa and causes colonic bacterial cessation of protein synthesis. The vesicle that contain the whole toxin ulceration. adhesin, This results in the damage of the intestinal cells leading transports the toxin to the golgi apparatus intimin. Fecal - oral route ○ Binding of to colitis and hemorrhagic diarrhea. In the golgi apparatus, the A subunit intimin to Tir dissociates from the B subunit and it Manifestation: - results in B. Pathophysiology: Hemolytic Uremic moves across the membrane of the polymerizatio Syndrome (HUS) vacuole and interact with a membrane ○ Fever, severe n of actin and protein (Gs) that regulates adenylate abdominal accumulation Endothelial cells appear to be particularly important cyclase cramps, of target cells for Shiga toxins. malaise and cytoskeletal The toxin is transported retrograde in Adenylate cyclase enzyme catalyzes the watery diarrhea elements membrane-bound vesicles to the endoplasmic transformation of adenosine triphosphate (with blood) beneath the reticulum. (ATP) to cyclic adenosine ○ Clinical attached The A subunit induces depuration in ribosomal RNA; monophosphate (cAMP). symptoms bacteria, loss this leads to cessation of protein synthesis and death of similar to of cell surface the cell. The effect of this interaction is an Shigella spp. integrity, and increase in cyclic adenosine (Must be death. Shiga-toxin binding to proteins on the surface of monophosphate differentiated glomerular endothelium and inactivating a (CAMP) level from Shigella Attachment is followed metalloproteinase called von species) by the active secretion The increased levels of cAMP stimulate of proteins into the Willebrand factor-cleaving protease (VWFCP). the intestinal cells to hyper secret fluids Virulence test: SERENY host epithelial cells and electrolytes into the lumen of the TEST through the type Ill Once the VWFCP is disabled, multimers of von intestine. secretion of Willebrand Factor (vWF) form and initiate platelet Similarities of EIEC ЕРЕС activation and cause micro thrombi formation. This results in watery diarrhea (similar with Shigella to cholera). Escherichia colil The arterioles and capillaries of the body become  CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS secreted protein F obstructed by the resulting complexes of activated B. Heat - stable (ST) toxin ○ Both are gram (EspF) - causes loss platelets which have adhered to endothelium via large negative, of intestinal barrier multimeric vWF ○ Binds to the transmembrane facultative function through guanylate peptide cyclase anaerobic and disruption of tight The growing thrombi lodged in smaller vessels destroy receptor found on the surface of non-spore-form junctions and induces red blood cells (RBCs) as they squeeze through the the intestine. ing bacilli host cell death through narrowed blood vessels, forming schistocytes, or ○ Binding results in an increased ○ Non-motile apoptosis. fragments of sheared RBCs This mechanism, known as accumulation of cyclic ○ Non-lactose microangiopathic hemolysis. guanosine monophsophate fermenters The consumption of platelets as they adhere to the (cGMP) which also causes ○ Negative for hyper-secretion of fluid and lysine thrombi lodged in the small vessels can lead to severe electrolytes. decarboxylatio thrombocytopenia ○ This results in watery diarrhea n Kidney Failure - Due to reduced blood flow leading to ○ Cross Ischemia and also due to the direct damage caused by Diagnosis reactions the toxin to the renal endothelial cells. -Characteristic symptoms such as watery between O Thus the triad symptoms of HUS - thrombocytopenia, diarrhea, abdominal cramps, antigens microangiopathic hemolytic anemia, and kidney failure. -Isolation of lactose fermenting Differentiation is done organisms on selective and differential thru: Laboratory Diagnosis media Stool culture with serotyping ○ Their infective Isolation using MAC using sorbitol instead of lactose -Biochemical tests dose Demonstration of a four-fold increase or greater in -Multiplex PCR ○ EIEC - has Shiga-toxin neutralizing antibody titer in patients' sera higher infective with HUS dose (106) ○ Shigella - as few as 10 organisms ○ Acetate or mucate utilization as carbon source EIEC is CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS positive MUG test (4-methylumbelliferyl-ß-D-glucoronide) ЕНЕС 0157H7 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 0157:H7 E.CHOLI (COLON BACILLUS) EXTRA-INTESTINAL INFECTIONS OTHER SPECIES UROPATHOGENIC E.COLI Meningitis - caused by E.coli (and also group streptococci!) E. hermannii Most common cause of UTI, mostly ○ Acquired in the birth canal during delivery ○ Formerly called E.coli atypical or enteric occurring among females ○ May also result if contamination of the amniotic group lI Originate from the large intestine as fluid occurs. ○ Yellow pigmented colonies resident biota, contaminate the urethra, ○ Isolated from CS, wounds and blood and ascend into the bladder and may Septicemia- originates from infections in the urinary tract or migrate to the kidney or prostate gastrointestinal tract CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS Pili - primary virulence factor which Encapsulated strains of E.coli (possess K1 Ag) - mostly E. vulneris uropathogenic strain used to adhere to associated with neonatal meningitis and septicemia epithelial cells and not be washed off ○ Isolated from humans with infected wounds with urine flow Bacteremia - E.coli is a clinically significant isolate in blood ○ Half of the strains produced cultures from adults. yellow-pigmented colonies Cytolysins - characterized as a Bacteremia in adults may result from a urogenital tract hemolysin that can lyse white blood infection or from Gl source. E. albertii cells and inhibit phagocytosis and Associated with diarrheal disease in children chemotaxis Aerobatin - allows uropathogenic strains to chelate iron CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS PART 2: ENTEROBACTERIACEA KLEBSIELLA 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) Capable of utilizing citrate as carbon source and can grow in KCN broth Unable to produce H,S All are lactose fermenter except K. ozaenae and K. rhinoscleromatis Further ferments glucose via the VP pathway Do not produce indole (except K. oyxtoca!) They possess O and K antigens Urease (+) and ornithine decarboxylase (-) Klebsiella pneumonia Klebsiella oxytoca Klebsiella granulomatis Klebsiella Klebsiella ozanae (Friedlander’s bacilli) rhinoscleromatis Most common isolated species Causative agent of Formerly called Causes Causative agent of chronic Has a polysaccharide capsule - antibiotic-associate Donovania granulomatis Rhinoscleroma a atrophic rhinitis called ozena. DISTINCT d hemorrhagic then chronic Ozena is a disease of the nose FEATURE colitis (AAHC). Calymmatobacterium granulomatous in which the bony ridges and Confers granulomatis before disease. mucous membranes of the protection Observed during being reclassified as It commonly affects the nose waste away against therapy with Klebsiella granulomatis nasal cavity and Transmitted via phagocytosis and antimicrobial amoxicillin-clavulanat nasopharynx, but it person-to-person contact. absorption. e, amoxicillin, Causes granuloma can also involve the penicillins, or inguinale - a larynx, trachea, -Responsible for the moist, ampicillin. granulomatous disease bronchi, middle ear, MUCOID, colonies affecting the genitalia and orbit. and inguinal area. Acquired via -Also tests positive for Neufeld Sudden onset of inhalation of Quellung test (capsular bloody diarrhea a Disease is called respiratory droplets CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS swelling) after 2 to 7 days of donovanosis containing the treatment with the causative agents. Disease Association oral antibiotics Transmitted through (mentioned in the unprotected Sexual Diagnosis of -Community - Acquired previous bullet). intercourse rhinoscleromatis Pneumonia depends on the (CAP), nosocomial Antibiotic therapy After prolonged identification of the disrupts the normal incubation of weeks to pathognomonic Opportunistic infections among gut microbiota and months, subcutaneous Mickulicz cells newborns, elderly patients, and promotes the nodules appear on the (MCs) which is most seriously ill patients. overgrowth of genitalia or in the prominent during Klebsiella oxytoca genital area. granulomatous phase. Wound infections, UTI and with subsequent bacteraemia production of Donovan bodies are Mickulicz cells (MCs)- cytotoxin called rod-shaped, oval foamy Carbapenemase - producing tilivallin that causes organisms that can be macrophages K.pneumoniae is an important mitotic arrest of seen in the cytoplasm of with cause of ventilator-associated host cells and mononuclear numerous pnuemonia. induces apoptosis. phagocytes or cytoplasmic vacuoles histiocytes in tissue containing viable and ○ Carbapenemase - AAHC resolves samples from patients nonviable Klebsiella these are versatile spontaneously with granuloma bacilli Beta-lactamases that within 2-3 days inguinale. can hydrolyze when the offending They appear deep penicillins, antibiotics are purple when stained with cephalosporins, discontinued. Wright's stain. monobactams, and carbapenems ○ Renders many beta lactam drugs inactive Laboratory Diagnosis -Gram stain - Gram negative bacilli CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS -Culture - large, moist, mucoid colonies on EMB, MacConkey and XLD 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 Enterobacteriacee 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 2. P. fluoroscens (pyoverdin) - Motile ○ Fluorescent ○ Growth at 35°C P. putida (pyoverdin) CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS pseudomon but not at 42°C ads ○ Oxidase (+) ○ Can grow at ○ OF medium: 4°C oxidize Glucose ○ associated ○ Pyocyanin: (-) w/ ○ Pyoverdin: (+) Nosocomial ○ Cetrimide & infections Acetamide: (-) (transfusion ○ Arginine: (+) associated ○ Resistant to sepsis) Carbenicillin ○ rarely ○ Susceptible to associated Kanamycin with opportunisti c infxns. P. fluorescens ○ Contamina nt ○ Rare cause of UTI & wound infns in humans P. putida ○ Isolated cases of septicemia, UTI, & pneumonia ○ CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS ○ ENTEROBACTER GENERAL CHARACTERISTICS DISEASE ASSOCIATION Inhabits soil and water and, to a lesser extent, the Enterobacter cloacae and Enterobacter aerogenes large bowels of man and animals Often confused with Klebsiella in terms of growth in ○ Two common isolates from the genus. MAC ○ Isolated from wounds, urine, blood, and CSF Also share biochemical reactions with Klebsiella spp: Pantoea (Enterobacter) agglomerans - gained notoriety with a nationwide (U.S) outbreak ○ Citrate positive of septicemia resulting from contaminated V fluids ○ Grows in KCN broth ○ Methyl Red test negative ○ LOA negative. A.K.A "triple decarboxylases" negative ○ Pantoea agglomerans HG XIlI - produce a yellow pigment, a plant pathogen. Growth on EMB - "fish eye" colonies Difference Klebsiella spp: -Enterobacter gergoviae - found in respiratory samples and is rarely isolated from blood cultures. ○ Positive for ornithine decarboxylase Cronobacter (Enterobacter) sakazakii - produces a yellow pigment and has been (Klebsiella are negative) documented as a pathogen in neonates causing meningitis and bacteremia often ○ Positive for motility (Klebsiella are coming from powdered infant formula. non-motile) 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. CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS SERRATIA GENERAL CHARACTERISTIC DISEASE ASSOCIATION Comprise of species that are opportunistic pathogens associated with Serratia marcescens - the most clinically significant species outbreaks in health care settings. ○ Frequently found in hospital-acquired infections of the urinary ○ Serratia marcescens, Serratia rubidea, Serratia plymuthica tract ○ Bacteremic outbreaks in nurseries and cardiac surgery and Pigment production at room temperature. "Prodigiosin" - pink-to-red in burn units. color Contamination of antiseptic solution used for joint injections has result in an epidemic of septic arthritis. ○ Serratia liquifaciens ○ Serratia odorifera - emits a dirty, musty odor resembling "rotten Serratia odorifera biogroups potatoes" ○ Serratia ficaria ○ S.odorifera biogroup 1 - isolated predominantly from the ○ Serratia fonticola respiratory tract and is positive for the fermentation of sucrose ○ Serratia entomophila and raffinose, and is positive for ornithine decarboxylation ○ S.odorifera biogroup 2 - isolated from blood and CS and is CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS All are sucrose fermenters negative for the fermentation of sucrose and raffinose, and negative for ornithine decarboxylation. Note: All Serratia species except for Serratia fonticola are late lactose ○ 50% of isolates are indole positive. fermenters, positive for ONPG. All are DNAse, Lipase, and Gelatinase positive (differential for other members of the family Enterobacteriaceae. Serratia species are known for their resistance to a wide range of antimicrobials. HAFNIA Hafnia alvei (Enterobacter alvei) Resembles Enterobacter but differentiated by its inability to ferment lactose, sucrose, sorbitol, and raffinose Differentiated from Serratia because it is DNAse and Lipase (-) Delayed positive citrate reaction - MAJOR CHARACTERISTICS OF Hafnia Clinical significance is still questionable despite being recovered from a number of human sources, including stools and wounds. Infrequent pathogen, but has been isolated in cases of bacteremia, respiratory tract infections, and gastroenteritis. CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS TRIBE PROTEEAE 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 GENUS PROTEUS PROVIDENCIA MORGANELLA Proteus mirabilis and Proteus vulgaris are Providencia species are lactose-negative, ​Morganella morganii - only species within the widely recognized human pathogens deaminase (PAD) positive, H2S-negative, and genus Morganella Isolated from urine, wounds, ear, and blood motile Biochemical characteristics - NLF, negative Responsible for 3% of UTI in the U.S Characterized by a pungent odor reactions for citrate, H2S, and LDC. Urease Ferment mannose, and do not swarm on SBA and deaminase (PAD) positive. ○ Ascend the urinary tract causing Five species: infections both in the lower and upper Opportunistic pathogen that causes tracts ○ Providencia alcalifaciens gastrointestinal disease, urinary tract, and ○ Can infect proximal kidney tubules ○ Providencia heimbachae wound infections. and can cause acute ○ Providencia rettgeri - the only glomerulonephritis particularly in urease positive Infections are more frequently seen in patients with urinary tract defects or ○ Providencia rustigianni CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS catheterization ○ Providencia stuartii immunocompromised patients and those ○ Urease activity of Proteus - lead to undergoing prolonged antibiotic therapy struvite kidney stones More serious infections include septicemia and Struvite Crystals - formed after the urine abscesses turns alkaline as a result of the conversion of 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) 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 Proteus mirabilis and Proteus vulgaris easily identified by their swarming colonies on culture Providencia-associated UTI - involved patients media. with predisposing urological problems Swarming - results form a tightly regulated Providencia-associated skin infections - are cycle of differentiation from standard often found in burn patients vegetative cells (swimmers) to hyper-flagellated, elongated, polypoid cells P.stuartii - associated with UTI in those with (swarmers) capable of coordinated indwelling catheters movement. Swarming ability - thought to play a role in the ascending nature of Proteus-associated UTI CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS Swarmers - produce a distinct "burnt chocolate" odor 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. NICE TO KNOW 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. Antigens: OX, 0X19, 0x2 CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS 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. 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 Composed of three species: E. tarda; E. hoshinae; E. ictaluri Isolated from cold blooded animals including reptiles, freshwater and aquarium fish, frogs, and turtles. CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS E.tarda - the only recognized pathogen. ○ Opportunistic ○ Isolated in GI infections All of them are negative for urea hydrolysis All of them are positive for: ○ Lysine decarboxylase ○ H2S, and indole All of them do not grow on Simmon's citrate PLESIOMONAS General Characteristics Formerly under the family Vibrionaceae Oxidase-positive (take note that Enterobacteriacee are oxidase negative!), glucose-fermenting, facultatively anaerobic, gram-negative bacilli Phylogenetically closely related to Enterobacteriacee, but it does not have the ability to produce gas from glucose Only oxidase-positive member of the family Enterobacteriaceae 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 Enterobacteriacee that have peritrichous flagella) Plesiomonas and Shigella share biochemical and antigenic features Plesiomonas shigelloides- appear to have much lower virulence 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. CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS PART 3: ENTEROBACTERIACEA 10. SHIGELLA GENERAL BIOCHEMICAL SPECIES SEROTYPES BASED MODE OF DISEASE PATHOPHYSIOLOGY CHARACTERISTICS CHARACTERISTICS ON THEIR O TRANSMISSI ASSOCIATION ANTIGEN ON -Non-lactose 1. Shigella Subgroup A - S. Humans and Shigella species cause Closely related fermenters sonnei – dysenteriae (Dysentery other large all types of bacillary to Escherichia – -Non-motile unique bacillus) primates are the dysentery - painful both belong to - Anaerogenic, except member of only known type of diarrhea the same tribe, Shigella flexneri the genus ○ Serotypes reservoir! characterized by Escherichieae -Do not hyrdrolyse - Ability to - Mod watery feces w/ 1-10 Shigella – not urea ferment ○ Ex. e of mucus, blood streaks & members of the -Do not produce H2S Shigella - attach to and invade the M cells located lactose S.dysenteriae trans pus cells GI microbiota, -Do not decarboxylate in Peyer patches. slowly (LLF) (shiga toxin) missi Infective dose: as low and are lysine -The organism secretes four invasion-plasmid producing on: as 100 bacilli are considered as -Do not use acetate or pink colonies Subgroup B - S. flexneri direct needed to initiate the antigen (Ipa) proteins into epithelial cells and “primary mucate as carbon on MAC agar (Strong's bacillus) perso disease in some macrophages that induce membrane ruffling on pathogens” source only after 48 n-to- healthy individuals. the target cell, leading to engulfment of bacteria. All species Subgroup C - S. boydii (S. hours of perso Predominant isolates: These proteins include: causes **Biochemically inert ambigua) dysentery incubation Subgroup D - S. sonnei n (bloody diarrhea (differentiate conta ○ S.sonnei - ○ ІраA with mucus, it with other ct, predominan ○ IpaВ Note: On selective ○ blood, and members of fecal- t in the U.S IpaC and differential leukocytes) the genus) oral-r and other ○ IpaD media used to All Shigella - ONPG oute industrialize selectively isolate species - positive with d countries Shigella - lyses the phagocytic vacuole and intestinal possess O replicate in the cytoplasm. pathogens, Shigella Shigella species – fragile carrie ○ S.flexneri - antigens, and appear as clear, and susceptible to rs as affects certain strains -The rearrangement of actin filaments in the host non-lactose various effects of LABORATORY the mostly possess K fermenting physical and chemical DIAGNOSIS sourc young cells propel the bacteria through the cytoplasm antigens colonies. agents: e. children. through the adjacent cells where cell-to-cell K antigens (capsular) if - Disinfectants Selenite broth - Leading passage occurs. present, - High concentrations of enrichment medium for May also be isolate of interfere with acids and bile the isolation of transmitted by gastroenteri ○ Cell-to-cell passage protects Shigella the detection of Salmonella spp., and flies, fingers, tis among from immune-mediated the O antigen Shigella species from and food and MSMs. HIV clearance. Note: Shigella are during serologic heavily contaminated water infections ○ They survive phagocytosis by inducing CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS grouping. specimens such as stool. contaminated by increases apoptosis. Apoptotic phagocytes susceptible to the May be Primary culture media: infected persons this risk. release IL1B that acts as a removed by acid pH of the stool. ○ S.dysenteri chemoattractant for PMNs into the boiling the Feces suspected EMB Personal ae type 1 infected tissues. This destabilizes the bacterial of containing Shigella spp., МАС hygiene plays a and integrity of the intestinal wall and suspension to must be XLD major role in the S.boydii are allows bacteria to reach the deeper remove heat immediately SSA transmission the most epithelial cells. labile K inoculated to НЕА common ○ Shigella dysenteriae - produces shiga antigen and to primary culture isolates in toxins, similar to the verotoxins expose the heat stable O media to increase developing produced by EHEC. antigen. recovery of the countries. ○ The B subunit binds to the host cell Non motile, do organisms. S.dysenteri glycolipid Gb3 receptor and facilitate not have H ae type 1 the transfer of the A subunit into the antigen remains the cell. most ○ The A subunit cleaves the 28S RNA in virulent the 60S ribosomal subunit, resulting in (significantl the disruption of protein synthesis y high resulting in cell damage and death. morbidity & Note: High concentrations of Gb3 mortality are found on intestinal villi and rate.) renal endothelial cells. This results in hemorrhagic diarrhea and hemolytic uremic syndrome (HUS) 11. SALMONELLA GENERAL ANTIGENIC CLASSIFICATION DISEASE ASSOCIATION SPECIMEN COLLECTION AND LAB CHARACTERISTICS STRUCTURES AND DIAGNOSIS VIRULENCE FACTORS  CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS O Ag: Stable to boiling Formerly, there are only 3 In humans, salmonellosis occurs in many Blood - 1st and 2nd week and acid alcohol known species: forms: Urine/Stool- 3rd and subsequent weeks H Ag: More stable Stool - can be used indefinitely as clinical - O and H are the ○ S.enteritidis; ○ Acute gastroenteritis or food specimen primary antigenic S.cholerasius; S.typhi poisoning characterized by NOTE: Culture is the best diagnostic procedure structures ○ S.enteritidis - also vomiting and diarrhea to identify Salmonella known as the ○ Typhoid fever, the most severe Vi Ag: Virulence Ag (K Gardner's bacillus; form of enteric fever, caused by antigen); occurs in S.typhi formerly S. Salmonella serotype Typhi, and Note for the following exceptions: Salmonella serotype typhosa is also known enteric fevers caused by other S.pullorum and S.gallinarum - Typhi and a few strains as the serotypes such as Salmonella Non-motile of Salmonella serotype Eberth's bacillus serotype Paratyhpi and Salmonella All are NLF -except for Cholerasius serotype Cholerasius. Salmonella Arizona -LLF - Plays a role in Genetic studies have shown All are aerogenic - except preventing that the three previously named -Mode of transmission - ingestion of the Salmonella Typhi phagocytosis species are genetically related organism in food, milk, and water All are H2S positive -except and that there are only 2 contaminated with human or animal excreta. Salmonella Paratyphi Surface Ag's - designated species; Except for Salmonella Typhi and invasiveness (enables Salmonella Paratyphi, Salmonella infect them to traverse the animals that serve as their reservoir ○ S.enterica (Type intestinal mucosa; Salmonella Typhi and Salmonella Paratyphi species of the genus); endotoxin, have no known animal reservoirs and Gram stain - Gram negative bacilli Many Salmonella serotypes are and with 6 enterotoxins, infections only occurs in humans. Carriers Culture: typically found in cold-blooded subspecies. intracellular capability are often the source of infection. animals as well as in rodents and in ○ ,bongori (rare isolate) birds, which serve as their natural - initially isolated ○ EMB/MAC - colorless Fimbriae - for adhesion A. Gastroenteritis hosts. from a lizard and is ○ SSA - colorless-black center (H2S+) Gram-negative, facultative anaerobic usually isolated from Source of infection have been attributed to ○ NA - maple leaf colonies w/irregular bacilli. cold-blooded poultry, milk, eggs, and egg products, as well margins On selective and differential media - animals and the as to handling pets. ○ XLD - slightly pink to white opaque Salmonellae produce clear, colorless, environment. Insufficiently cooked eggs and domestic fowl, colonies non-lactose fermenting colonies. such as chicken, turkey, and duck and common sources of infection. S. Typhi: "Metallic colonies with a black ring" Outbreaks associated with consumptions of on BSA ○ Colonies with black foodstuffs such as peanut butter, cantaloupe, centers are seen in media puffed rice and wheat cereals, corn-and with sodium thiosulfate vegetable-coated snacks, vegan cheese, such as alfalfa sprouts, cucumber, and raw tomatoes. HEA, SSA, and XLD Cross -contamination through cooking ○ Black precipitates indicate utensils also occur H2S production. B. Enteric Fever Also known as typhoid fever - a febrile CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS 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 Widal Test Cholerasius Serologic test that detects the presence of antibodies in the person's serum against O and H 1st Week of infection antigens of Salmonella Typhi. When organisms are ingested - they are Agglutination test. Performed on 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. Tube Method to Determine Antibody Titer to Organisms multiply intracellularly inside Salmonella Antigens phagocytes and are released in the blood stream for the second time. The titer is the highest dilution that gives a This results to febrile episode and the visible agglutination organism can be easily isolated from blood Interpretation: 2nd and 3rd Weeks of Infection Titer of 1:100 or more for 0 agglutinins and 1:200 or more for H agglutinin should be considered Patient generally sustained fever with clinically significant as these indicate active prolonged bacteremia infection. The organism invade the gall bladder and the Peyer's patches of the bowel. They also reach the intestinal tract via the biliary tract.  CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS "Rose-spots" - blanching rose-colored papules around the umbilical region also appear during the second week of fever. Involvement of the biliary system initiates Gl 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 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. YERSINIA GENERAL CHARACTERISTICS YERSINIA PESTIS YERSINIA ENTEROLITICA YERSINIA PSEUDOTUBERCULOSIS Three human pathogens: The causative agent of plague - a primary disease Similar to Yersinia pestis for being a in rodents that are spread to humans by bites of Found in a wide variety of animals, including domestic disease primarily of rodents, ○ Yersinia pestis - causative agent of fleas. swine, cats, and dogs. particularly guinea pigs.  CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS plague ○ Yersinia pseudotuberculosis Xenopsylla cheopis (oriental rat flea) - flea vector Mode of transmission - contact with household pets. Birds are natural reservoirs. ○ Yersinia enterocolitica (most common and effective vector of Yersinia Transmission via pigs has been greatly emphasized in pestis) Europe Disease is characterized by caseous Implicated in sporadic cases of gastroenteritis, Parasite of the rodent Rattus rattus - a black rat, swelling called "pseudo-tubercles" mesenteric lymphadenitis especially in children, as the main rodent host of Yersinia pestis Human infections - most often occurs after the and generalized septicemic infections in ingestion of contaminated food, often pork, and Human infections are rare, and are immunocompromised hosts. Yersinia pestis (Plague bacillus) vacuum packed deli meat, lamb, chicken, and associated with close contact with Microaerophilic, gram negative, short, plump possibly milk and water. infected animals, or their fecal Yersinia enterocolitica - has been a cause of bacillus material, or ingestion of diarrhea outbreaks in numerous communities Shows marked bipolar staining when stained with The ability of the organism to survive in cold contaminated drink or foodstuff. and it has been seen to mimic appendicitis. "Wayson Stain"- cells having a characteristic temperatures - is a potential risk of transmitting "Safety Pin" appearance infections via consumption of refrigerated foods Morphology - resembles Yersinia MAC - colonies are small, pinpoint colonies; (refrigeration is ineffective as a measure to control pestis Non-motile at 37C, but motile at 25C, except the infection) Yersinia pestis. Differences from Yersinia pestis - Optimal growth is at 25-30C In transfusion medicine, reports of the transmission of motility at 18C - 22C, urease TSI - Acid/orange (A/K) in 24 hours due to Yersinia enterocolitica from contaminated packed production, and ability to ferment weak acid reaction in the slant with no change red blood cell components have been recorded. rhamnose. in the butt - suspected of Yersinia Clinical manifestations: In culture, the organism prefers a temperature between 25C to 30C, though it can still grow at ○ Acute gastroenteritis 37С. ○ Appendicitis - like syndrome Non-motile both at 25C and at 37C ○ Less frequent manifestations such as Catalase positive septicemia arthritis, and erythema Oxidase negative nodosum. Plague - occurs in three distinct forms: Resembles other Yersinia species 1. Bubonic plague - most common form of Gram-negative coccobacilli, with bipolar staining the disease, results from the bite of a Optimal growth at 25C-30C flea, or by direct inoculation of an open Clearly motile at 25C, but non-motile at 35C. skin would by plague-infected material. Cold-enrichment - used to increase the recovery in Buboes, lymph nodes filled with fecal samples. inflammatory cells that can ulcerate, can be several in diameters in centimeters. 2. Secondary septicemic plague - results Cold enrichment of fecal specimens for Yersinia from the hematogenous dissemination of enterocolitica the organism, following the damaged of CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS the lymph nodes in patients with bubonic plague. A. Fecal material is inoculated into isotonic ○ Primary septicemic plague - occurs in saline and kept at 4C for 1-3 weeks with patients without buboes as primary weekly subculture to selective agar for infections. Yersinia. ○ Symptoms - similar to bubonic plague, B. Subculture can be performed using but then progressively worsen as the Cefsulodin -Irgasan-Novobiocin (CIN) - bacterial endotoxins set off an selective for Yersinia that contains immunologic cascade of events, cefsulodin, irgasan, novobiocin, bile salts, ultimately leading to multiple organ and crystal violet as inhibitory agents failure, respiratory distress, and DIC, C. CIN - better than MAC in inhibiting noted by petechiae and gangrene in the normal colon microbiota and provides extremities (fingers, toes, and noes). better opportunity for the recovery of 3. Secondary pneumonic plague - results Yersinia enterocolitica in feces. form the dissemination of the organism to D. YSA - Yersinia-selective agar, a modified other organ systems such as the lungs, version of CIN that contains mannitol as brain, liver, and spleen, with days-near differential agent. 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. "Bull's Eye" colonies of Yersinia enterocolitica on CIN medium. Results from the fermentation of mannitol and production of acids that cause the pH indicator neutral to turn red at the center of the colony and precipitate bile. \ CLINICAL BACTERIOLOGY BSMLS-3I MLS 411 | LEC | FINALS LABORATORY DIAGNOSIS OF ENTEROBACTERIACEAE SPECIMEN COLLECTION, DIRECT MICROSCOPIC CULTURE SCREENING FOR STOOL TRANSPORT, AND EXAMINATION CULTURES FOR PATHOGENS PROCESSING A. Enterobacteriacea are facultative Isolated from wide variety of Generally gram-stain is not anaerobe and grow at optimal A. Fecal specimens should be clinical specimens valuable sinc