Gram Positive BacilliAerobe and Facultative Anaerobe Flashcards
Why are gram positive bacilli significant
-not highly pathogenic
-associated with soil and water
-common lab contaminants and part of normal F
-can be aerobic or facultative anaerobic
Two groups
1-with endospores inside
2-Cell shape (regular, irregular, or branched_
size large or small
non spore forming
What group 1 endospores like
environmental spores
usually single spores
-in unfavorable conditions the cells disintegrates
and releases the spore which can be in the environment for years
-vegetative cells can produce ONE single spore and one spore can germinate into a vegetative cell
-on a gram stain the spores are wide
-can be terminal or subterminal
-will not take up stain so it will look clear
as the cultures get older the cell wall deteriorates and they loose the ability to hold onto CV/Iodine complex
What are group 2 GPB
Cellular size
-Large or small
Cellular shape
-regular- long sides straight and parallel
-irregular- long curved sides not parallel (small irregular are Vs and pallisades)
-branched - tree like and elongated
Habitat and condition for Bacillus and Clostridium
- part of family bacillaceae
-clostridium likes anaerobic best and its catalase neg
Bacillus is found as
-psychrophiles - likes cold temps
-thermophiles - likes warm or hot temps
-soil and aquatic areas as spores like to move as the soil and water moves
-areas with high acidity and alkalinity
-NF in humans and animals
environmental
how can you grow bacillus and what are two example species
-grows on BA
-non pigmented colony
-Catalase Positive
-form endospores in O2
-common as lab contaminants seen as one or two colonies away from the streak lines and not in quadrants
-larger colony more then GN bac
-grey ground glass appearance
B. anthracis: Anthrax
Obligate pathogen of animals and humans
Often as a spore outside of animal or human host
B.cereus: Food-poisoning
Other Bacillus species: Opportunistic infections
What are some virulence factors of bacillus anthracis
-capsule has glutamic avid
-3 part protein as exotoxin
BOTH ARE CARRIED ON PLASMIDS
-Anthrax toxin has 3 proteins seperately they arent toxic but together act synergistically to damage cells
PA - protective agent
EF - edema factor that is adenylate cyclase to increase CAMP in host cells
LF- lethal factor - protease that kills cells
LF + PA = cell death
EF + PA = edema
What is anthrax
-used to be leading cause of death in herbivores like cows because they would eat plants with spores but now we have a vaccine
-life threatening in humans, transmission from accidental or occupational exposure
-black necrotic ulcerations that can lead to MENINGITIS
-recovery is immunity
-was seen in Sask - bison
-using cow hide to make drums
-reservoir in soil
endemic
What is the cutaneous form of anthrax
Cutaneous
Intestinal
Inhalation/ pulmonary
Injectional
most common
- enters through skin breaks caused by cut, bites
-then pimple forms
-then Vesicle forms erythematous ring where the center gets black and dries forming depressed necrotic area (eschar)
-heals in 2 weeks
-falls off and scar forms
What is the Intestinal form of anthrax
Cutaneous
Intestinal
Inhalation/ pulmonary
Injectional
-when you eat contaminated meat
-diarrhea, vomiting, fever, anorexia
-higher fatality than cutaneous because of challenges in diagnosis
What is the Inhalation/ pulmonary form of anthrax
Cutaneous
Intestinal
Inhalation/ pulmonary
Injectional
-spore inhalation into pulmonary parenchyma
-macrophages in lung take them up and spread through lymphatic system if untreated can go into the bloodstream to cause fatal septicemia and toxemia
-can cause respiratory distress and disorientation
-if untreated 100% mortality
What is Woolsorters disease?
pulmonary anthrax
-rag pickers disease
-sheep shearing
-handling animals fibres and hides that have spores in them
-through skin or inhalation of spores
-can cause all 3 forms of anthrax
starts off an non specific illness and then turns serious
pleural effusion coma
What is injectional anthrax
-spores injected into soft tissue ; drug use
-like cutaneous anthrax with no black eschar more serious
-large mortality rate
-necrotizing fasciitis, meningitis, organ failure and shock
What type of lab testing is done for anthrax bacillus species
-collect using swabs for cutaneous anthrax
-collect sputum -pulmonary
-cultures if severely ill -pulmonary
Grows on
-BA w/ 5%SB grow in RT or CO2, less growth in ANO2
-MAC: NG
-Bacillus species grow on PEA agar (B. anthracis does not)
-
how does Bacillus anthracis grow differently then regular bacillus
-on culture FLAT GREY NH, irregular margin “MEDUSA HEAD” with chains from surface
-holds onto agar tightly - tenacious
-edges lift up like you just beat an egg
-shape of bamboo rod on gram stain
-chains of large GNB with central unstained spore
-NH
-NON MOTILE
-NG ON PEA
-MAKES LECITHIN
What is Bacillus cereus
food poisoning and opportunistic
-food poisoning from food with spores in it )meat fish pasta)
-spores germinate during storage and bacteria produces enterotoxins and hurts the gut
-since the condition occurs because of the toxin and not the bacteria it is called “food intoxication” and does not need antimicrobial treatment but supportive therapy like hydration
non gastrointestinal
Eye infections: keratitis with abscess formation, penetrating ocular trauma (eye swabs)
Meningitis, septicemia
Treatment: vancomycin with aminoglycoside
What are the two forms associated with Bacillus cereus
Diarrheal
-LONG incubation 8-12 hours from eating fruits or veggies /meat that didnt get stored properly
-enterotoxin is HEAT LAIBLE
-tummy pain, diarrhea
-done in 36 hours
Emetic
-SHORT incubation 1-5 hours after eating COOKED or STORED FRIED RICE
-heat STABLE enterotoxin
-done in 10 hours
how can Bacillus cereus cause opportunistic infections
-bacteremia
-endophthalmitis - eye trauma or from seeding bacteremia can cause loss of sight
-osteomyelitis, Necro fas
-risk for infection is higher if you have burns wounds where bacteria can enter, on dialysis , indwelling catheters
-opportunistic because it is NF or common contaminant
What will you see in lab testing of Bacillus cereus
-3-8mm raised, grey green frosted glass appearance
-large zone BH
-large GPB - parallel sides and regular
What do the Bacillus species look like
-GPB short or long = spore forming
-if they are short they are just dividing
- spores can be elliptical, round or egg shaped and can be central, terminal or sub terminal
-use spore stain Wirtz-Conklin
-over decolorizes very easy but look for large box car shape
how to distinguish GBP from GNB use the KOH string test
-mix isolate with 3% KOH and GNB will be sticky and thick (pos) and GPB will stay in solution (NEG)
-FLAT GREY DULL ON BA
-MOTILE
-MAKE LECITHINASE
-BH
-G ON PEA
What is the lecithinase test- egg yolk test
- Lecithinases or phospholipases are enzymes released by bacteria that can destroy animal tissues
-usually found as emulsifying agents in tissues, serum or egg
-lecithin is part of the egg yolk and lecithinases can break it down causing a white halo around the colony on egg yolk agar
pathogenic B cerues and B anthracis is pos while the other no pathogenic bacillus spp are not
Is AST done on Bacillus anthracis or cereus ?
what is the treatment for them
-no CLSI breakpoints
B. anthracis is S to peni but can become R if beta lactams are produced
-B cereus - R to penicillin and Beta lactams
-use ciprofloxacin , doxycycline to start then orally as well same
-cattle given the vaccine
-human vaccine only for at risk and military
What are some REGULAR NON SPORE-FORMING GRAM POSITIVE BACILLI and characteristics
Listeria, Erysipelothrix, Lactobacillus
-small GPB bacilli
-regular straight parallel sides - long
-no endospore production
What is Listeria and where is it found
-only monocytogenes species associated with human disease
-found in soil and rotting vegetables, water, vac packed meat
-can make people and animals sick
-asymptomatic carriers in both
What are the virulence factors and risk factors for listeria
Virulence
-Hemolysin: listeriolysin O – damages phagosome membrane preventing macrophage from killing Listeria
-Catalase, superoxide dismutase, phospholipase C, surface protein
-Flagella
Risk
-pregnant women, transplant pts, neonates , alcoholics and pts on corticosteriods
How is listeria transmitted
-eating contaminated foods like coleslaw, cabbage, cheese, ice cream, vac packed meat
-survives in cold temps
-you can get cutaneous listeriosis by handling infected animal tissue
-mom to baby in utero
-nocosomial sometimes the source isnt found
What is the clinical significance of Listeria
-infection un common usually from neonate (early via infected amniotic fluid or late after birth - less fatal) or pregnant women (3rd trimester- seeds uterus and fetus, young children
-infect as food borne illness or outbreaks via contaminated meat
-manifest as septicemia and meningitis with high fatality rate
-invasive in elderly
-can cause still birth or spontaneous abortion **most common case
-in neonates it copies GNB septicimia
how do you diagnose Listeria
-blood, csf, (correlates with sepsis and meningitis) amniotic fluid, placenta and fetal tissue
-found in low numbers need 10 ml of CSF
-grows on BA with 5 % SB in RT, CO2 or ANO2-
-SMALL GRY/WHT SMALL ZONE BH
-if samples are non sterile put in an enrichment broth and hold at 4 and let the listeria grow to have enough numbers
-if you do a direct gram it looks like GBStrep - GPC
how to differentiate Listeria from GBS
Listeria is
catalase positive, motile- tumbling on wet prep** and small GPB
Can be differentiated from Erysipelothrix sp which is catalase negative and non-motile
Bile Esculin hydrolysis: positive
6.5% NaCl: Growth
Camp test: positive (block shape)
Semi-solid media: “umbrella motility on SIM best at RT
at 35 its not motile
Where is lactobacillus found and what is the clinical significance
-NORMAL in mouth, intestine, vagina
-v common
-opportunist for immuno comp
-keeps other bacteria from colonizing vagina byt fermenting glycogen and keeping acidic environment pH 3.5-4.5
–most are Facultative Anaerobes but some are strict aerobes
-not pathogenic very rare to be one
What does lactobacillus look like
-long slender GPB, non spore forming can be coccoid, pleomorphic (spiral and chain)
-grows on CHOC and BA
-small AH or NH
-on BA - SMALL GRY AH, FLAT, PPT, ROUGH
Catalase: negative
Motility: negative
H2S production: neg
Esculin hydrolysis: negative
Vancomycin: R while most GPB are Van S
part of NF dont want to get rid of it so no AST
What is Erysipelothrix or E. rhusiopathiae
-E. rhusiopathiae is only known human pathogen
-associated with pigs
-septicimia and arthritis in animals
-occupational disease causing lesions in people that handle meat, poultry, fish and fertilizer
-Erysipeloid (not to be confused with human erysipelas-GAS) –cellulitis –purple red hands & fingers- fish handlers can become systemic bacteremia
What is the CMI for Erysipelothrix
-ppt after 24 hours
-after 48 it has 2 types small, smooth regular and large, rough, flat AH or NH
-after 72 long filaments form
Catalase: negative
non-motile
Esculin: variable (about 50% pos)
H2S positive (from TSI) (differentiate from Listeria and Lactobacillus which are both negative)
What are some IRREGULAR NON SPORE-FORMING GRAM POSITIVE BACILLI
Corynebacterium species, Arcanobacterium, Gardnerella vaginalis
-curved
-no spores
PATHOGENIC SPECIES ARE FACULTATIVE
NON PATH ARE AEROBES
What is Corynebacteria and where is it found
Coryneform or diphtheroid” – club-shaped (depending on nutrients)
-small GPB
-seen in vees and palisades
-Skin & mucous membranes of humans and other animals -C jeikeium NORMAL on skin and Hosp (bone marrow transplants or heart surgery infections)
-significant C. diphtheriae
What is C diptheriae and where is it found
-found only in humans (upper Resp tract )
-spread by droplet or h/m
-rare
-included in DPT vaccine
What are the two types of C diptheriae
mode of transmission
treatment
virulence factor
systemic effects
prophylactic
Two Types
-Respiratory (can lead to systemic infections) deadly leads to systemic problems
-Cutaneous (associated with tropics)
mode of transmission - aerosols from convalescent and carriers - incubate for 2-5 days (then expresses exotoxin on endothelial cells)
treatment - Antitoxin , penicillin , erythro
virulence factor-diphtheria toxin (blocks protein synthesis)
Systemic effects: exotoxins that cause myocarditis , heart failure affecting the peripheral nervous system
-creates a pseudomembrane on the trachea causing suffocation
prophylactic -immunization with toxoid (vaccine)
What does C diptheriae manifest as and how to sample
-local effect
-Cell necrosis, exudate formation
-Thick, gray-brown pseudomembrane grows over mucosa of UPT triggering inflammation
-BULL NECK -can cause suffocation
-sore throat, dysphagia, malaise
take sample of throat or nasoph swab
-pseudo membrane culture incubate at 35 O2
CMI of C diptheriae
-well on BA
-Tinsdale or hoyles or loefflers agar with potassium tellurite and cystine to diff between C diphtheriae and the diphtheroids found in the upper respiratory tract.
-C Dip makes black colonies with brown halo but diptheroids cannot on Tinsdale agar
-the halo due to H2S formation because of cysteine and tellurite salt mix
Facultative anaerobe
Catalase positive
Non-motile
Uses lactose, mannitol, xylose
Esculin hydrolysis negative
Gelatin hydrolysis negative
Urease neg
Elek Test for toxigenicity**WHAT IS IT FOR>
What is the ELEK Test for toxigenicity
-done only at ref labs
-virulence test to see if isolate makes the toxin
-uses filter paper with antitoxin put in media and let solidify
-Inoculate organism and known control organisms perpendicular to antitoxin strip
–antitoxin diffuses away from paper
-toxin produced by organism diffuses away from growth
At “zone of equivalence” a line of precipitin forms (indicates the organism produces toxin)
What is the significance of Corynebacterium jeikeium
-OPPURTUNISTIC
-joint infections, endocarditis , prosthetic valve endocarditis
-infection limited to those with prosthetic devices, have central line catheters, have undergone invasive procedures
What types of specimen do you need to test for Corynebacterium jeikeium
what is its ID
What is its treatment
Specimen type: IV tips, blood culture
Strict aerobe
Non-hemolytic, urease and nitrate NEGATIVE
Treatment
-highly resistant
-VAN - S
What is C.minutissimum-erythrasma
-superficial skin infection
-inflammatory scaling of skin folds
-lesions range from pin point to palm-sized
-fluoresce coral red under UV light
What is C.urealyticum
-causes urinary tract infections (rare)
-strongly urease positive, Nitrate negative
What is C.ulcerans-
-ulcerans-diphtheria-like sore throat and skin ulcers
-Nitrate negative (differentiates it from C. diphtheriae), Urease positive
ONLY ONE THAT IS BH
What are Diptheroids and what does it look like
-non pathogenic NF - skin and URT
-can contaminate cultures and wounds
-Opportunistic
-dry colonies that can be pushed around on the agar or can be cut in half
-sml, dry and grey OR med yellow
What is Arcanobacterium
4 species but A.haemolyticum most recognized
-associated with pharyngitis
-Pseudomembrane” resembling diphtheria in some cases
-natural habitat not established
What does Arcanobacterium look like
-BH
-Small (<0.5mm after 48 hours)
-Rough (from resp. tract) or smooth (wounds)
-“Pitting” of the agar
-Catalase negative
-Thin, irregular, GPB –”V” forms
What is Gardnerella vaginalis
irregular shape
-best known for role in bacterial vaginosis (BV) ONE SPECIES IN GENUS
-NF in urethra
-almost never causes a UTi
-Pleomorphic, non-spore forming Gram variable (positive and negative) coccobacilli
-Catalase and oxidase: negative
-Associated with Mobiluncus spp. found in vaginal secretions of patients with BV (Mobiluncus spp. not found without G.vag
-also produces a pseudo membrane
BV is diagnosed by finding a higher pH and large bacteria number
-can be caused by bubble baths in kids
how do we diagnose BV
-Gardneralla Vaginalis
-Gram stain on vaginal secretion
-lactobacillus is NF but problems start when it gets replaced
-Absence of lactobacilli, presence of cells resembling G.vaginalis and Mobiluncus spp
-turns score from normal flora to alter flora
-Clue cells also suggest bacterial vaginosis; vaginal epithelial cells that are covered with bacteria including G.vaginalis, Mobiluncus,
-ectopic pregnancy, pelvic disease PID
-caused by bubble bath, douching , poor hygiene
********NEED TO KNOW
How are clue cells formed
-dont CULTURE JUST A GRAM
-Decrease in Lactobacilli causes increase in pH allowing anaerobes to flourish including G. vaginalis
-Anaerobes degrade proteins in vagina = foul smelling amine byproducts
-Amine + organic acids = exfoliation of vaginal epithelial cells (no inflammation)
-Alkaline pH allows bacteria to attach tightly onto the epithelial cells = CLUE CELLS
in a stain CLUE cells will show stippling
how do we make a clinical diagnosis of BV
MUST HAVE ATLEAST 3
-clue cells present in vagina sample see in wet prep
-clue cells and Gardnerella on Gram stain is the confirmatory test
-pH of vagina fluid increased over 4.5 favors anaerobic growth
-watery non inflammatory exudate - physically see
-discharge is like milk attached to vagina
-exudate smells - smell after mixing sample with KOH
KOH + amine = fishy smell
How to treat BV
Metronidazole (aka Flagyl) (G. vaginalis)
Clinda
-“Restoration” of the Lactobacilli balance by probiotics
What are the BRANCHING FILAMENTOUS AEROBIC GRAM POSITIVE BACILLI
Nocardia species
-Aerobic Actinomycetes
-fungus like
-produces spore by fragmentation of hyphae
-branching filamentous - like fungi
found in soil and plant material
-Pulmonary or cutaneous infections
how is Nocardia species seen in lab
-do a presumptive ID and sent to public health
-aerobic fungi like
-good on non selective media
-produce branched beaded cells and filamentous hyphae that form spores or reproduce by fragmentation of hyphae
-hyphae can penetrate surface and project in air
-slow growing 2-4 weeks so it has to be specifically requested for testing
-dry colonies because of the hyphae with SOIL like smell
-use Modified Acid Fast stain with less harsh decolorizer
Modified Acid-fast Positive
Growth at 50°C
Metabolism of glucose
Maldi - not the best ID due to cell wall impermeability , PCR
what is in an acid fast stain
Ziehl-Neelsen and Kinyoun stains
Use carbolfuchsin as the primary stain, and methylene blue as the counterstain. In the traditional method, use an acid-alcohol as the decolorizer. For the “modified stain” –use a weaker acid.
Ziehl-Neelsen –uses heat -Kinyoun’s is a “cold” stain.
