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Medical Microbiology Exam 3
dmr6p9

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Gram-negative Cocci and Coccobacilli
Learning Objectives


For the following G-:
  • Neisseria meningitidis
  • Neisseria gonorrhoeae
  • Haemophilus influenzae
  • Bordetella pertussis
  • Pasteurella multocida
You should know:
  • general characteristics
  • major virulence factors
  • epidemiology
  • pathologenesis and disease
  • laboratory diagnisis
  • treatment and prevention



N. meningitidis vs N. gonorrhoeae


N. meningitidis


  • Glucose +

  • Maltose +

  • Capsule +

  • causes meningitis

  • coffee bean morphology


N. golorrhoeae


  • Glucose +

  • Maltose -

  • Capsule -

  • Causes gonorrhea

  • coffee bean morphology





N. gonorrhoeae


N. gonorrhoeae Major virulence factors


  • Pili: mediate attachment; undergo antigenic morphology (change morphology of pili so that the host has difficulty developing an immune response)
  • Por protein: prevention of phagolysosomal fusion
  • Lipo-oligosaccharide (LOS) = endotoxin (similar to LPS)
  • IgA protease: common in many bacteria; break up IgA which is present on mucous membranes



N. gonorrhoeae Epidemiology



  • Humans are the only host

  • Second most common STD

  • High prevelance in adolescents (62% of cases between ages 15-24)

  • Asymptomatic carriage: women > men

  • Disease: men > women




Pathogenesis and Clinical Manifestations of N. gonorrhoeae


  • primary site: Urethra, vagina
  • Men: Urethritis
  • Women: Inflammation of the endocervix, purulent vaginal discharge, irregular intermenstrual bleeding, infection of fallopian tubes>PIS>Scarring>INfertility
  • Babies: Ophthalmia neonatorum




Gonococcal Conjunctivitis (Ophthalmia neonatorum)



gonococcal urethritis



disseminated gonococcal infection


other sites of infection N. gonorrhoeae


  • anus/rectum: Protitis
  • Throat: Pharyngitis
  • Joints: Arthritis, especially in young, sexually active females (particularly in the knees and ankles)




Laboratory Diagnosis of N. gonorrhoeae


  • gram stain of the urethral/vaginal discharge

  • culture: Thayer-Martin agar (VPN)

  • Glu+, mal-, sucrose-

  • DNA probe

  • picture (a) chocolate medium overgrowth (grows all rectal bacteria)

  • picture (b) Thayer-martain Medium grows Neisseria only: chocolate agar + antibiotic that kill off normal rectal flora




N. gonorrhoeae Treatment and Prevention



  • Ceftriaxone--3rd generation cephalosporin

  • Mixed infection with Chlamydia trachomatis--add tetracycline

  • Prevention: nonspecific (e.g., condoms)




N. meningitidis: Major Virulence factors


  • Capsule = a polysaccaride that coats bacteria in order to evade immune response: antigen that defines 13 serogroups (epidemic serogroups A, B, C, W135, X and Y); antigen detected in CSF; antigen included in vaccine
  • Outer membrane protein (OMP)
  • Endotoxin (LOS--Lipooligosaccharide, similar to enteric LPS): major cause of infection
  • IgA protease--evasion of mucosal immunity



N. meningitidis Epidemiology


  • Asymptomatic carriage in the nasopharynx: ~5-10% (unclear as to why): lower in children; 25% in teenagers (particularly common on college campuses
  • children (6 mo-2yrs) are more susceptible
  • Serogroup B: the most prevalent in the U.S.
  • 2001: 1,094 cases



N. meningitidis Pathogenesis


  • Capsule and LOS
  • LOS-mediated damage to ciliated epithelial cells--generation of pro-inflammatory cytokines
  • invasion of submucosa--Type IV pilus (like a hook they can project and reel back in), N.m. adhesion A (NAD)



N. meningitis associated diseases



  • Meningitis, bacterium crosses the BBB: Fever, Headache, Stiff Neck, INcrease PMNs in the CSF (fatal in 20% of humans)

  • Meningococcemia: DIC (diffuse intravascular coagulopathy; as it travels through blood, LOS is relased), shock, waterhouse-friderichsen syndrome (very deadly, typically die within hours of symptoms appearing)





meningococcemia



meningococcemia



meningococcemia



Wterhouse-Friderichsen Syndrome (WHS): N. meningitidis evaded the adrenal glands; if a patient reaches this point of infection, he will almost certainly die because he can no longer secrete hormones



Laboratory Diagnisis of N. meningitidis:
  • Specimen: CFS (blood)
  • Direct exam: G-, kidney-shaped diplococci
  • Rapid detection: latex (bead) agglutination
  • Culture: BAP, CHOC
  • Ox+, Glu+, Mal+



Treatment and Prevention of N. meningitidis





  • Penecillin + 3rd generation cephalosporin

  • Quadrivalent Vaccine with group A, C, Y, and W135 polysaccharides (4/6 epidemic strains):

  • Meningococcal conjugate vaccine (MCV4)--Unvaccinated college freshman

  • Meningococcal polysaccharide vaccine (MPSV4)--2years of age and adolescents

  • group B is excluded due to low immunogenicity




Haemophilus influenzae general characteristics


  • gram-nevative
  • plomorphic
  • nonmotile cocobacillius
  • X (hemin) and V (NAD) factor requirement
  • two major categories:l encapsulated and unencapsulated (non-typable) strains




Haemophilus influenzae Gram-stain


Haemophilus influenzae Virulence factors



  • LPS: highly diverse glycoform structure--contributes to both commensal and pathogenic lifestyle

  • outer membrane proteins (OMPs)

  • adhesion proteins (allow it to stick to epithelial cells)

  • capsule: antigen that defines serotypes--6 major classes; antigen detected in CFS; antigen included in vaccine

  • IgA protease--probably does not play a major role in this particular case




Epidemology of Haemophilus influenzae


  • part of normal microbiota
  • incidence in the past: 8000 cases/year
  • incidence in 2008: 2,886 cases
  • Children < 5 y-old: 437 cases
  • decreased dramatically due to Hib vaccine




Pathogenesis and Disease of H. influenzae:
From nasopharynx H. flue spreads to:
  • middle ear - otitis media
  • sinus - sinusitis
  • bronchi - bronchitis
  • alveoli - pneumonia
  • epiglottis - epiglottitis
  • meninges - meningitis



Non-Respiratory Diseases (H. Influenzae)


  • cellulitis
  • arthritis



Laboratory Diagnosis of H. Influenzae


  • Culture: X and V factor requirement--H. influenzae is a difficult organisim to culture--reduced sensitivity of detection
  • Catalase+, Oxidase+
  • Antigen detaction--latex particle agglutination teat (LAT)
  • Polymerase chain reaction (PCR) (most sensitive)--used in clinics; most reliable



Treatmant and Prevention--H. Influenzae


  • Otitis media and sinusitis: Ampicilli, Augmentin, Trimethoprim-sulfamethoxazole
  • Meningitis: 3rd Generation Cephalosporin, i.e., ceftriaxone, cefotaxime
  • Hib Vaccine (very effective): Polysaccharide-protein conjugate vaccine; expensive, limited usage in the Developing World



Gram-negative Coccobacilli


  • Pasteurell multocida
  • Bordetella pertussis




Pasteurella multocida:
  • relatively uncommon
  • Short coccobacillus
  • encapsulated
  • grows well on BAP or CHOC at 37 degrees C
  • Gram negative, stains bipolarly
  • part of the normal flora in the mouths od domestic dogs and cats
  • transmission: through bites



Virulence Factors Pasteurella multocida


  • capsule
  • endotoxin
  • exotoxin (PMT): disrupts cellular metabolison in the skin, connective tissue, fat, bane (causes cells to become hyperactive vs. killing them)



Clinical Manifestations of Pasteuraella multocida


  • Cellulitis at the bite site--not to be confused with cat scratch disease (Nartonell app.)
  • Osteomyelitis--PMT causes bone degradation



Pasteurella multocida


  • bipolar staining
  • Indole+
  • "Sick" triple sugar iron (TSI) test



Pasteurella multocida


  • penicillin
  • fluoroquinolones
  • tetracyclines




Bordetella pertussis:
  • small coccobacillus
  • encapsulated
  • obligate human pathogen
  • Transmission: via aerosol
  • Highly contagious
  • worldwide distribution
  • occurs primarily in infants and young children (adults are carriers but are usually asymptomatic)
  • In the US, 2008: 13,278 cases



Virulence Factors: Bordella pertussis


  • Filamentous hemagglutination (FHA)
  • Pertacin
  • Pertussis toxin --> eukaryotic Adenylate cyclase increase --> cAMP increase
  • Adenylate cyclase toxin --> cAMP increase
  • Tracheal cytotoxin --> inhibits cilia --> promotes coughing fits --> expels bacteria
  • does not want to kill  us because they (bacteria) would die as well



Pathogenesis of Bordella pertussis


  • attachment to the cillia of teh epithelial cells in teh upper airways: with the help of FHA Pertactin
  • Tracheal cytotoxin
  • Non invasive
  • Pertussin toxin causes: mucuc secretion; lymphocytosis--increase in the number or proportion lymphocytes in the blood
  • Adenylate cyclase/Pertacin: Helps prevent invasion/uptake of teh bacterium



Bordella pertussis Clinical Manifestations


  • Tracheobronchitis
  • Severe proxysmal cough
  • Inspiratory "whoop" and vomiting after coughing--halmarks of pertussis infection
  • Duration: 1-4 weeks
  • Death ususally due to pneumonia, caused by secondary bacterial infection



Bordella pertussis Lab Diagnosis


  • Nasopharyngeal Swabs
  • Bordet-Gengou medium--selective for Bordetella spp., small pearl-like colonies
  • agglutination in specific antiserum
  • DFA--Firect Fluorescent antibody test (bacteria glow green if they are present)



Bordella pertussis treatment and Prevention


  • Erythromycin (sensitive to wide variety of patients but only antimicrobial that actually works in patients)
  • Amoxicillin, tetracycline, fluoroquinolones and cephalosporins--god in intro activities, not suitable for in vivo treatment
  • DaPT (Diptheria, acellular Pertussis and Tetanus) Vaccine: Acellular pertussis vaccine; mixture on 5 antigens (generally modified pertussis toxoid etc.)



Case Study 1: A 10-year old girl is brought in to Green Meadows Clinic because of redness, pain, and swelling of the left hand. According to her mother, the day before the onset of her symptoms, the child was accidentally bitten by her cat. Physical exam reveals temperature of 37.8 degrees C, enlarged axillary lymph nodes and erythematous and swollen left had that was hot to touch. The pain now has spread upward and involved the upper arm. An atending physician notics early sins of abcess formation and accumulation of pus at the bite site. A presumptive diagnosis is made and the purulent exudate sent for culture.
  • What is the most likely diagnosis?
  • What is the most likely cause?



  • Cat-bite cellulitis
  • Pasteurella multocida



Case 2: A 2-month-old male infant was brought to the peditrician because of coughing and chocking spells for two weeks. At the end on the coughing spell, teh baby often vomited and sometimes seemed to have difficulty catching his breath. The nurse in the clinic noted that the cough was paroxysmal. The mother related that she had had coughing spells followed by choking and near vomiting for three weeks. She had recieved a course of amoxicillin withour relief. ON evaluation, the baby's temperature was 37.7 degrees C (↑); pulse, 200/min (↑); respiration 72/min (↑); WBC, 140,000/mm3 (↑↑↑) of which 50% were lymphocytes (↑↑). Chest  x-ray revealed upper lobe infiltrates bilaterally and patchy infiltrates in teh middle lobes consistent with pneumonia. The infant was admitted to pediactric ICU where he was intubated and placed on a mechanical ventilator.
  • What is the most likely diagnosis?
  • What is the most likely cause?
  • What are virulence factors?
  • What is the treatmet choice?
  • How can teh disease be prevented?



  • Whooping cough (pertussis)
  • Bordetella pertussis
  • filamentous hemagglitinin, adenylate cyclase, pertussis toxin
  • Erythromycin
  • Immunization (DaPT)




Haemophilus influenzae:
  • small, grayosh, mucoid, dewdrop-like colonies
  • "Musty basement" odor
  • Pleomorphic--many forms
  • Fastisious--requires A (hemin) and V (NAD) factors, present in choclate agar




Identification of H. influenzae--XV test



Satellite test is poor mans version of the XV test



Bacillus anthracis general characteristics:
  • in culture: "bamboo-like rods" large gram+ rods (squared ends) with centrally, poorly stained oval endospores
  • in tissue: short chains, capsule, NO spore
  • colonies: rough, comma shaped projections from colony edge: "medusa head"



bacillus anthracis virulence factors


  • encoded on two plasmids (essential)
  • capsule (poly-D-glutamic acid, protein, NOT polysaccharide)
  • anathrax toxin (tripartite toxin): lethal factor (LF, protease); edema factor (EF, adenylate cyclase); protective antigen (PA)
  • PA binds and helps EF and LF enter the cell
  • PA + EF = edema toxin, causes edema
  • PA + LF = lethal toxin, (dominant virulence factor) causes cell death




Anthrax toxin:
  • EF + CaM + ATP --> ↑cAMP --> edema
  • LP --> ↓MAPKs --> necrosis hypoxia



Epidemiology (bacillus anthracis)


  • B. a. is a saprophytic organism found in the soil worldwide
  • Anthrax is a zoonotic disease (has the ability to infect many animal types)--cattle and humans prior to the 20th century
  • Biological WMD: Japan, UK, US, Iraq and Russia
  • Persian Gulf War: Iraq had bombs and missles laden with anthrax spores



Pathogenesis and Clinical Aspects (bacillus anthracis)


there are three common forms forms of anthrax:
  • cutaneous anthrax
  • gastrointestinal anthrax
  • pulmonary anthrax



cutaneous anthrax


spores through breaks in the skin --> "malignant pustule" (localized llesion at site of infection) --> 20% death if untreated


gastrointestinal anthrax


contaminated meat -->
  • nausea, vomiting, anorexia, fever
  • abdominal pain, bloody diarrhea
  • toxemia and shock
  • death within 2 to 5 days (high mortality rate)



pulmonary anthrax


Inhalation of spores --> macrophages rapidly take up and carry the spores to the lymph nodes --> patient is non-infectious
  • spores germinate and lyse macrophages to liberate B. anthracis
  • B. anthracis invades the lymph nodes and cause hemorrhagic necosis, edema, and mediastinitis
  • non specific symptooms: malaise, fever, and cough
  • difficulty breathing (due to edema filling the lungs), profuse sweating, bloody vomitus
  • bacteremia, sepsis (primary cause of death), meningitis
  • septic shock, death in 2 to 5 days




inhalational anthrax: macrphages engulf in alveoli and take to lymph nodes



inhalational anthrax chest x-ray: mediastinal widening and pleural effusion on chest x-ray in inhalation anthrax; and filling with edema


outbreak of Inhalational Anthrax in Sverdloovsk, Russia


  • 1979: Among people who lived within 2.5 miles from a Soviet biological warfare facility
  • Livestock died within 30-mile zone
  • 1992: Soviet officials admitted that an accident happaned and anthrax spores were released
  • 77 cases and 66 deaths
  • cause of death: inhaltional anthrax



Ameritrax attack--2001


  • Sep-Nov 2001
  • Intentional spread of weapons grade anthrax spores via US Postal System
  • Targets included Members of Congress and Federal employees
  • Postal workers unintentionally exposed
  • a total of 22 cases: 11 cutaneous anthrax; 11 inhalational anthrax; 5 deaths (all due to inhalational anthrax)



pop quiz: Inhalational anthrax is contagious. (T/F)


false



"Giving the Finger to Bioterrorists"
  • A 30-year assistant at New York Post, developed an "itchy bug-bite-like bump" on her middle finger
  • Within next several days, the llesion evolved to (picture)
  • went to walk-in clinic: recieved Augmentin
  • Two days later: couldn't bend the finger, felt throbbing
  • Went to ER: doctors seemed puzzled but, knowing she was on strong medicine, sent her home
  • Next day: back in th ER because of increaseing pain in the finger and fever. The decision was made to biopsy the lesion. culture of the lesion reveled the following: (look @ "lab diagnosis" NC)



lab diagnosis (bacillus anthracis)


  • "Medusa head" colonies
  • Non-hemolytic, nonmotile
  • smears: large gram positive rods in chains
  • biopchemical testing: subsequently, strain was subjected to DNA sequencing by CDC--Ames isolate, allowed FBI to track the source of the spores




bacillus anthracis culture on BAP



gram stain bacillus anthracis in tissue (single or bacilli with capsule)



gram stain bacillus anthracis in culture (chain of rods with spores)


anthrax treatment


  • IV Penicillin
  • If allergic to penicillin, IV Ciprofloxacin
  • (Administer radifly and in high dose)
  • Altermatively, IV Doxycycline
  • Suspected biological warefare: Ciprofloxacin, 500mg p.o. (by mouth) twice a day for 6 weeks; OR Doxycycline, 100 mg p.o. twice a day for 6 weeks



anthrax prevention


  • treatment of imported animal materials with formaldehyde
  • decontamination of environmental surfces in wool processing factories with paraformaldehyde
  • vaccination--only those @ high risk (painful!!)
  • Select Agent Program (US): monitor those with access



vaccination (anthrax)


  • vaccine containing Prootective Antigen (PA)
  • 92.5% effective
  • Mandatory (since 1998): members of US Army Forces
  • Reecommended: "Wool sorters", Veterinarians, Biomedical researchers



Bacillus cereus General Characteristics


  • colonies resemble those of B. a.
  • No capsule
  • Hemolytic
  • Motile
  • Some strains are virulent in humans



Bacillus cereus Epidemiology


  • Portal of entry: GI tract
  • Transmission: spores are heat stable
  • Rice reheated --> germination of spores --> proliferation --> enterotoxin production --> food poisoning "Fried Rice Syndrome"



Bacillus cereus Virulence Factors


  • Heat-stable enterotoxin (Cereulide--cyclic peptide similar to vancomycin) → Emesis
  • Heat-labile toxin (necrotic toxin) → diarrhea; eye and wound infections
  • cereolysin (hemolysin) → eye and wound infections
  • Phospholipase C → eye and wound infections



Bacillus cereus Diagnisis, RX, Prevention


  • Lab DX: rarely done, disease is self-limiting and relatively mild
  • Treatment: Normally supportive, oral rehydration, IV rehydration where necessary
  • Prevention: Avoid reheated rice



Corynebacterium diphtheriae general characteristics


  • gram-positive, non-motile
  • arranged as "chinese character"
  • metachromatic polar granules




C. diphtheriae: chinese letter morphology


corynebacterium diphtheriae major virulence factor



Diphtheria Toxin (this toxin is the only thing that cuases disease, strains that lack toxin do not cause disease)
  • encoded by bacteriophage
  • A-B toxin ("Active-Binding toxin")
  • ADP-ribosylating enzyme
  • inactives elongation factor 2 (EF-2)
  • Stops protein synthesis resulting in cell death




diphtheria toxin modifies EF-2, inhibiting protein synthesis


Diphtheria Epidemiology


  • Reservoir: Humans are the only host
  • Transmission: via respiratory droplets; direct contact (skin diphtheria)
  • Seasonality/Geography: Winter/Temperate Climate
  • Carriage: 3-5% in endemic areas (where vacciniation in incomplete or unavaliable)
  • U.S.: No nw cases reported in last 3 years



Diphtheria Pathogenesis and Cliniacl Aspects:


  • No invasion--located on epithelia of upper respiratory tract
  • Entirely toxin mediated:
  • sore throat
  • malaise
  • low grade fever
  • diphtheritic membrane
  • airway obstruction
  • systemetic diphtheria: cardiac, CNS (spreading of toxin to cardiac muscle is most common COD)




Diphtheritic Membrane (damages lining of trachea → this can grow and block air passage)



"Bull Neck"--caused by Diphtheria Toxin


Dipphtheria Lab Diagnosis


  • Loeffler: for smears to demonstrate metachromatic granules
  • Tinsdale: (selecctive/diffferential medium): highly diagnostic; black colonies; brown halo
  • toxin demonstration: in vitro (immunodiffusion); in vivo (guinea pigs)




C. diphtheriae on Tinsdale agar (small black colonies with brown halos)



toxin precipitation (Elek Probe)
  • center: paper soaked in anti-toxin antibodies
  • pos: has toxin--bacterial growth +ppt
  • test: patient sample--has toxin in this case--bacterial growwth +ppt
  • neg: no toxin (bacterial growth, no ppt)



C. Diphtheria Treatment and Prevention


  • Erythromycin
  • Antitoxin
  • DaPT (produce antibody response to active form): Diphtheria toxoid; B. pertussis antigens; tetanus toxoid



Listeria moncytogenes general characteristics


  • pleomorphic
  • resembles corynebacteria amd streptococci
  • BAP: narrow zone of beta hemolysis; indistinguishable from GBS
  • CAMP positive
  • Motality at room temperature
  • Intracellular bacterium (invades our cells and multilys inside host)--highyl virulent (only need to be exposed to 100 of fewer organisims to establish infection)




CAMP tast for Listeria monocytogenes (L. m. makes CAMP factor that ↑ beta-hemolysis region


Listeria monocytogenes Major Virulence Factors


  • Adhesion-important for inital attachmetn
  • Invasins-Internalins (InlA and InlB)
  • Listeriolysin (LLO, pore forming toxin): cytotoxic; facilitates phagosomal escape
  • Phospholipase C (PLC)
  • Actin nucleator, ActA: promotes transfer between cells



Listeria monocytogenes Epidemiology


  • Ubiquitous--found in soil, silage, water sources, natural host may be amoeba
  • Facultative pathogen--GI (5-10% carriage rate; disease (immunocompromised people or pregnant women)
  • food-borne pathogen
  • US: 759 cases in 2008



Listeria monocytogenes Pathogenesis


  • Bacterial actively invade internal epithelial cells (non-APCs) through internalin proteins
  • Bacteria also invade/phagocytosed by macrophages
  • Bacteria uitilize Phospholipases/LLO to escape from membrane compartments
  • Bacteria replicate in host cytoplasm
  • Bacteria stimulate "actin comet" formation to move within the cell and penetrate adjacent cells




Listeria monocytogenes Life Cycle:
  • LLO and PLCs (escape vaccule)
  • ActA (move to next cell)



Clinical Aspects Listeria monocytogenes


  • Enters the bloodstrem: bacteremia/sepsis
  • Crosses the BBB: meningitis in infants (transvaginal transfer)
  • Crosses the placenta: Granulomatosis infantiseptica



Listeria monocytogenes: Lab Diagnosis


  • Hemolysis on BAP: narrow band
  • Catalase positive
  • Motility at room temp
  • CAMP positive



Listeria Treatment and Prevention:


  • Antibiotics: Penecillin, Ampicillin, Erythromycin, Chloramphenicol
  • Pregnant women: Avoid raw mailk, Avoid soft cheese




  • the ocular has a magnification of 10X
  • 4 objectives: 4X, 10X, 40X, 60X
  • Total Magnification: ocular x objective
  • Always focus on teh lowest power FIRST
  • ONLY use lens paper to clean the lenses
  • do NOT use paper towel as it may scrctch the lenses



Bacterial Staining


  • To provide the contrast so the cells can bee seen more easily
  • To study their microscopic properties
  • To divide bacteria into sppecific groups for diagnostic purposes
  • Simple staining: direct stain (stains the bacteria); negative stain (stains the background)
  • Differential staining: seperation into groups; visualization of structure




  • Rationale: Differentiate bactria into G+ and G-
  • Crystal violet (CV)binds to peptidoglycan (PPG)
  • Iodine (I-, aka mordant) complexes with CV
  • Ethanol (EtOH, aka de-colorizer) dehydrates and shinks G+ cell walls; leaves lipid-rich G- cell walls permeable
  • H2O washes out the CV-I complex from G- but nut G+ cell walls
  • Safranin (counterstain): stains "insivible" G- cells



Culturing bacteria


  • growth medium: a mixture of substances on or in which bacteria grow: solid medium (agar plate, agar slant); liquid medium
  • Inocculum
  • Inoculation
  • Culture
  • Subculture



Selective media


  • promotes growth of certain bacteria and inhibits others--don't want fast bacteria to grow and take over
  • example: Lowenstein-Jensen agar



Differential media


  • allow certain bacteria to be distinguished from others by the apperance of their colonies
  • contains compounds that will be used by some, but not other bacteria; allows you to see differences between bacteria
  • example: blood agar



Selective-Differential media


  • select and differentiate between certain species at the same time
  • example: MacConkey agar



Blood Agar Plate (BAP)


  • Differential
  • contains 5% sheep RBCs
  • based on hemolysis
  • alpha hemolysis = imcomplete: partial destruction of RBCs; appears dark and greenish, can't see through
  • Beta hemolysis = complete: total lysis of RBCs; cleat or transparent, can see through



Chocolate agar (CHOC)


  • neither selective nor differential
  • contain RBCS which have been lysed
  • Hemin and NAD from RBCs in media
  • This allows fastidious bacteria to grow because the nutrients are there already lysed from RBCs waiting for them!



MacConkey Agar


  • Both selective and differential
  • contains 3 ingredients: Lactose (D); Bile (S); Crystal Violet (S)
  • Lactose + colonies are PINK
  • Lactose - colonies are COLORLESS
  • G-GI pathogens have NO problems growing here



Pure Cultures


  • contain only one type of organism and are suitable for the study of their cultural, morphological, and biochemical properties
  • colony: individual, macroscopically visible masses of microbial growth on a solid medium surface, each representing the multiplication of a single cell




Streaking a medium:
  • to isolate single bacterial colonies and study their cultural and biochemical charateristics
  • Hemolysis
  • Size
  • Pigmentation
  • Form
  • Elevation



General Rules for Reading a Demo Plate


  • Determine media used to culture organism: blood agar (look for hemolysis); chocolate (consider fastidious organisims); MacConkey (determine lactose use)
  • Describe colony morphology: size, pigmentation, form
  • Determine if there is a notable odor




Staphylococcus aureus:
  • Staphyle = clusters ("grape-like clusters")
  • Large golden colonies
  • Creamy appearence on plate
  • Narrow beta-hemolysis zone around colonies
  • large golden colonies




Staphylococcus epidermidis:
  • Hemolysis = none
  • Medium white colonies
  • Staphyle = clusters
  • part of the normal microbiota of human skin
  • "prosthetic devices"--catheters, heart valves, joint replacements
  • opportunistic pathogen




Streptococcus pyrogenes (GAS):
  • Large beta-hemolysis zone around colonies (destroys RBCs)
  • Small "pinhead" colonies
  • strep = chains (long slender chains)
  • NOT part of normal flora




Steptococcus agalactiae (GBS):
  • Narrow beta-hemolysis zone, usually under colonies
  • medium white colonies
  • step = chians (shorter chains than GAS)
  • "B" mnemonic: Birth relates; Baby's Brains--causes neonatal meningitis; Breathing




Streptococcus pneumoniae:
  • alpha-hemolysis with greenish bruising
  • small colonies
  • lancet-shaped diplococci (not chain)





Enterococcus faecalis:
  • Usually no beta-hemolysis but sometimes weak alpha hemolysis
  • medium white/greyish colonies
  • normally found in the intestinal tract



gram+ vs. gram- bacteria


Gram+
  • Cocci
  • Stain PURPLE
  • ~40 layers of PPG which makes is easy fo Crystal Violet (CV) to bind
  • No lipopoly saccharide protective layer
  • no outer membrane
Gram-
  • rods
  • stain RED
  • ~3 layers of PPG, CV can't bind as easily therefore it washes out!
  • lots of lipid, easy for CV to rinse out



WHY Crystal Violet?


  • binds well to PPG (which G+ cocci have a lot of, but soth G+ and G- apppear purple after this step)



WHY Iodine?


  • complexes with CV (forms CV-I complex) and helps locks color into G+
  • at this point, both G+ and G- bacteria are both purple because nothing has washed away the CV from the G- yet
  • So if you didn't use Iodine, the G+ will become decolorized in the next step (and in the end will appear red just like the G-, not allowing you distinguish between them)



WHY Ethanol?


  • dehydrates and shrinks G+ cells and leaves G- lipid rich walls permeable
  • G+ are now permanently purple, while G- lis invisible because it washes the CV out of the cell
  • If you skip this step all bacterial will appear purple like gram+ in the end because there is nothing to decolorize the G-, thus it will not counterstain red in the step



WHY Safranin Red?


  • counterstains G- bacteria red



Miss CV or I?


the bacteria all stain red


Miss EtOH?


the bacteria will all be purple


Know why we streak for isolation


  • So that we can seperate single bacterial colonies and observe their colony morphology
  • Like: hemolysis (alpha or beta or none), size, color, odor, etc.



Nasopharyngeal Micobiota


  • Alpha-hemolytic (viridans) streptococci
  • Nesseria species (saprophytic Neisseriae)
  • Moraxella catarrhalis
  • Corynebacterium species (diphtheroids)
  • Streptococcus pneumonia
  • Staphylococcus aureus
  • Staphylococcus epidermidis
  • Candida albicans (fugus/yeast)
  • Haemophilus influenzae
  • Anaerobic bacteria (Fusobacterium, Antinomyces etc.)



Asymptomatic Carriage


  • an infection with with no noticable symptoms
  • streptococcus pyrogenes (GAS)
  • Nisseria meningitidis



Identifying Bacteria Tests of choice


  • catalase
  • coagulase
  • mannitol fermentation (on mannitol salt agar)
  • baciracin sensitivity
  • CAMP
  • Optochin
  • Bile esculin
  • 6.5% NaCl




staphylococcus aureus BAP



staphylococcus aureus OR staphylococcus epidermidis gram stain



staphylococcus epidermidis BAP


catalase test


  • Rational: To differentiate staphylococci (Cat+) from streptococci (Cat-)
  • Catalase breaks down hydrogen peroxide into water and oxygen, resulting in bubbles: 2H2O2 → 2H2O+H2




  • Principle: Coagulase promotes conversion of fibrinogen into fibrin → clotting
  • Rational: to differentiate S. areus from other staphylococci
  • Procedure: Inocculate plasma with S. aureous.
  • expensive, takes hours
  • S. aureus uses this enzyme to protect itself from the immue system--forms a fibrin clot aroud itself so that the immune system cannot see it




Clumping Test as a substitute for coagulase test
  • on slide: inexpensive, takes seconds
  • when mixed with plasma (we used rabbit plasma), S. aureus cells clump and produce "snowflakes" (+)
  • if it is milky, then it is negative
  • clumping test tells indirectly that coagulase is present




Mannitol Salt Agar (MSA):
  • MSA = selective/differential
  • for primary isolation and differentiation of S. aureus
  • selective (salt), inhibits most G+ and G- bacteria
  • differential (mannitol): S. aureus ferments mannitol, resulting in acid production → yellow
  • differential S. aureous from other staphylococci




streptococcus pyogenes (GAS) BAP



streptococcus pyogenes (GAS) gram stain



GAS is sensitive to Bacitracin:
  • Rationale: Group A strep is susceptible to the antibiotic bacitracin
  • If an organisim is susceptible to the drug, a zone of growth inhibition appears where the drug diffuses out
  • Purpose: to differentiate S. pyrogenes from other Sbeta-hemolytic streptocci




Streptococcus agalactiae (GBS) BAP



streptoccus agalactiae (GBS) gram stain



GBS is CAMP positive:
  • GBS produces a factor that enhances the lysis of RBCs in teh presence of S. aureus
  • POsitive: "arrowhead" forms at junction of teh 2 organisims
  • purpose: to differentiate GBS from other beta-hemolytic streptococci




steptoccus pneumoniae BAP



streptococcus pneumoniae gram stain



s. pneumoniae is sensitive to optochin:
  • if an organism is susceptible to Optochin, a zone of growth inhibition appears where the drug diffuses out
  • purpose: to differentiate S. pneumoniae from other alpha-hemolytic (viridans) streptococci




E. faecalis and S. bovis:
Bile Esculin Agar:
  • rational: both E. f. and S. b. hydrolyze esculin to esuletin
  • positive: the plate tuns black
  • purpose: to differentiate E. f. and S. b. from other streptococci
6.5% NaCl growth test:
  • Differentiate between E. f. (+) and S. b. (-)




Neisseria meningitidis BAP and CHOC agar



Neisseria meningitidis gram stain of CSF


Minimum diagnostic criteria corynebacterium dipheriae



  • Oxidase +
  • Glucose +
  • Maltose +




Haemophilus influenzae



satellite test


Listeria monocytogenes


  • G+ rod
  • the "hot dog bug"--loves many temperatures and food, such as milk, soft cheses, lunch meats, etc.
  • EXTREMELY hazardous during pregnancy--causes stillbirths
  • distinctive motality: if grown @ 37°C organisims are nonmotole cocci; if grown @ 25°C organisims are motile rods
  • small grey-white colonies
  • identical twin of GBS (S. agalactiae): CAMP+; narrow beta hemolysis



Differentiation of L. monocytogenes from GBS


  • motility test: listeria is motile, GBS is non-motile
  • gram stain: Listeria is rod-shaped, GBS are cocci
  • catalase test: listeria is cat+, GBS is cat-



Corynebacterium diphtheria


  • small, white colonies on BAP
  • Black colonies on Tinsdale agar
  • Distinctive garlic odor
  • gram stain: distinctive "Chinese character", or "X-Y" arrangement
  • Definitive test: toxin demonstration




C. diphtheriae methylene blue stain



toxin demonstration (Elek Test)--C. diphtheria ppt


Strep can be divided into 3 groups


  • beta, alpha, and non-hemolytic
  • in order to differentiate between the different streps, it is important to look at hemolysis
  • beta-hemolytic is the MOST pathogenic
  • alpha-hemolysis does not always meano pneumo, it can also be various other viridans (also strepto but don't have to remember the names!)
  • beta = GAS, GBS
  • alpha = S. pneumo, viridans
  • non-hemolytic = E. fae, S. bovis (GDS)



viridans


  • streptococci
  • viri-green
  • normal flora in throat (nasopharyngeal)
  • if it moves through the blood dtream and lodge in heart, can cause sub-acute endocarditis
  • important clinical relevance: "clenced-fist syndrome"--hit someone in the mouth, fingers have little padding, therefore bane makes contact with teeth. If teeth have viridans bacteria and they get into bone, osteomyeletis can occure
  • why it is important to perform Optochin test and differentiate between S. pneumo and alpha-hemolytic viridans streptococci



Staphylococcus aureus


  • G+ cocci
  • on a BAP media--colony morphology: NARROW zone of beta-hemolysis around colonies; LARGE GOLDEN colonies; creamy appearance on plate
  • microscopic morphology: staphy = clusters
  • TEST OF CHOICE: coagulase/clumbing test (catalase +/coagulase+); Mannitol Salt Media (S. aureus ferments mannitol, changing the pH, plate turns yellow)



Staphylococcus epidermis


  • G+ cocci
  • on BAP media--colony morphology: MEDIUM sized WHITE colonies; NO hemolysis
  • Microscopic Morphology: staph = clusters
  • part of normal flora on human skin
  • LOVES prosthetic devices
  • opportunistic pathogen
  • catalase +
  • coagulase -



streptococci pyogenes (GAS)


  • G+ cocci
  • on a BAP media--colony morphology: LARGE zones of beta-hemolysis; SMALL PIN-HEAD colonies
  • Microscopic Morphology: strep = chains; GAS = LONG, slender schains
  • NOT part of the normal flora
  • catalase -
  • TEST OF CHOICE = Bacitracin Test (BAC): BAC is anantibiotic that GAS is susceptible to, therefore a zone of growth inhibition grows aroud BAC disc in the BAP plate
  • To help yor remember: both middle letters are A or GAS comes out your BAC



streptococci agalactiae (GBS)


  • G+ cocci
  • on a BAP--colony morphology: NARROW beta-hemolysis UNDER colony; MEDIUM WHITE colonies
  • microscopic morphology: step = chains; GBS = shorter chains that GAS
  • TEST of choin = CAMP Test (GBS is CAMP +): GBS produces hemolysin in a BAP plate; this hemp;ysin enhances the lysis of RBCs in the presence of S. aureus; arrowhead forms at the junction of teh two
  • to help you remeber: Ypu are going to take your Baby to CAMP Arrowhead



Streptococcus pnumoniae


  • G+ cocci
  • on a BAP--colony morphology: alpha-hemolysis; small colonies; mucoid, crater like colonies (FYI: mucoid = encapsulated bacteria = #1 virulence factor)
  • microscopic morphology: lancet-shaped DIPLOCOCCI--NOT chain
  • TEST OF CHOICE = Optpchin Test: Optochin is a detergent that causes the autolysins in S. pneumo to work to dissolve itself; ZOne of growth inhibition around the optpchin disc in the BAP media; Bile is another alternative to optochin disk; thisis how we differentiate s. pneumo from other viridans



Enterococcus faecalis (E. fae) aka Streptococcus faecalis


  • G+ cocci
  • on a BAP media--colony morphology: NO hemolysis; MEDIUM GRAY-sh colonies
  • microscopic morphology: strep = chains
  • nosocomial--hospital acquired infections
  • E. fae and S. bovis (GDS) are similar in appearance on plate
  • clinical relevance of distinguishing of differentiating between the 2: S. bovis is linked to colon cancer; of you get colon cancer you will inevitably get S. bovis; if you have S. bovis they should check you for colon cancer
  • TEST OF CHOICE = Bile Esculin Agar: Both E. fae and S. bovis will hydrolyze esculin to esculetin therefore turning the plate black--then do: 6.5% NaCl growth test: differentiates the two bacteria; E, fae will grow (+) (because it is used to being in low pH of the GI tract) where as S. bovis will not (-)



cornebacterium diphtheriae


  • G+ rod
  • on a BAP media: SMALL, WHITE colonies
  • microscopic morphology: distinctive "CHINESE CHARACTER" or "X-Y ARRANGEMENT"; on special stainL METACHROMATIC GRANULES
  • GARLIC odor
  • definitive test: toxin demonstration (Elek Test)
  • on tinsdale madia: C. diphtheriae produces black colonies with brown halo
  • diagnostic criteria: Oxidase +, Glucose +, Maltose +



Listeria monocytogenes


  • G+ rod
  • on a BAP media: SMALL GREY-WHITE colonies; narrow beta-hemolysis (looks exactly like GBS)
  • "hot dog bug": loves milks, soft cheeses, lunch meats, etc.; hazardous during pregnancy because can cross the placenta and cause still birth
  • distinctive motality: non-motile cocci when not at room tep; MOTILE RODS when grown at ROOM TEMP
  • CAMP+ (like GBS)
  • differentiation between L. monocytogenes from GBS = motality lest (listeria is motile at room temp. GBS is NOT); catalase test (listeria = catalase +, GBS = catalase- (because it is a strep)



Haemophilus influenzae (H. flu)


  • G- pleomoph
  • media: chocolate agar
  • colony morpholgy: SMALL, GRAY-ish, MUCOID; DEWDROP-LIKE colonies
  • microscopic morphology: PLOMORPHIC--same as poymorphic - different shapes
  • "MUSTY BASEMENT ODOR"
  • FASTIDIOUS: needs X and V factor (X = blood = hemin; V = sugar = NAD); on chocolate media both and X and V factor are present
  • TEST OF CHOICE = XV test; Satelite Test (poor man;s XV test): S. aureus straked on a BAP (after H flu is streaked) lyses RBC's releasing both the X and V factors, therefore H. flu cna grow; H. flu colonies only grow right around the S. aureus streak becasue only close to S. aureus are the RBCs lysed and X and V released



Nesseriae: N. meningitidis and N. gonorrhoeae


  • look similar to H. flu; both N. are identical to one another on CHOC
  • G- diplococci
  • both can grow on CHOC
  • N. men. can grow on BAP; N. gono can't
  • TEST OF CHOICE = sugar test: N. meningitidis (uses malose and glucose mg=mg); N. gonorrhoeae uses glucose only (g=g)



plates streaked with S. aureus


always streak GBS of H. flu first, than S. aureus
  • CAMP Test: GBS
  • Satalite Test: H. flu



Mannitol Salt Agar (MSA)


  • selective/differential
  • selects (salt) inhibits most G+/G- bacteria from growing
  • differentail (mannitol) mannitol can fermented by S. aureus, so this media differentiates S. aureus from other staph (becuase it is the only on that can ferment mannitol)
  • mannitol fermentation results in acid production, pink → yellow
  • when you streak for isolation in this media, a section of it will remain pink, why? becuase the isolated colonies don't have enough bacteria to produce enough acid to perform a color change like on the rest of the plate, or colonies simply did not grow there at all



test break down


  • S. sureus = mannitol and coag/cat test
  • GAS = BAC
  • GBS = CAMP
  • S. pneumo = optochin
  • H. flu = satalite



Medically Important Enterobacteriaceae (Lab)



  • All members are G- rods, ferment glucose, reduce NO3 to NO2

  • Clinical Relevance: The most common cause of UTI in females is E. coli. Numerous other bacteria can cause UTI's though, so in order to differentiate what bacteria it is, doctors will measure the NO2 in the urine. Because it is formed by E. coli, the patient's will have high level in their urine if they have a UTI caused by E. coli.

  • Look essentially the same on blood agar

  • On MacConkey agar: Lac+(pink) and Lac-(colorless)

  • Produce distinctive patterns on TSI, Indole, and Urea tests (T-I-U)




First, look at colony Morphology and patterns of Lactose use on MacConkey agar



  • E. coli (lac+/-)

  • Klebsiella pneumoniae (lac+)

  • E. coli 0157:H7 (lac+)

  • Shigella sp. (lac-)

  • Proteus sp. (lac-)

  • However, colony morphology alone is not enough to ID the organisms




T-I-U


  • 3 tests: Triple Sugar Iron (TSI); Indole (Tryptone); Urease (Urea)
  • It is necessary to observe the patterns of all three tests (T-I-U) in order to make an accurate diagnosis.




  • Intact T-I-U
  • Each organism has its own distinct pattern on T-I-U tests
  • Allows presumptive ID on most Enterobacteriaceae members




Test #1 Triple Sugar Iron (TSI) contains:


  • The contents of the tube are red before adding that bacteria because it is made up of peptone (peptides) which is a Nitrogen source

  • Triple sugars: glucose, sucrose, lactose

  • Nitrogen source peptone (peptides)

  • Sulfur source: sodium thiosulfate which can then be converted to H2S and bind to iron (if H2S is produced, remember the butt turns black)

  • Iron: H2S indicator

  • pH indicator: phenol red, turns yellow in acidic conditions (when the bacteria ferments the sugar)--Remember: if it turns yellow, it is fermenting that sugar, therefore it is positive for that sugar




TSI section A



  • The slant = TOP

  • Indicates if the bacterium can ferment lactose in addition to glucose

  • If the organism can't lactose, it uses peptides after using up glucose → NH3+ → makes slant alkaline (pink)

  • The production of bubbles is due to CO2: This may be hard to see on a test. This is important when dealing with Klebsiella, which has lots of gas produced.




TSI section B


  • The butt = BOTTOM
  • Indicates if the bacterium ferments glucose only
  • If the butt is yellow, bacteria fermeted glucose
  • If butt is black, H2S is produced as well as glucose fermentation




TSI Rational and Appearance:
YELLOW = Acidic
RED = Alkaline (basic: peptone → ammonia NH3)
  • YELLOW on BOTTOM: bacterium ferments glucose
  • YELLOW on BOTTOM and TOP: Bacterium can utilize both glucose and lactose
  • RED on TOP: bacteria cannot ferment lactose; digests peptides instead after using up glucose
  • RED on BOTTOM and TOP: bacterium cannot use either sugar
  • BLACK on BOTTOM: bacteria can convert thiosulfate in H2S




Test #2-Indole Test
  • AKA tryptone
  • indicates the ability of bacteria to conver tryptophane → indole
  • KOVACS REAGENT is  addded to detect indole → red lining
  • All E. coli, by definition, are indole positive




Test #3-Urease Test:


  • Indicates the ability of the bacteria to produce the enzyme urease, which converts urea → NH3 (ammonia) + CO2

  • Color change due to pH change (alkaline)

  • Yellow (acidic) to Rose (alkaline) or Magenta (alkaline)

  • Strength of pink color is dependent upon how much ammonia is produced




E. coli can be Lac+ or Lac-


  • This makes a big differences on the Mac plate and in the TSI patteren you see
  • Most E. coli are Lac+
  • The one that is Lac- is EIEC
  • 4 different types to know: EIEC, EHEC, ETEC, EPEC
  • ALL are indole +
  • ALL are urease -
  • EIEC: ONLY one the is lac-, TSI: red/yellow (lac-/glu+)
  • EHEC (aka E. coli 0157:H7): ONLY one that is sorbitol- (grows colorless on a sorbitol half plate), clinical relevance: HUS-shigatoxin damages the inside of blood vessels (capillaries) of the glomerulus in the kidney, therefor sends patient into KIDNEY FAILURE
  • EHEC, ETEC, EPEC: TSI: yellow/yellow (lac+/gluc+)




E. coli0157:H7
  • Another exception
  • Test: Streak on a Sorbitol-MacConkey biplate
  • This is the only E. coli that is colorless on sorbitol
  • Regular E. coli will appear pink on both sides of the Sorbitol-MacConkey biplate, while 0157:H7 will be pink ONLY on the MacConkey agar side
  • E. coli 0157:H7 = EHEC




T-I-U for Lac+ E. coli
  • TSI: Yellow/Yellow
  • Indole: always positive
  • Urease: negative
  • For Lac-, the ONLY diffference is TSI: Red/Yellow




T-I-U for Klebsiella pneumoniae
  • TSI: Yellow/Yellow (lac+/glu+) often with lots of gas
  • Indole: Negative
  • Urease: Weak Positive that results in rose color
  • MacConkey: PINK because lac+




T-I-U for Shigella
  • TSI: Red/Yellow (lac-/glu+)
  • Indole: Negative
  • Urease: Negative
  • MC: COLORLESS bacuse lac-




Proteus spp.
  • Proteus will not form any single colonies on a BAP
  • Instread colonies produce "SWARMING" (key word)
  • Swarming happens only on BAP and ONLY with Proteus spp.




T-I-U for Proteus spp.
  • TSI: Red/Black (lac-/glu+/H2S+)
  • Indole: depends on species--p. vulgaris (+); p. mirabilis (-)
  • Urease: Strong Positive that results in magenta color
  • MC: COLORLESS because lac-
  • PUTRID, terrible, NASTY smell




T-I-U for Salmonella:
  • TSI: Red/Black (Slight yellow in the bottom of butt) (lac-/glu+/H2S+)
  • Indole: Negative
  • Urease: Negative
  • MC: COLORLESS because lac-




Klebsiella and Proteus are both urease positive:
  • Can differentate on color intensity:
  • Proteus = Magenta
  • Klebsiella = Rose



which 2 pathogens are urease +


  • Kllebisella (rose)
  • Proteus--both kinds (magenta/hot pink)



which two pathogens are indole +


  • E. coli (all)
  • Proteus vulgaris



EIEC?


lac-


EHEC (O157)


sorbitol-


all E. coli?


indole+, urease-


LAC-?


SSEP (shigella, salmonella, proteus, e. coli)



LAC+?


KE (klebsilla, and e. coli)


GI Pathogens:


For the GI Pathogens:
  • Campylobacter jejuni
  • Shigella spp.
  • E. coli
  • Salmonella
You should know:
  • General Traits
  • Key virulence factors
  • Epidemiology
  • Pathogens
  • Treatment and Prevention




General characteristics for Campylobacter jejuni:
  • Motile
  • Comma or spiral-shaped
  • Gram (-)
  • Oxidase (+)--grows poorly in O2
  • Microaerophilic (grow at 5% O2, 10% CO2)
  • Prefers 42°C (normal avian body temp)




C. jejunji of on Skirrow medium" Selective blood agar plate supplemented with antibiotics and antimycotics to inhibit other intestinal organisms



C. jejunji gram stain


C. jejunji Virulence Determinants


  • Invasiveness: Flagella and Adhesin
  • Survival witin the phagosome
  • LPS = Endotoxin
  • Entertoxin: MOA similar to Cholera Toxin → Adenylate Cyclase ↑↑↑ → cAMP ↑↑↑
  • Cytotoxin → Tissue destruction and abcess formation



C. jejunji Pathogenesis and Clinical Aspects


  • Chicken (avian spp.) is common source os C jejuni
  • 104 Organisms (relativiely low virulence--most die in teh stomach acid) → EnteritisSite: Small and Large Intestines
  • Action: Mucosal invasion; Enterotoxin invasion
  • Results: Ulcerations (release of toxin at the site of infection)
  • Symbtoms: Fever, abdominal pain, bloody diarrhea, usually self limiting (usually do not have to prescribe antibiotics)



C. jejuni Postinfection Sequelae


  • Who? Persons with HLA-B27 type (those with a predisposition)
  • When? ~3 weeks pos C. jejuni infection
  • What? Reiter syndrome (urethritis, conjunctivits, arthritis--"can't see, can't pee, can't climb and tree"); Guillain Barre syndrom (systematic peripheral neuropathy)



C. jejuni Laboratory DX


  • Selective media: Skirrow's, Campy-BAP
  • Incubation: microaerophilic at 42°C (this temperature selects against most other flora)
  • 48 hours: mucoid, coalescing colonies
  • Identification" corkscrew-like motility, oxidase+, catalase+, hippurate+



C. jejuni Treatment


  • Fluid replacement therapy (FRT)--most people resolve in 5-10days
  • Erythromycin (more severe cases)



Enterobactiaceae Family


  • Large family of Gram-negative rods
  • Ferment glucose
  • Reduce NO3 to NO2--reduce nitrate
  • only a few cause disease in humans



Shigella spp. cause bacterial dysentery


  • bloody diarrhea
  • fever and abdominal cramps
  • strainf and painful defecation
  • WBCs in stool specimen (fecal leukocytes+)
  • mucus in the stool



Shigella spp. General Characteristics


  • Enteric Pathogen
  • Non-motile
  • Based on O antigen: 4 groups
  • S. sonnei is the most common in the U.S.



Shigella spp. Major Virulence Factors


  • Adhesins
  • Hemolysin = similar to Listeriolysin O
  • Shiga toxin (S. dysenteriae)--key virulence toxon: A-B toxin; N-glycoside activity; inhibits protein synthesis



Shigella spp. Pathogenesis


  • <100 organisms → Dysentery (Highly virulent)
  • resistance to stomach acid
  • enterotoxin → profuse watery diarrhea
  • invasion of the colonic epithelial cells → horizontal spread into adajacent cells → inflammation →  cell death and gross ulceration (causing blood to leak into the GI-track)




Colonoscopy in Dysentery--Mild dysentry: light bink color, a few lesions, a bit of blood



Colonoscopy in Dysentery--Sever Dysentry: much more serious as the disease progresses


shigella spp. Pathogensis


  • evasion of immune system via phagosome escape
  • destruction of mucosa → malabsorption → bloody diarrhea
  • neurological symptoms (e.g., seizures in kids)




shigella spp. lfe cycle:
  • moves from cell to cell causing death of old host/ ulceration
  • escapes lysosomes



Shiga toxin


  • produced by shigella dysenteriae
  • MOA: Nicks adenine residue form the 28S rRNA in teh 60S → Inhibition of protein synthesis
  • Damages the endothelial cells lining blood vessels (blood and lymph invade actual kidney tissue)→ Hemolytic uremic syndrome (HUS) or kidney failure



Shigella spp. Post infectious Sequelae


  • What? Reiter syndrome (urethritis, conjuctivitis, arthritis)
  • Who? Afflicts primarily persons with HLA-*27 type
  • When? ~3 weeks post infection



Shigella spp. Lab DX


  • Primary isolation on selective/differential media
  • Subculture on TSI (acid in the butt, no gas), indole(-), urease(-), motility(-)
  • slide agglutination in specific antisera



Shigella spp. treatment


Use ACT mnemonic:
  • Azithromycin
  • Ciprofloxacin
  • 3rd generation Cephalosporins (Ceftriaxone)
  • Trimethoprim/sulfamethoxazole
  • Oral rehydration salt (ORS)--to replace water and ions lost in diarrhea



Eterocolitis-Associated E. coli


  • Enterotoxigenic E. coli (ETEC)
  • Enteroinvasive E. coli (EIEC)
  • Enteropathogenic E. coli (EPEC)
  • Enterohemorrhagic E. coli (EHEC)



ETEC


  • major cause of traveler's diarrhea (because you have not developed resistance to strain in new H2O) and infant death in developing counntries
  • pathogenesis resenbles V. cholerae
  • mediated by heat-labile (LT) and heat-stable (ST) enterotoxins→ cAMP/cGMP → electrolyte secretion (diarrhea)



EIEC


  • causes shigellosis-like disease
  • does not produce shiga toxin (so no kidney coplications)



EPEC


  • causes childhood diarrhea
  • adherence → depolymerization of actin → collapse of cytoskeleton → loss of absorptive capacity by mucosal cells → dirrhea



EHEC = E. coli O157:H7


  • numerous outbreaks associated with consumption of hamburgers contaminated with E. coli O157:H7
  • from mild uncomplicated  dirrhea to hemorrhagic colitis and hemolytic uremic syndrome (HUS) or acute renal failure
  • Resembles Shigella-dysentery: EHEC produces Shiga toxin
  • Shiga toxin damages the endothelial cells lining the blood vessels → HEmolytic uremic syndrome (HUS) or kidney failure



salmonella enterica: General Characteristics


  • Enteric pathogen
  • motile
  • lactose non-fermenter
  • H2S producer
  • 2,000-3,000 serotypes



Salmonella enterica Epidemiology


  • Salmonella enterica
  • the 2nd most common cause if infectious dirrhea in the US
  • poultry and  milk products are the major source of food-bourne infections



Salmonella Major Virulence factors


  • Adherence and Invasion
  • PRevention of phgolysosome fusion is mechanisom of survival in the macrphages
  • can enter the bloodstream and cause LPS-mediated septic shock
  • Vi antigen (surface polysaccharide)




Salmonella:
  • (1) Epithelial cells lining small intestine
  • (2) Salmonella trigger phagocytosis
  • (3) Salmonella multiply within phagocytic vacoule (vs. in the cytoplasm)
  • (4) Salmonalla kills hos cell, inducing diarrhea
  • (5) Bacteremia: Salmonella moves into the blood stream




Salmonella


Pathogenesis and clinical aspects of Salmonella


  • Salmonella → invasion od absorptive mucosal cells and mucosa associated macrophages → decreased absorptive function of mucosal epithelial cells → diarrhea
  • Diease: mild, self-limiting dirrhea (immunoconpetent persons); systemic disease (infants adn immunocompromised); typhoid fever by S. typhi. any can get it but rare



Typhoid Fever


  • (1) S. enteric Tyhpi (always goes systemic) → intestinal wall where it multiplies → bloodstream → spleen, liver and gall bladder (immune system cannot control infection here) where it continues to multiply
  • (2) large number of S. Typhi relased into the bloodstream
  • (3) symptoms: high fever, chills, rash around abdominal area, and neurological signs
  • (4) mechanism: LPS-mediated release of inflammatory cytokines
  • usually causes death in 10% of people; many people are carriers



Salmonella Postinfectious Sequalae


  • What? Reiter syndrome (urethritis, conjunctivitis, arthritis)
  • Who? Afflicts primarily parsons with HLA-B27 type
  • When? ~about 3 weeks post infection



Salmonella Lab DX


  • Culture: MacConkey, Salmonella-Shigella (SS), and Bismuth-sulfite agar media
  • Subculture on TSI (acid and gass production from glucose, H2S+) indole(-), urease(-)
  • slide aglutination



Salmonella treatment


  • mild uncomplicated diarrhea: NONE
  • systemic salmonallosis and typhoid fever: use ACT (Ampicillin, Azithromycin, Chloramphenicol, Ciprofloxacin, 3rd generation Cephalosporins (Ceftriaxone), Trimethoprim/sulfamethoxaole)




Helicobacter pylori (only found in humans) General characteristics:
  • microaerophilic (lives in low O2 environments--5% vs. 20%), gram negative comma or spiral shaped organsims (similar to camplyobacter spp.)
  • multiple polar sheather flagella -- highly motile with rapid corck screw motion
  • to colonize the stomach, H. pylori must survis acidic pH of the lumen and use its flagella  to burrow into the mucus to reach its niche, close to the stomach's epithelial cell layer



H. pylori virulence factors


  • motility → penetration of gastric mucus allows access to gastric epithelial cells (swims away from the acidic contents of the lumen)--swims away from the proton gradient
  • Adhesins--BabA binds to Lewisbantigen on cells
  • produces  urease enzyme: 100X more potent that other bacterial ureases
  • Usease convers ures to NH3 and CO2 (NH3 + H+ → NH4+ -- raises local pH at the gastric mucosa, H. pylori survives at pH 2-3)
  • Hemolysin (Vacuolating toxin, VacA) → epithelial tissue damage
  • Cytotoxiin-associated gene A (CagA) → alters cell cytoskeleton; stimulates IL-8 production → neutrophil recruitment → tissue damage



Pathogenesis and clinical Aspects of H. pylori


  • Urease splits urea → ammonia toxic to tissues)
  • Mucinase → degrades mucin proteins → exposes epithelial cells to  stomach acid
  • Phospholipases → degrade tissue membranes




Pathogenesis og H. pylori:
  • (1) Bacteria invade mucus and attach to gastric epithelial cells
  • (2) Helicobacter, its exotoxin, and inflammation cause the mucus layer to become thin
  • gastric acid destroys epithelial cells and underlying tissue




Peptic ulcer:
  • H. pylori, gastric acid, pepsin, and NSAINDS enter



H. pylori and gastric Carcinoma


  • H. pylori carriers → 10-20% lifetime risk of peptic ulcers, 1-2% lifetime risk of gastric cancer
  • Oathomechanism: H. pylori → chronic inflammation → destruction of surface epithelial cells → exposure of immature cells to carcinogens in diet → damage the DNA → mutation → Gastric Cancer
  • No association between H. pylori and duodenal cancer




H. pylori culture on skirrow's media



H. pylori Endoscopic Biopsy: staining with gram adn warthin-starry (silver stain)


H. pylori Lab diagnosis


  • culture: antibiotic containing media, i.e., Skirrow's
  • Urea breath test: 14C-labelled urea is injected and 14CO2 is detected
  • Serology--bllood + fecal samples (look for antibody response)
  • these are not always accure--the are likely to give a flase negative




Urea breath test--injest radioactive urea adn look for radioactive CO2


H. puylori combination therapy


First choice:
  • Bismuth
  • Methronidazole
  • Tertracycline
  • control pH the kill bacteria
Second choice:
  • Metronidazole
  • amoxicillin (or clarithropmycin)
  • omeorazole  = H+/K++-ATP-ase Antagoonist
  • also kills bacteria and controls stomach pH




Vibro cholerae:
  • Gram (-), comma-shaped
  • Optimum pH 8.0-9.5 (one of the only bacteria the like a basic pH)
  • Motile
  • Oxidase positive



Vibro cholerae Antigenic Structure


  • LPS → lipid A, core oligosaccharide, O-antigen → 139 O-serotypes
  • V. choleraeO1 and O139 → Choler toxin → Epidemic chlolera
  • V. choleraeO1 further divided ito two biotypes: classical (chollerae) adn El Tor (current pandemic biotype)



V. cholerae Major Virulance Factors


  • Type IV Pilus (Toxin co-regulated pulus, tcp)
  • enterotoxin (CTx): A-B toxin; ADP-ribosylating toxin → Adenylate cyclase ↑ → cAMP ↑ → wtery diarrhea
  • Zot (zona occludes toxin)--disrupts tight junctions
  • Ace (accessory echolera enterotoxin)



V. cholerae Pathogenesis


  • Bacteria adhere to mucosal cell layer--tcp and chemotaxis proteins
  • CT, zot and ace produced
  • Toxins promote efflux of Na+ and Cl- ions
  • Water follows by osmosis
  • profuse diarrhea



V, cholerae Epidemiology


  • Halophilic--prefers high salt environments
  • Grows at 10°C-30°C (relatively low temp)
  • Transmission: fecal-oral (shellfish are carriers as well as contaminated water/common in flooded areas)
  • geographic distribution: developing world
  • Pandemics cased by V. vholeraeO1
  • 1992: Epidemic of O139 Begal cholera




Hyperactivation:
  • Cholera A-B toxin binds to Ganglioside receptor
  • increased adenylate cyclase activity
  • increase cAMP
  • loss of cell nutrients (Na+ and Cl-) which leads to osmitic upset causing Diarrhea



V. cholerae Clinical Aspects


  • Wtery diarrhea ("rice water" stool)
  • no abdominal pain
  • symptoms related to severe dehydration: cardiac failure, renal failure, acidosis, and hypokalemia
  • mortality rate without treatment: 50%




V. cholerae Lab diagnosis:
  • lac(-)
  • oxidase(+)
  • TSI: acid/acid
  • aggllutination in specific antisera




V. cholerae culture on TCBS (Thiosulfate Citrate Bile Sucrose): large yellow colonies are indicative in Vibro cholerae


V. cholerae Treatment and Prevention


  • ORS - salt, gllucose, citrate solution
  • Doxycycline or tetracycline
  • Trimethoprim/Sulfamethoxazole for children
  • Clean water and food supply
  • No perfet vaccine



vibrio parahemolyticus


  • marine organism
  • contaminated seafood
  • rare in the US
  • common in japan
  • enterotoxin-mediated
  • nausea, vomiting
  • abdominal cramps
  • diarrhea
  • fever
  • usually self-resolving



vibrio vulnificus



  • fulminant necrotizing woun infections
  • septicemia following consumption of shellfish
  • mortality rate: 50%
  • elevated case number post-Katrina




TCBS
  • V. vulnificus
  • V. parahaemolyticus
  • V. cholerae




V. vulnificus Necrosis


Pseudomonas aeruginosa


  • opportunistic gram(-) rods
  • strict anerobe--needs O2
  • Non-fermneter
  • oxidase positive
  • can grow i water
  • pigment producing




Pseudomonoas aeruginosa pigmented culture



pseudomonas aeruginosa gram stain


P. aeruginosa major virulence factors


  • adhesins
  • alginate capsule
  • elastase
  • multi drug resistance
  • endotoxin
  • Exotoxin A: MOA-DT-like; tissue damage
  • Exoenzyme U (ocular infection): Phospholipase A
  • Exoenzyme S (associated with cystic fibrosis)--MOA: Exotoxin A- and DT-like



P. aeruginosa Pathogenesis


  • normal flora
  • prefers moist areas of the body
  • one of the leading causes of nosocomial infections--because of multidrug resistance
  • opportunistc pathogen,causes disease in: immunosuppressed (cancerpatients, HIV co-infection); patients with burn injury; CF patients



P. aeruginosa Clinical Aspects


  • UTI
  • Wound infections
  • Pneumonia in CF patients
  • swimmers ear
  • malignant otitis externa in diabetics
  • nail infection
  • folliculitis
  • osteochondrisis
  • corneal ulceration
  • endocarditit
  • septicemia with a mortality rate of > 50%
  • Ecthyma gangrenosum




P. aeruginosa Nail Infection (Onycholysis)--pigmented; especially commmon in immunosuppressed



folliculitis caused by P. aeruginosa infection



corneal ulcer caused by P. aeruginosa--leads to blindness



ecthyma gangrenosum caused by P. aeruginosa infection--this is a rare presentation and only found in severly immuno supressed patients


Lab Diagnosis of P. aeruginosa


  • lac-
  • pigment pyocyanin
  • fluorescein
  • pleasant fruity odor
  • oxidase +



P. aeruginosa treatment and prevention


  • Aminoglycosides + beta-lactams: nebulized Tobramycin for treatment of CF patients with lung infections--recieve as a prophylaxis; must treat as early as possible
  • early ddetection is critical for CF patients
  •  Infection Control



Legionella pneumophila general characteristics



  • common cause of community and hospital-acquired pneumonia
  • commonly present in A/C cooling tower, shower and other water systems
  • NO person-to-persoon transmission
  • slender
  • gram(-)
  • motile
  • nutritionall fastidious (L-cysteine required)
  • chlorine resistant



legionella pneumophila pathogenesis


  • lung tropism
  • coiled phagocytosis by macrophages
  • prevention of phagolysosmal fusion
  • proliferation in the lung → cell death → necrotizing multifocal pneummonia



Legionnaire's Disease


  • Who? Immunnocomprromised, smokersm patients with chronic lung diseasee
  • Myalgia, headache, fever and chills, dry cough (contains bacteria containing mucus → but this does not spread disase)
  • chest pain, vomiting, diarrhea, confusion, and delerium
  • MOSF (multiorgan system failure) → shock → death
  • mortality rate: 50%



Other legionelloses


pontiac fever:
  • milder and less common than Legionnaires' d.
  • acute infection, spontaneous resolution



ligonella pneumophila lab diagnosis


  • lung aspirate and bronchial washing then culture on:
  • medium: buffered charcoal yeast  extract (BCYE)
  • groud-glass like colonies
  • IFA
  • serology




gram stained smear of legionella pneumophila


treatment and prevention of legionella pneumophila


  • resp. tract quinolones (levofloxacin, moxifloxacin, gemifloxacin)
  • Macroliddes (azithromycin, clarithromycin, roxithromycin)
  • Erythromycin: efficient in controlling spread of infection
  • decontamination of water supplies




staphylococcus aureus gram stain



staphylococcus aureus culture on BAP


S. areus surface antigens


  • Protein A: binds to the Fc region of IgG and makes it unavaliable to the receptor site on the phagocyte (helps evade immune response)
  • Clumping Factor: promotes adherence to tissues, catheters etc. (helps bind and colonize)
  • Teichoic Acid: mediates adhesion by binding to tissue fibronectin (helps bind and colonize)



S. aureus Extracellular enzymes


  • Coagulase: prevents opsonization and phagocytosis by using bodies own fibrin
  • Lipase: plays a role in oathogenesis of boils and acne
  • Hyaluronidase: hydrolyzes hyaluronic acid
  • Nucleases: cleave DNA and RNA-entrapment in NETs (neutrophil extracellular trap)



S. aureus Toxins


  • Hemolysin--pore forming toxin (gives us hemolysis on BAP)
  • Enterotoxin (SE)
  • Epidermolytic/Exfoliative Toxin (ET)
  • Toxic Shock Syndrome Toxin (TSST-1)



S. aureus Epidemiology


Transmission:
  • person to person via direct contact or by contaminated clothing and bed linens
  • Ingestion of contaminated food → food poisoning
  • Aspiration of nasopharyngeal secretions → pneumonia



S. aureus Inflammatory Disease


  • Folliculitis
  • Furunclosis and Carbunculosis (Boils)
  • Impetigo
  • Osteomyelitis
  • Arthritis
  • Pneumonia
  • Endocarditis




Impetigo by S. areus (can also be caused by strep. pyr.)
  • blisters filled with pus; easy to burst--leave a reddish raw-looking base (in infants)
  • Itching blisters: fliied with yellow or honey-colored fluid; oozing and crusting over
  • Skin lesions on the face, lips, arms, or legs, that spread to ther areas
  • Swollen lymph nodes near the infection (lymphadenopathy)



Toxin-Mediated Diseases


  • Staphyloccal Scaled Skin Syndrom (SSSS)
  • Food poisoning: incubation perios (2-6 hrs); s. aureus in food produces toxin → toxin injested → interacts with mucosa → vomiting, diarrhea
  • Toxic Shock Syndrome (TSS): S. aureus grows in tampon → TSST-1 enters the blood stream → fever, rash, shock (death rate 3%)




  • Epidermolytic Toxins (ET-A, ET-B)
  • Toxins cleave a desmosomal cadherin that mediates cell-cell adhesion
  • Superficial epidermis is relased forming blisters/peeling skin



Staphylococcal enterotoxin (SE)


  • disrupts intestinal mucosa to cause disease symptoms
  • intoxication NOT infection → bacteria is not replicating or even present at this point



Toxic Shock Syndrome (most severe/lethal)


  • fever
  • septic shock
  • diffuse erythematous rash
  • desquamation ofplams and soles
  • vomiting and dirrhea
  • septic shock




  • interactions of SAgs with host cells iffer from conventional antigens in teh SAgs:
  • 1. Directly bind outside the peptide-binding groove of MHC class II
  • 2. Exert biological effects without internalization or "proteolytic processing" by APC
  • 3. Are not MHC-restricted



S. aureus lab dignosis


  • colony morphology: large, creamy (golden), opaque colonies, usually beta-hemolytic
  • biochemical testing: catalase+, mannitol/salt+, coagulase+ (clumping test)



S. aureus treatment and prevention


  • beta-lactamase-resistant penicillins: methicillin, nafcillin, cloxacillin, etc.
  • if methicilli-resistant S. aureus (MRSA): Vancomycin (VRSA), Quinupristin-dalfopristin, linezolid, daptomycin
  • Non-specific prevention, i.e., washing hands, disposable aprons and gloves, etc.



Staphylococcus epidermidis


  • part of the normal skin microbiota--esily transmitted by hand to hand contact
  • produces biofilm--colonizes prosthetic devices such as urinary catheters and replacement heart valves
  • the biofilm erodes the plastic surface and/or breaks off and disseminates
  • difficult to treat



S. epidermidis treatment


  • 40% of coagulase-negative stphylocci are resistant to beta-lactamase resistant antibiotics
  • antibiotic of choice is Vancomycin
  • Rifampin and gentamicin are considered in some cases



Staphylococcus saprophyticus


  • an important cause od UTIs--second only to e. coli
  • up to 30% of UTI in young, sexually active women
  • coagulase-negative, resistant to novobiocin (differentiates from S. epidermidis)
  • often ignored



streptococcus spp. group characteristics


  • gram-positive cocci in chains (single axis of cell division)
  • catalase negative
  • nutritionally fastidious
  • hemolysis: alpha, beta, or non
  • metabolisim: aerotolerant anarobes




s. pyogenes (GAS) gram stain



s. pyogenes (GAS) culture on blood agar


streptococcus pyogenes (GAS) importnat surface antigens


  • capsule (hyaluronic acid)--blocks phagocytosis by neutrophils
  • M protein--highly anti-phagocytic, major target of adaptive immune response
  • LTA (Lipoteichoic acid)--attachment



s. pyogenes (GAS) toxins and enzymes


  • Streptolysin O (SLO)
  • Streptolysin S (SLS)
  • Streptokinase--dissolves clots within the body and allows bacteria to spread throughout the body
  • nucleases (DNAse and RNAse)
  • Hyaluronidase
  • Streptococcal Pyrogenic Exotoxin (Spe), superantigens (Spe A, B, C, aka erythrogenic toxins)



s. pyogenes (GAS) Epidemiology


  • Most streptocci are part of normal microbiota
  • HUman throat: S. pneumoniae, S. mutans, viridans streptococci
  • intestines: E. faecalis, S. Bovis, etc.
  • (women) genital tract: S. agalactiae
  • S. pyrogenes is NOT part of normal microbiota!--true pathogens




Pathogenesis and Clinical Manifestation of S. pygenes disease


pyogenic diseases


  • impetigo--not similarity to S. aureus infection
  • cellulitis
  • erysipelas
  • streptococccal pharyngitis




Erysipelas--inflammatory disease associated with s. pyogenes



strep throat--inflammatory disease associated with s. pyogenes (most common presentation)


toxigenic diseases (s. pyogenes)


  • scarlet fever: caused by spe--the same class of toxins that cause TSS
  • Streptococcal Tixic Shock Syndrome: S. pyogenes grows in infected wound → Bacteria enter the blood stream and produce SPE A (superantigen) → fever, rash, shcok (death rate 30%)




Perioral Pollar and strawberry tongue in a child with scarlet fever (s. pyogenes)



Necrotizing fasciitis (s. pygenes)



Toxic Shock Syndrome (s. pyogenes)--note the vascular compomise in streptoccal TSS


Streptococcal and staphylococcal TSS can be differentiated based on...?


isolation of s. pygenes in the blood stream


Immune-Mediated Diseases


  • acute rheumatic fever (ARF): anti-streptococal antibodies cross-react with epitopes on heart tissues leading to congestive heart failure
  • acute glomerulonephritis (AGN): Immune complexesdeposit in kidney nephrons



s. pyogenes lab diagnosis


  • catalase test (-ve), differentiates from staph. spp.
  • beta-hemolysis
  • sensitivity to bacitracin



s. pyogenes treatment


  • penicillin in most cases
  • clindamycin in TSS--inhibition of toxin synthesis



steptococcus agalactiae (GBS)


  • beta-hemolytic streptococcus
  • causes:
  • neonatal meningitis--transfer from the birth canal
  • RDS (Respiratory Distress Syndrome)
  • Sepsis