Gram Positive BacilliAerobe and Facultative Anaerobe

Question 1

Why are gram positive bacilli significant

Answer 1

-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

Question 2

What group 1 endospores like

Answer 2

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

Question 3

What are group 2 GPB

Answer 3

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

Question 4

Habitat and condition for Bacillus and Clostridium

Answer 4

• 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

Question 5

how can you grow bacillus and what are two example species

Answer 5

-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

Question 6

What are some virulence factors of bacillus anthracis

Answer 6

-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

Question 7

What is anthrax

Answer 7

-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

Question 8

What is the cutaneous form of anthrax

Cutaneous
Intestinal
Inhalation/ pulmonary
Injectional

Answer 8

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

Question 9

What is the Intestinal form of anthrax

Cutaneous
Intestinal
Inhalation/ pulmonary
Injectional

Answer 9

-when you eat contaminated meat
-diarrhea, vomiting, fever, anorexia
-higher fatality than cutaneous because of challenges in diagnosis

Question 10

What is the Inhalation/ pulmonary form of anthrax

Cutaneous
Intestinal
Inhalation/ pulmonary
Injectional

Answer 10

-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

Question 11

What is Woolsorters disease?

Answer 11

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

Question 12

What is injectional anthrax

Answer 12

-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

Question 13

What type of lab testing is done for anthrax bacillus species

Answer 13

-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)
-

Question 14

how does Bacillus anthracis grow differently then regular bacillus

Answer 14

-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

Question 15

What is Bacillus cereus

Answer 15

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

Question 16

What are the two forms associated with Bacillus cereus

Answer 16

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

Question 17

how can Bacillus cereus cause opportunistic infections

Answer 17

-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

Question 18

What will you see in lab testing of Bacillus cereus

Answer 18

-3-8mm raised, grey green frosted glass appearance
-large zone BH
-large GPB - parallel sides and regular

Question 19

What do the Bacillus species look like

Answer 19

-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

Question 20

What is the lecithinase test- egg yolk test

Answer 20

• 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

Question 21

Is AST done on Bacillus anthracis or cereus ?
what is the treatment for them

Answer 21

-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

Question 22

What are some REGULAR NON SPORE-FORMING GRAM POSITIVE BACILLI and characteristics

Answer 22

Listeria, Erysipelothrix, Lactobacillus
-small GPB bacilli

-regular straight parallel sides - long
-no endospore production

Question 23

What is Listeria and where is it found

Answer 23

-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

Question 24

What are the virulence factors and risk factors for listeria

Answer 24

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

Question 25

How is listeria transmitted

Answer 25

-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

Question 26

What is the clinical significance of Listeria

Answer 26

-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

Question 27

how do you diagnose Listeria

Answer 27

-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

Question 28

how to differentiate Listeria from GBS

Answer 28

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

Question 29

Where is lactobacillus found and what is the clinical significance

Answer 29

-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

Question 30

What does lactobacillus look like

Answer 30

-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

Question 31

What is Erysipelothrix or E. rhusiopathiae

Answer 31

-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

Question 32

What is the CMI for Erysipelothrix

Answer 32

-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)

Question 33

What are some IRREGULAR NON SPORE-FORMING GRAM POSITIVE BACILLI

Answer 33

Corynebacterium species, Arcanobacterium, Gardnerella vaginalis

-curved
-no spores
PATHOGENIC SPECIES ARE FACULTATIVE
NON PATH ARE AEROBES

Question 34

What is Corynebacteria and where is it found

Answer 34

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

Question 35

What is C diptheriae and where is it found

Answer 35

-found only in humans (upper Resp tract )
-spread by droplet or h/m
-rare
-included in DPT vaccine

Question 36

What are the two types of C diptheriae
mode of transmission
treatment
virulence factor
systemic effects
prophylactic

Answer 36

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)

Question 37

What does C diptheriae manifest as and how to sample

Answer 37

-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

Question 38

CMI of C diptheriae

Answer 38

-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>

Question 39

What is the ELEK Test for toxigenicity

Answer 39

-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)

Question 40

What is the significance of Corynebacterium jeikeium

Answer 40

-OPPURTUNISTIC
-joint infections, endocarditis , prosthetic valve endocarditis
-infection limited to those with prosthetic devices, have central line catheters, have undergone invasive procedures

Question 41

What types of specimen do you need to test for Corynebacterium jeikeium
what is its ID
What is its treatment

Answer 41

Specimen type: IV tips, blood culture

Strict aerobe
Non-hemolytic, urease and nitrate NEGATIVE

Treatment
-highly resistant
-VAN - S

Question 42

What is C.minutissimum-erythrasma

Answer 42

-superficial skin infection
-inflammatory scaling of skin folds
-lesions range from pin point to palm-sized
-fluoresce coral red under UV light

Question 42

What is C.urealyticum

Answer 42

-causes urinary tract infections (rare)
-strongly urease positive, Nitrate negative

Question 43

What is C.ulcerans-

Answer 43

-ulcerans-diphtheria-like sore throat and skin ulcers
-Nitrate negative (differentiates it from C. diphtheriae), Urease positive

ONLY ONE THAT IS BH

Question 44

What are Diptheroids and what does it look like

Answer 44

-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

Question 45

What is Arcanobacterium

Answer 45

4 species but A.haemolyticum most recognized
-associated with pharyngitis
-Pseudomembrane” resembling diphtheria in some cases
-natural habitat not established

Question 46

What does Arcanobacterium look like

Answer 46

-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

Question 47

What is Gardnerella vaginalis
irregular shape

Answer 47

-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

Question 48

how do we diagnose BV

Answer 48

-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

Question 49

How are clue cells formed

Answer 49

-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

Question 49

how do we make a clinical diagnosis of BV

Answer 49

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

Question 49

How to treat BV

Answer 49

Metronidazole (aka Flagyl) (G. vaginalis)
Clinda
-“Restoration” of the Lactobacilli balance by probiotics

Question 49

What are the BRANCHING FILAMENTOUS AEROBIC GRAM POSITIVE BACILLI

Answer 49

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

Question 49

how is Nocardia species seen in lab

Answer 49

-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

Question 50

what is in an acid fast stain

Answer 50

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.