AEROBIC GRAM-POSITIVE

BACTERIA

Chapter 16
Objectives
 Compare general characteristics of this group of bacteria
 Know the clinical significance associated with these bacteria (i.e.
disease, symptoms, anatomical location)
 Be able to differentiate these bacteria from each other in the lab
 Describe microscopy morphology and colony appearance
 Aerobic GPB
 Nonspore forming  Spore-forming
 Corynebacterium  Bacillus
 Arcanobacterium
 Rhodococcus
 Listeria
 Erysipelothrix
 Gardnerella
 Actinomyces
 Norcardia
Watch out for Lactobacillus trying to join this group . . .
Sneaky little Gram positive bacilli
KNOW 16.1

morphology

Major tests:
Catalase
TSI
Motility
Bile esculin
GPB NON-
BRANCHING,
CATALASE
POSITIVE
Corynebacterium
Listeria
 Corynebacterium Loeffler
agar
 Human, animal, and plant pathogens and
saprophytes
 Normal flora of skin and mucous membranes

 100 species with ~50 of clinical importance

 Most need 16S rRNA sequencing for proper
identification
 Divided into lipophilic and non-lipophilic
 Lipophilic: fastidious and need 48h on standard
media
 Gram stain: slightly curved, “club ends” -
diptheroid
Corynebacterium diphtheriae
 In 1921 (USA): 206,000 cases  15,520 deaths
 DPT Vaccine: 2 cases in USA from 2004-
2017. . . Vaccines important?
 2 forms of disease: respiratory and cutaneous
 Respiratory: uncommon in US
 Cutaneous: gray-membrane ulcer; Tropics

 Humans ONLY natural host

 Spread: aerosol and hand-to-mouth
 Symptoms: low-grade fever, malaise, sore throat
 Gray-to-white pseudomembrane -> suffocation
 Toxin-induced via necrosis and exudate
 Corynebacterium diphtheriae lab
diagnostics KNOW!
 Facultative anaerobe Black colony with brown halo
 Small β-hemolytic (?)
 Selective on CTBA-halo
 Clinical detection
dependent on toxin
production
 Elek test
 Elek test
 Clinical detection dependent
on toxin production

March 1997Journal of Clinical Microbiology 35(2):495-8

Other Corynebacterium
 C. amycolatum: most frequently recovered within genus
 Normal microbiota of skin
 Nonlipophilic: colonies flat, dry, waxy appearance

 C. jeikeium: most common Corynebacterium-caused prosthetic valve

endocarditis in adults
 Lipophilic, strict aerobe, urease (-), no tinasdale halo, nitrate reduction (-), gamma
hemolysis
 C. pseudodiphthericum: NF nasopharynx, clinical infections in
immunocompromised
 C. pseudotuberculosis: veterinary pathogen, clinical cases associated with sheep
contact
Other Corynebacterium
 C. striatum: NF skin and nasopharynx; frequent isolate
 Device related infection, endocarditis, etc

 C. ulcerans: diphtheria-like illness, HOW do you differentiate?

 Urease +, nitrate reduction (-)
 Infections acquired through interactions with animals, ingestion of unpasteurized milk

 C. urealyticum: most common associated UTI

 Pinpoint, nonhemolytic, white colonies; diphtheroid morphology
 C231: wild type
 Toxminus: pld-mutation
 pTB111: pld complement
 Antonie Van Leeuwenhoek. 2019 Feb 15. doi: 10.1007/s10482-019-01240-4.
 Detection and virulence potential of a phospholipase D-negative Corynebacterium ulcerans
from a concurrent diphtheria and infectious mononucleosis case.
 Abstract
 Diphtheria by Corynebacterium ulcerans is increasingly occurring in children, adolescents and
adults. In addition to diphtheria toxin (DT), phospholipase D (PLD) is considered a virulence
factor of C. ulcerans. In the present study, a first case of concurrent diphtheria by a PLD-negative
C. ulcerans and infectious mononucleosis (IM) was verified. Clinical and microbiological
profiles and binding properties to human Fibrinogen (Fbg), Fibronectin (Fn) and type I collagen
(col I) biotinylated proteins and virulence to Caenorhabditis elegans were investigated for C.
ulcerans strain 2590 (clinical isolate) and two control strains, including PLD-positive BR-AD22
wild type and PLD-negative ELHA-1 PLD mutant strains. MALDI-TOF assays and a multiplex
PCR of genes coding for potentially toxigenic corynebacteria identified strain 2590 as non-DT
producing. Interestingly, strain 2590 did not express PLD activity in the CAMP test although the
presence of the pld gene was verified. PLD-negative 2590 and a PLD-positive 210932 strains
showed similar affinity to Fbg, Fn and type I collagen. C. elegans were able to escape from C.
ulcerans strains, independent of PLD and DT production. Higher mortality of nematodes was
verified for PLD-negative strains. Additional studies concerning multifactorial virulence potential
of C. ulcerans, including environmental conditions remain necessary.
Know major tests!
 Two year-old male child experienced an upper respiratory infection 2 weeks
prior to hospital admission. Four days prior to admission, anorexia, and lethargy
were noted. The patient had a 39.9dC fever 3 days before admission. Physical
examination revealed a clear chest, exudative pharyngitis, and bilaterally
enlarged cervical lymph nodes. A throat culture was taken and a course of
penicillin was begun. The child’s course worsened, and he became increasingly
lethargic; he developed respiratory distress on the day of admission. Throat
culture from 3 days ago, showed no GAS. On examination, the patient was
febrile to 38.9dC and had an exudate in the posterior pharynx that was described
as a yellowish, thick membrane which bled when scraped and removed. Patient’s
medical history revelaed that he had no immunizations. Fig. shows the organism
recovered from patient’s throat culture on special medium and fig 2 Gram stain
What microbe was it?
What special test is necessary to
prove that it can cause disease?
What special medium is used?
Common in the USA? World?
Drug of choice
 Diphtheriae: treat with anti-toxin right away
 Penicillin to combat bacteria

 Rest of Corynbacterium spp. : Vancomycin due to high resistance

except
Listeria monocytogenes
 Recent outbreaks
 Habitat
 Environmental
 Raw milk, cheese, processed meat, cantaloupe

 Virulence factors
http://textbookofbacteriology.net/Listeria_2.html
 Listeriolysin O
 Ability to survive and replicate in phagocytes
 Ability to survive and grow at low temperatures

 Transmission: Foodborne
 Vertical – mother to infant
Listeria monocytogenes
 Infections
 Bacteremia and Meningitis: high
mortality rate
 Newborns and elderly most
common
 Pregnant women
 3rd trimester
 Can cross placenta barrier
 Responsible for septic abortion and
still birth
 Newborns
 Disease similar to Group B strep https://mbio.asm.org/content/8/3/e00949-17
 Listeria monocytogenes Fig 16.6

 Cold enrichment
 Specimens incubated at 4℃ for several weeks,
subculture periodically
 Grows well on blood and choc
 Ground glass appearance
 Small zone beta-hemolysis
 Very similar to Group B streptococcus
CAMP-like
 Catalase positive
 Hippurate positive
 CAMP test positive
 “block” type pattern

 Motile
Differentiating Listeria
monocytogenes
GPB, NON-
BRANCHING,
CATALASE
NEGATIVE
Erysipelothrix
Gardnerella
Arcanobacterium
Ersipelothrix rhusiopathiae
 Only species to cause human disease
 Habitat
 Commonly found in animals

 Infections:
 Wound: Erysipeloid
 Bacteremia/endocarditis
 Rare clinical isolate
 Transmission: Occupational exposure through cuts/break in skin
 Ersipelothrix rhusiopathiae
 Pleomorphic rod, form long filaments
 Catalase negative
 Small slow growing non-hemolytic or alpha
hemolytic colonies
 H2S production in TSI
 Arcanobacterium haemolyticum
 Habitat: Skin
 Infections: Pharyngitis
 Young adults
 Laboratory
diagnosis/isolation/identification
 Slow growing
 Pinpoint beta-hemolytic (large zone)
 Catalase negative
 Reverse CAMP positive
 Inhibition of hemolysis

www.icjournal.org
 Gardnerella vaginalis
 Habitat: NF of vagina
 Infections
 Associated with bacterial vaginosis – most common vaginal infection (15-44yrs old)
 Decrease in Lactobacillus
 Allows for BV organisms to proliferate
 BV associated with preterm labor
 BV does not generally have a inflammatory response

 Laboratory diagnosis/isolation/identification
 Gram variable bacilli: thinner peptidoglycan layer
 “wet mount” - Presence of Clue cells
 Short, pleomorphic rod,
 Catalase negative
 Hippurate positive
 Small slow growing non-hemolytic colonies: ~ 48h
NON-SPORE
FORMING,
BRANCHING
GPB Norcardia
Streptomyces
Aerobic Gram positive bacilli
 Aerobic Actinomycetes
 Nocardia spp
 Actinomadura
 Streptomyces
 Gordonia
 Tsukamurella
 Rhodococcus
 Tropheryma whipplei
 Nocardia
 General characteristics
 Found in soil
 Aerobic
 Branching
 Beaded-Gram positive bacilli
 Weakly (modified) acid fast
 Slow growing
 Most common species: N. brasiliensis, N. cyriacigeorgica, N.
farcinica, and N. nova
 Clinical Nocardia
 Pulmonary infections
 Generally in immunocompromised patients
 Steroid therapy
 Transplant patients
 Inhalation from dust or soil
 Most common etiologic agent: N. cyriacigeorgica and N. farcinica https://wellcomecoll
ection.org/works/v5
 Confluent bronchopneumonia – symptoms typically months qr3scw
 Chest x-ray variable and unable to distinguish from other agents
 High mortality rate: ~40% diagnosed at autopsy
 Lung abscesses form and may spread to other organs, including
brain
 Minimal inflammatory response
 No encapsulation of abscess
 No granuloma formation
 Clinical Nocardia (norcardiosis)
 Cutaneous
 Occurs in immunocompetent host
 Etiologic agent: N. brasiliensis
 Normally seen in hands and feet
 Inoculation of skin: starts as a local abscess
 Highly invasive and transformed into a lesion known as actinomycotic
mycetoma
 Mycetomas: swelling, draining sinuses, and granules
 Can extend to underlying bone
 May discharge “sulfur” granules
Nocardia diagnosis/identification
 Gram positive beaded, branching, bacilli
 Can be seen in direct Gram stain of sputum or aspirate
 Must be careful to differentiate from Gram positive cocci
 Partially acid-fast
 Granules in pus: 0.5-1mm white-cream
 Slow growing, 3-6 days
 Chalkly, matte, velvety, or powdery appearance
 White, yellow, pink, orange, peach, tan, or gray
 Dry crumbly appearance
 Aerial hyphae – dissecting microscope

 Wet mount: reveals granules

 Masses of filamentous organisms held together by calcium
phosphate
 Often appear yellow or orange: “sulfur” granules
Nocardia
SPORE-FORMING, NON-
BRANCHING,
CATALASE-POSITIVE
BACILLI
Bacillus
 Bacillus spp.
 General characteristics
 >100 species
 Found widely in the environment
 Form endospores
 highly resistant dormant structures formed in response to
adverse conditions
 Grow well on blood and chocolate
 No growth on CNA
 Catalase positive
 Most species considered laboratory contaminants
 Can be confused with aerotolerant Clostridium spp.
Bacillus cereus group
Most medically relevant:
B. anthracis
B. cereus
B. thuringiensis
B. mycoides
Bacillus anthracis
 Bioterrorism agent
 Capsule
 3-part protein exotoxin
 Not individually toxic
 Protective antigen (PA)
 Binding molecule for EF and LF
 Allows for binding to host cells
 Edema factor (EF): Cause edema when combined with PA
 Lethal factor (LF): Causes cell death when combined with PA
Bacillus anthracis

 Clinical disease - RARE

 Common in livestock
 Vaccine preventable
 Human infection usually associated with animal or animal product exposure
 Four common forms
 Cutaneous: black eschar
 Papule appears 2-3 days after exposure
 Inhalation/Pulmonary (woolsorter’s disease)
 Possible mortality within 24h
 Respiratory distress
 Gastrointestinal
 Injectional
Bacillus anthracis
 Morphology
 Large Gram positive encapsulated bacilli
 Gram variable with age
 May chain together in “bamboo” forms
 Culture growth
 Large, gray, flat, irregular margins
 Non-hemolytic
 “medusa head”
 Tenacious consistency
 Sticky, stand when manipulated

South Dakota Department of Health - State of South Dakota

Bacillus anthracis identification
 Identification
 Catalase positive
 Facultative anaerobe
 Non motile
 Penicillin susceptible

 Once suspected – all work must take place in biological safety cabinet
 Sentinel laboratory testing
 Non hemolytic
 Catalase – positive
 Motility - negative
Bacillus anthracis
 Identification
 Public Health laboratory testing
 Direct flourescent antibody (DFA) assays
 Historical test
 Inoculate in penicillin containing agar
 Incubate 3-6 hours
 Examine microscopically
 B. anthracis will for chains of spherical bacilli
 “string of pearls”
Clinical infections by Bacillus
cereus
 Food poisoning
 Caused by enterotoxin
 Diarrheal
 6-18 hours post ingestion
 Abdominal pain and diarrhea
 Self-limiting: average 24 hour
 Associated with meat, poultry, vegetable, and pasta
 Emetic
 1-5 hours post ingestion
 Nausea and vomiting
 Self-limiting: Average 9 hour
 Source: typically fried rice
Clinical infections by Bacillus
cereus
 Infection
 Non gastrointestinal
 Ocular – poor visual outcome
 Meningitis
 Septicepmia
 Endocarditis
 Osteomyelitis
 Rare, but serious
 IV drug users
 Neonates
 Immunosuppressed
https://www.dovepress.com/endophthalmitis-following-pars-plana-vitrectomy-a-
literature-review-of-peer-reviewed-fulltext-article-OPTH
Bacillus cereus
 Laboratory diagnosis
 Culture of suspected food (> 10^5 CFU/g)
 Not normally performed in clinical laboratories
 > 10^5 CFU/g feces

 Morphology
 Large Gram positive bacilli
 Gram variable with age
 May be able to visualize spores
 Culture growth
 Large frosted glass
 beta-hemolytic
 Motility positive

 Treatment
 Not normally indicated for diarrheal and emetic forms
 Serious infections
 Vancomycin or clindamycin – with or without aminoglycoside
B. anthrax vs B. cereus