Spirochetes

Leanne Jessa G. Gumahad, RMT

• Family Leptospiraceae
• Leptospira
• Family Spirochaetaceae
• Borrelia
• Treponema
Spirochetes
• Slender, flexuous, helically shaped, unicellular bacteria, with one
or more complete turns in the helix
• Flexible cell wall which several fibrils are wound
• Exhibit various motions in liquid media
• Free-living, or survive in association with animal & human hosts as
normal biota or pathogens
• May be anaerobic, facultatively anaerobic, or aerobic
Leptospires
• Tightly coiled, thin, flexible spirochetes
• Spiral are very close together (may appear as chain of
cocci)
• Highly motile with hooked ends
• Electron microscopy: 2 periplasmic flagella
• Obligately aerobic
• Species: L. interrogans & L. biflexa
• Virulence factor: reduced phagocytosis in host, soluble
hemolysin produced by some strains, cell mediated
sensitivity to leptospiral antigen by the host, small
amount of endotoxin
https://www.ncbi.nlm.nih.gov/books/NBK441858/bin/leptospira.jpg
Leptospira Infections
• Incubation period: 10-12 days (3-30 days)
• Clinical illness is usually abrupt, with nonspecific, influenza-like
constitutional symptoms
• May result in hepatic, renal, and CNS involvement
• Major renal lesion is an interstitial nephritis w/ associated
glomerular swelling & hyperplasia that does not affect glomeruli
• Severe systemic disease: Weil Disease/ Syndrome
• Renal failure, hepatic failure, intravascular disease leading to death
• 1-3 weeks
Leptospira Infections
• Acute phase (first week): blood or CSF
• Urine is a better specimen after the first week of
illness
• Isolation of leptospira is by direct inoculation of 1-2
drops of blood or CSF to media
• Incubation in dark, room temperature
• Serology test for antigen detection: ELISA, radio
immunoassay (RIA), immunomagnetic capture
• Antigen detection in tissue: Immunofluorescence &
immunohistochemistry
• Reference method: Microscopic agglutination using
living cells
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Borrelia
• Highly flexible organism which are less tightly coiled than
leptospira
• Stains easily & can be seen in brightfield microscope
• EM: long, periplasmic flagella coated w/ sheaths of
protoplasm & periplasm
• Composed of 3-10 loose coils & actively motile
• 15-20 axial filaments & 2 insertion disk
• Cultivated in Kelly medium
• Species: B. recurrentis, B. dutoni, B. hermsii, B. turicatae,
B. parkeri, B. afzelii, B. burgdorferi, B. garinii

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Epidemiology
• Lyme disease
• Hard ticks (Ixodes)
• Tick borne (endemic relapsing fever)
• Soft ticks genus Ornithodoros
• Transmits disease through bites
• Louse borne (epidemic relapsing fever)
• Pediculus humanus
• Humans- reservioir
• Transmits disease when infected lice are
crushed & scratched into the skin

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B. recurrentis & similar Borrelia
• Louse-borne/ epidemic/ European relapsing fever
• Causes relapsing fever with acute febrile episodes that subside
spontaneously but reoccur after weeks
• Vector: Louse
• Incubation: 2-15 days

• B. dutoni, B. hermsii, B. turicatae, B. parkeri

• Agents of tick borne relapsing fever/ endemic/ American relapsing fever
Antimicrobial susceptibility
• Tetracyclines drug of choice
• Reduce relapse rate
• 39% of cases with antimicrobial agents experience fever, chills,
headache, & myalgia
• Caused by the sudden release of endotoxin which is referred to as
Jarisch-Herxheimer reaction
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Borrelia burgdorferi
• VF: ability to bind plasminogen & urokinase type
plasminogen activator to surface, binds to factor H
• Lyme borreliosis
• First stage
• Presence of erythema migrans (red, ring-shaped
lesion with a central clearing or bull-eye like
lesion at the site of tick bite)
• Second stage
• Weeks to months after infection
• Neurologic disorders (meningitis) & nerve palsy
• Third stage
• Acrodermatitis chronica atrophicans &
demyelination of neurons with symptoms of
Alzheimer’s disease & multiple sclerosis
Borrelia
• Relapsing fever: Blood
• Lyme disease: Blood, CSF, & biopsy specimens
• Tissue: Warthin-Starry stain
• Blood/CSF: Acridine orange. Geimsa stain
• Culture: Barbour-Stoenner-Kelly medium or chick embryo
• Serology
• Relapsing Fever- increased titers to Proteus OXK
antigens
• Lyme disease- IgG & IgM antibodies (ELISA & IFA)
• PCR of B. burgdorferi to detect DNA in urine

https://www.cdc.gov/relapsing-fever/images/socialmedia/tbrf-smear600x300.jpg?_=12766
Treponemes
• Thin, motile with graceful flexuous movement, spiral
organism & difficult to visualize under bright field
microscope
• Spirals are regular & angular, with 4-14 spirals per
organism, 3 periplasmic flagella, & 1 insertion disk
• Ends are pointed & covered with sheath
• Species: T. pallidum subsp. pallidum, T. pallidum
subsp. endemicum, T. pallidum subsp. pertenue, T.
carateum

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T. pallidum subsp. pallidum
• Causative agent of syphilis
• VF: ability to cross intact mucus membrane & placenta,
disseminate throughout the body, & infect any organs
• Transmission: Sexual contact with an individual who has active
primary or secondary syphilitic lesion, non-genital contact with a
lesion, or transplacental transmission
• Site of inoculation: Vagina, cervix, penis
 https://d12qz6jssicxsf.cloudfront.net/images/herpes_vs_syphilis_2.jpg

Syphilis
• Primary Stage
• Appearance of hard chancre (hunterian)- single erythematous lesion w/c
is non tender but firm with a clean surface & raised border; highly
infectious
• Secondary Stage
• Appearance of symptoms and rash
• Highly infectious but mild & maybe unnoticed by patient
• Latent stage
• Disease becomes subclinical but not necessarily dormant (w/in 1 year)
• Tertiary Stage/ Late Syphilis
• Non-infectious
• Development of granulomatous lesions (gumma) in skin, bones, & liver

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Treponemes
• Specimen: Skin lesions
• Oral lesions should not be examined
• Demonstration of motile treponemes from chancre specimen is
diagnostic for primary stage
• Serology is the primary method used for laboratory diagnosis of
syphilis
 Serologic test of https://i.pinimg.com/736x/d4/86/4d/d4864d789c4b1dbeac23456c21729a38.jpg

Syphilis
• Nontreponemal test
• Detect reaginic antibodies that develop against lipids released
from damaged cells
• Commonly used for treatment monitoring
• 2 types of test:
• VDRL (Venereal Disease Research Laboratory) https://microbenotes.com/wp-content/uploads/2018/07/Rapid-Plasma-Reagin-RPR-Test.jpg

• Cardiolipin antigen that is mixed with patient’s serum or

CSF
• Flocculation occurs in a positive reaction & observed
microscopically
• RPR (Rapid Reagin Plasma)
• More common; uses carbon particles & read
macroscopically
• When mixed with a positive serum on a disposable card,
black charcoal particles clump together with the
cardiolipin-antibody complexes
Serologic test of Syphilis
• Treponemal test
• Detect antibodies specific for treponemal antigens
• Historically used as a confirmative test for nontreponemal tests
• 2 Types:
• Indirect Fluorescent Antibody Test (FTA-ABS)
• Overlaying of whole treponemes that are fixed to a slide with
a heated serum from possible syphilis patient
• Used to confirm positive nontreponemal
• (+) Fluorescence of treponemes
• Treponema pallidum particle agglutination (TP-PA)
• Gelatin particles that are sensitized with T. pallidum
antigens
• (+) Agglutination in the presence of anti-TP antibodies

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Other Treponemes
• T. pallidum subsp. endemicum
• Causative agent of endemic non-venereal syphilis or
bejel (found in the Middle east)
• Can be transmitted by direct contact or sharing
eating utensils
• T. pallidum subsp. Pertenue
• Causative agent of yaws
• MOA: direct contact through skin breaks w/ an
infected lesion
• T. carateum
• Causative agent of pinta
• Lesions begin as scaling, painless papules & are
followed by an erythematous rash that becomes
hypopigmented overtime
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Chlamydia, Rickettsia, &
Similar Organisms
Leanne Jessa G. Gumahad, RMT
 Chlamydia
• Family Chlamydiaceae
• 2 genera:
• Chlamydia: C. trachomatis
• Chlamydophila: C. pneumoniae, C. psittaci, C. pecorum
• Non-motile, small, gram-negative cell wall
• Obligate, intracellular organisms that require living cells for growth
2 Distinct Forms
• Elementary body (EB)
• Infectious form
• Extracellular form of Chlamydia and resembles a gram-negative bacilli
• Infects host cell by inducing active phagocytosis
• Has 2 components: Major outer membrane protein (MOMP) &
lipopolysaccharide antigen
• Reticulate body (RB)
• Intracellular & metabolically active form of Chlamydia
C. trachomatis
• Divided into 3 biovars (based on MOMP antigenic difference)
• Trachoma
• Serovar A-K
• Serovar A, B. Ba. & C- severe eye infection trachoma
• Serovar D-K, Da, Ia, & Ja- inclusion conjunctivitis, & urogenital infection
• Lymphogranuloma venereum
• Serovars L1, L2, L2a, L2b, L3- invasive urogenital tract disease
• Mouse pneumonitis (renamed C. muridarum)
C. trachomatis
• Carries 10 stable plasmids with unknown function
• Causes chronic eye infection trachoma
• Scarring & continual abrasion of the cornea as the eyelid turns downward
toward the cornea
• Causes LGV (STD)
• Bacteria enters lymph nodes near genitals & produce strong inflammatory
response and bubo formation & rupture of lymph nodes
• Associated with Reiter syndrome
• Can travel through the birth canal & infect infants during birth
• Associated with infertility & ectopic pregnancy
• Most common sexually transmitted bacterial pathogen in US
 C. trachomatis
• Type of specimen for laboratory processing depends on symptoms &
clinical presentation of patients
• Regardless of source, specimen should be from infected cells & not from
exudate
• First void urine & vaginal swab specimens are excellent for detecting
infection
• Transport medium: Dacron, cotton, & calcium alginate swabs
• Swabs with plastic or metal shaft are preferred because wooden shaft are toxic to
cells
• Direct Microscopic examination
• Direct Fluorescent Antibody (DFA) used for endocervical or urethral scrapings
• Characteristic fluorescence of EB is only suggestive, but verification must be done
• Sensitivity 80-85%
• Used as an immediate quality control of presence of columnar epithelial cells
• Cell culture
• Was considered a gold standard before PCR assays
• McCoy, HEp-2, HeLa, & Buffalo green monkey kidney
• Immunoassay
• Most used rapid antigen assay for detection – Enzyme immunoassay (EIA)
• EIA detects the outer membrane LPS or MOMP
• Not recommended for urine/ vaginal swabs
• Positive result must be considered preliminary & should be verified
• CDC considers EIAs as substandard for detection & not recommended for
diagnosis
• Nucleic Acid Hybridization & Amplification Assay
• Preferred diagnostic method in genital infections
• Specimens: endocervical swabs, urethral swabs, urine
• Not approved for conjunctival, oropharyngeal, rectal specimens
• Antibody detection
• Micro immunofluorescence (MIF)
• Response of a specific IgM to a different serovar
• Diagnostic of systemic C. trachomatis infection in infants
• Detects antibodies to chlamydial serovars
• (+) IgM titer of 1:32 titer
• Complement fixation
• Used to diagnose LGV
• Not useful in non systemic chlamydial conjunctivitis or routine urogenital tract infections
• (+) Greater than or equal to 1:64 titer
Chlamydophila pneumoniae
• Former name: Chlamydia sp., strain TWAR
• Important respiratory pathogen (pneumonia & pharyngitis)
• Risk factor for GBS
• Specimen: sputum, bronchial lavage fluid, nasopharyngeal
aspirates, throat washings, throat swabs
• Culture media: HeLa cells or Hep-2 cell lines
• Preferred method: Micro immunofluorescent assay
 Chlamydia psittaci
• Causative of psittacosis among birds (AKA: ornithosis or parrot fever)
• Cause feline conjunctivitis, rhinitis, abortion, & respiratory infections
• MOA: Inhalation of infected aerosols from dried excreta or handling of
infected birds
• Isolation of bacteria is not recommended
• Commonly used test: Complement fixation with a titer of greater than
1:32 to be considered as acute illness
Rickettsia
• Arthropod-borne, obligately intracellular pathogens that can grow
only in cytoplasm of host cells
• Short, nonmotile, intracellular parasite, gram-negative bacilli
• Culture: require living cells for growth
• Have the simplest bacterial form and are considered as
transitional organisms between bacteria & viruses
• Multiplication via binary fission
• Humans are accidental host
• Agent of bioterrorism: R. prowazekii & R. ricketsii
 3 Groups of Rickettsia
• Spotted Fever Group
• R. rickettsii causes the most severe form of infection- Rocky mountain Spotted
Fever (RMSF)
• Infection by tick bites- Dermacentor variabilis & Dermacentor andersoni (vector & reservoir)
• Flu-like symptoms & rash which begin as erythematous patches on ankles & wrists & may
extend to the palms of hands & soles of feet
• Causes vasculitis in blood vessels of lungs, brain, heart leading to pneumonitis, CNS
manifestation, & myocarditis
• Also, may lead to disseminated intravascular coagulation
• Boutonneuse Fever
• AKA: Mediterranean spotted fever by R. conorii (France, Spain, & Italy)
• R. conorii causes Kenya tick typhus, South African tick fever, Indian tick typhus
• Clinically like RMSF but rash may include the face, but difference is the presence of taches
noires (black spots) at the primary site of infection
• Typhus Group
• R. typhi- Endemic typhus (Murine typhus)
• Vector- Oriental rat flea, cat flea
• Unlike RMSF, does not produce rash for about 50% of cases
• R. prowazekii- Epidemic Louse-borne typhus & Brill-Zinsser Disease (AKA: Recrudescent
typhus)
• Vector: Human louse, squirrel flea, squirrel louse
• Reservoirs: Human & flying squirrels
• Similar progression to RMSF
• Brill-Zinsser Disease is seen in patients who has previous history of epidemic louse-borne typhus
• Bacteria will lie dormant in the lymph nodes until reinfection happens
• Transitional group
• Rickettsial pox- R. akari
• Reservoir: Common house mouse
• Vector: Mouse mite
• Like RMSF but milder form & enter by mite (chigger) bite
• Incubation is 10 days which a papule forms- progresses to a pustule & then to an
indurated eschar
• Rash appears in face, trunk, extremities only
Orientia
• Orientia tsutsugamushi- Scrub typhus
• Previously a Rickettsia but separated due to the
absence of lipopolysaccharide and peptidoglycan, &
the presence of 54-to 58 Kda major proteins
• Replicates in the cytoplasm of host cells & released
through a process that involves pinching off the host
cell
• Vector: chigger
• Main reservoir: rat

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 Diagnosis of Rickettsiaceae
• Specimen: Biopsy of skin, peripheral blood, CSF
• Immunohistology of skin biopsy is sensitive & specific for Rickettsia in
tissue sections
• Visualization of morulae is by Giemsa stain
• Rickettsia, Erhlichia, & Anaplasma can be isolated from humans in an
antibiotic-free, centrifugation-enhanced shell vial culture
• C. burnetti- lung tissue cell medium
• Serologic test
• Indirect Immunofluorescent antibody test
• Reference method for Q fever
• Weil-Felix reaction
• Presumptive test for Rickettsia
• Cross-reaction of serum positive Rickettsia patients with Proteus vulgaris antigens
and produces agglutination
• Micro immunofluorescent dot test
• Excellent sensitivity for detecting antibodies to Rickettsia
• Used for early diagnosis of RMSF after onset of symptoms
Cell Wall Deficient Bacteria
Leanne Jessa G. Gumahad, RMT
Cell wall deficient bacteria
• Belongs within the class Mollicutes
• 2 genera: Mycoplasma & Ureaplasma
• Slow-growing, highly fastidious, facultatively anaerobes requiring
complex media containing cholesterol & fatty acids for growth
• Mycoplasma spp. Often grow embedded beneath surface of solid
media; some species form colonies w/ slightly raised centers
giving the fried egg appearance
• Common parasite of genital tract & transmission is related to
sexual activity
 Mycoplasma pneumoniae
• Causes Primary atypical pneumonia (AKA: Walking pneumonia)
• Not a normal flora
• Risk population: PDL, college students, military personnel
• MOA: inhalation of contaminated aerosol droplets
• Initiation of disease: attachment to respiratory mucosal cells, evasion
from phagocytosis, & modulation of the immune system
• Culture: SP4 broth- colonies are yellow color
Mycoplasma hominis &
Ureaplasma species
• Both may play a role in bacterial vaginosis
• Frequently isolated from asymptomatic sexually active individuals
• Causes Nongonococcal urethritis, pyelonephritis, pelvic
inflammatory disease (PID), postpartum fevers
• U. urealyticum- common organism isolated from tracheal
aspirates of low-birthweight infants w/ respiratory disease
(caesarean section)
• Culture:
• M. hominis- “fried egg” appearance in solid medium
• U. urealyticum- dark-brownish lumps on A7 or A8 agar medium
 Diagnosis
• Specimen: Body fluids, wound aspirates, nasopharyngeal, cervical, &
vaginal swabs
• Extremely sensitive to drying & heat-isolation at bedside is recommended
• Transport medium: SP4, Shepard’s 10B broth, 2SP
• Swabs: Dacron or calcium alginate
• If plating not available w/in 24 hrs, specimen should be frozen at -70 deg C.
• Culture
• M. pneumoniae- SP4 broth & agar; requires biphasic culture system &
incubation up to 21 days in a chamber with 5-10% CO2
• M. hominis- A8 agar; only species capable of growing in BAP/CAP
• Ureaplasma spp.- require medium pH near 6.0 (Shepherd’s 10B arginine broth)
• Serodiagnosis
• Previously, serum should be collected at onset of symptoms and 2-3
weeks later for acute & convalescent measurement
• ELISA is most used
• Detection of specific IgM in a sample is diagnostic of the acute infection
by M. pneumoniae
• Manganese chloride urea test
• Rapid ID of U. urealyticum
• (+) dark brown precipitate of manganese oxide around the colonies
Mycobacterium species
Leanne Jessa G. Gumahad, RMT
 Mycobacterium species
• Most familiar species: MTB & M. leprae
• Slender, aerobic, curved or straight, rod-shaped, non-motile, non-spore
formers, non-encapsulated
• Catalase positive
• Produce Much’s granule
• Cell wall has a high lipid content & N-glycolylmuramic acid- resist
staining
• 2 groups: (1) MTB complex: M. tuberculosis, M. bovis, M. africanum, M.
canettii, M. micrtoti; (2) NTM
Staining Method
• Staining method
• Ziehl Nelsen Method
• Kinyoun Method
• Auramine-rhodamine fluorochrome staining
• sensitive, reliable, & specific method
• Auramine is more sensitive than carbol fuchsin
• AFB are examined at 250x & 400x magnification using a fluorescence microscope
• (+) bright yellow-orange bacilli against black background
Mahon, C., Lehman, D., & Manuselis, G. (2015). Textbook of Diagnostic Microbiology, 5th edition (5th ed.). Saunders Elsevier.
Source from DOH NTP Manual 6th Ed.
Culture Media
• Staining method is Ziehl Nelsen Method, Kinyoun Method, Auramine-
rhodamine fluorochrome staining
• Egg-based (whole or yolks)
• ATS medium
• Petragnani medium
• Wallenstein medium
• Lowenstein-Jensen
• Preferred basic nonselective media
• Inhibitor: Malachite green
• Egg suspension: provide fatty acids & protein required for metabolism of mycobacterium
• Agar based
• Middlebrook 7H10 or 7H11
• Mitchson 7H11
• Liquid media
• BACTEC 12B (MB 7H12) & BACTEC 13A (MB 7H13)
• BACTEC- automated method for detecting mycobacteria
• Growth enhancer: Polyoxyethylene stearate
• Growth indicator: Release of CO2
• Can also be used for susceptibility testing of M. tuberculosis
• Septi-check AFB
• Biphasic medium that is used for rapid growth & identification of mycobacteria
• Middlebrook 7H9
• Reduces turnaround time average 10 days
• Used in BACTEC system for continuous monitoring in automated systems
M. tuberculosis
• AKA: Koch’s bacillus
• Causative agent of pulmonary tuberculosis & extrapulmonary TB
(spleen, liver, lungs, bone marrow, kidney, adrenal glands, eyes
• Also causes Pott disease (skeletal TB)
• Longest replication time among mycobacteria species
• Virulence factor: cord factor
• Colonies: nonpigmented, described as being buff colored, cauliflower-
like (Lowenstein-Jensen)
• (+) Niacin & nitrate reduction
• (-) for thiophene-2-2carboxylic acid hydrazide (T2H)
M. bovis
• TB in animals & humans
• Closely resembles PTB
• Attenuated strain is used for vaccination (BCG vaccine)
• Grows very slowly on egg-based media- small, granular, rounded,
nonpigmented colonies with irregular margins after 21 days of
incubation
• Middlebrook 7H10 medium- colonies similar to M. tuberculosis
• (-) Niacin, nitrate reduction, & T2H
(Table 1 . Group Characteristic of the Mycobacteria and The..., n.d.)
https://www.researchgate.net/profile/Vivek-Kashyap-3/publication/324964339/figure/tbl1/AS:631595685920828@1527595554890/Group-characteristic-of-the-mycobacteria-and-the-representative-species.png
 Non-tuberculous Mycobacteria (NTM)
Slow Growers
• M. avium complex (MAC)
• Most common cause of pulmonary infection in humans that is like TB
• M. avium- cause of disease in poultry & swine
• Can be isolated from sputum, blood, & bone marrow aspirate
• Microscopy: pleomorphic, short, coccobacilli w/out beading
• (+) PAS staining
• M. kansasii (Yellow bacillus)
• Second to MAC that causes NTM lung disease
• Slow-growing organism that appears long rods with distinct crossbanding
• Middlebrook 7H10 agar- smooth to rough, w/ characteristic wavy edges & dark centers
• Ability to hydrolyze Tween 80, (+) nitrate reduction, catalase, pyrazinamidase production
• M. marinum
• Typical presentation of a tender red or blue-red subcutaneous nodule, or swimming
pool granuloma in the elbow, knee, toe, or finger that occurs when an open wound
encounters contaminated chlorinated water
• Long rods with cross barrings
• Colonies are smooth to rough, wrinkled, & yellow (photochromagen)
• M. scrofulaceum
• Associated with cervical lymphadenitis in children
• Uniformly stained, acid-fast, medium to long rods
• Slow grower; incubation: 4-6 weeks at 25- 37 deg C
• Colonies are smooth with dense centers and pigmentation from light yellow to
deep orange
• Scotochromogenic
• (-) Tween 80, nitrate reduction
• M. simiae
• Isolated from lymph nodes of monkey
• Appear as short coccobacilli
• Egg medium: smooth colonies in 10-21 days
• Middlebrook 7H10: thin, transparent, & filamentous
• Photochromogenic (in prolonged incubation)
• (+) niacin test
• (-) nitrate reduction
• M. szulgai
• Most common manifestation is pulmonary disease like PTB
• EPTB- lymphadenitis & bursitis
• Medium to long rods with some cross barring
• Egg-based medium: smooth & rough colonies
• 37 deg C- yellow to orange pigmentation 7 intensifies upon light exposure
• 22 deg C- non pigmented or buff in the absence of light
• Slow hydrolysis of Tween 80
• (+) nitrate reduction
• Inability to grow in 5% sodium chloride
• M. ulcerans
• Rare cause of mycobacteriosis (AKA: Buruli ulcer)
• 3 most common Mycobacteria
• Disease: painless nodule under the skin after previous trauma
• Moderately long, w/out beading or cross banding
• 6-12 weeks incubation at 30-33 deg C
• Colonies: smooth or rough & nonpigmented or lightly buff
• M. xenopi
• Has been recovered from hot & cold-water taps including water storage tanks of hospitals
• Long filamentous rods
• Middlebrook 7H10: small, w/ dense centers & filamentous edges
• Cornmeal-glycerol agar (at LPO) round colonies w/ branching & filamentous extensions
(aerial hyphae seen in rough colonies)
• Young colonies: Bird’s nest appearance w/ characteristic sticklike projections
• Optimal temp.: 42 deg C
• Non-photochromogenic
• (-) niacin, nitrate reduction
• (+) arylsulfatase & pyrazinamidase
• M. gordonae
• AKA: Tap water bacillus
• Culture: colonies are smooth& yellowish –orange colored
• Scotochromogens
• (+) Tween 80 hydrolysis
• (-) nitrate medium
• M. terrae complex
• Species: M. terrae, M. triviale, M. nonchromogenicum
• Short to medium coccobacilli
• Culture
• M. terrae- smooth
• M. triviale- rough & dry
• M. nonchromogenicum- smooth to rough & white to buff
• (+) Tween 80 hydrolysis, growth in 5% NaCl for M. triviale
 NTM- Rapid Growers
• M. chelonae
• Belongs to the Mycobacterium abscessus Group
• Rapidly growing mycobacteria most likely isolated from disseminated cutaneous
infection in immunocompromised patients
• Greater antibiotic resistance than M. fortuitum
• Microscopy of young colonies: strongly acid-fast, with pleomorphism ranging from
long, tapered to short, thick rods
• Culture: rough or smooth, non-pigmented to buff colonies w/in 3-5 days at 37 deg C
• (+) 3-day arylsulfatase test
• (-) nitrate reduction
• Can grow in MacConkey agar w/out crystal violet
• M. fortuitum Group
• Species: M. fortuitum, M. peregrinum
• Associated with localized cutaneous infection, long use of intravenous & peritoneal
catheters, injection sites, & surgical wounds following mammoplasty & cardiac bypass
procedures
• Microscopy: colonies with branching filamentous extensions & rough colonies w/ short
aerial hyphae
• Cells are pleomorphic, ranging from long & tapered to short thick rods
• Old cultures: decolorize & appear partially acid-fast with any acid-fast staining
• M. smegmatis Group
• Species: M. smegmatis, M goodie
• Cause pulmonary, skin, & bone infection
• Cells are long-tapered or short rods with irregular acid fastness
• Rods are curved w/ branching or Y-shaped forms; swollen, w/ deeper staining, beaded, or ovoid
• Egg-medium: rough, wrinkled, or coarsely folded/ smooth, glistening after 2-4 days
• Middlebrook 7H10- heaped & smooth/ rough with dense centers
• Can grow in MacConkey agar w/out crystal violet
• (-) 3- day arylsulfatase test
• (+) nitrate reduction
Non-cultivable NTM
• M. leprae
• Causative agent of Hansen’s disease (AKA: Leprosy)
• Tuberculoid leprosy
• Skin lesions & nerve involvement & loss of sensation
• Can produce cell-mediated response
• Lepromatous leprosy
• Skin lesions & progressive, symmetric nerve damage
• Cannot produce cell-mediated response
• Cannot be cultivated in any synthetic media
• Microscopy: rod-shaped, “cigar-pocket”/ “pocket-fence”
• Culture: exhibit growth in living tissues of footpads of mice & armadillos
• Skin test: Fernandez & Mitzuda reaction
Specimen for Mycobacteria
• Sputum
• Gastric lavage
• Urine
• Bronchoscopy specimen
• Body fluids
• Blood
New Tool for PTB
Diagnosis
 Decontamination
• Biosafety cabinets: Class I negative-pressure * Class II vertical, laminar flow
cabinets
• Digestion-Decontamination process
• Liquefy sample through digestion of proteinaceous material
• Allow chemical decontamination agent to contact & kill NTM
• Sodium Hydroxide (2-4%)
• Digestant & decontaminant
• N-acetyl-L-cysteine
• Digestant & decontaminant
• Oxalic acid (5%)
• Used to decontaminate specimens contaminated w/ P/ aeruginosa in sputum specimens
with cystic fibrosis
• Benzalkonium chloride (AKA: Zephiran with Trisodium phosphate)
• Zephiran-bacteriostatic for tubercle bacilli
References
• Ciulla, A. P., & Lehman, D. C. (2009). Success! in Clinical Laboratory Science: A Complete Review.
Prentice Hall.
• Graeter, L., Hertenstein, E., Accurso, C., & Labiner, G. (2014). Elsevier’s Medical Laboratory Science
Examination Review - E-Book. Elsevier Health Sciences.

• Mahon, C., Lehman, D., & Manuselis, G. (2015). Textbook of Diagnostic Microbiology, 5th edition (5th
ed.). Saunders Elsevier.

• Rodriguez, M. T. (2016). Review Handbook in Diagnostic Bacteriology. In Review Handbook in

Diagnostic Bacteriology. C & E Publishing.

• Tille, P. (2017). Bailey & Scott’s Diagnostic Microbiology Fourteenth Edition.

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