GRAM NEGATIVE INTESTINAL PATHOGENS (NON ENTERIC) CAMPYLOBACTER

- Vibrio - May appear like V. cholera

- Aeromonas - Wings of seagulls
- Helicobacter - appears as S-shape Bacilli
- Campylobacter - Motile with single polar flagellum with darting motility
- Requires 5% 02 and inc. CO2 10%
GENUS VIBRIO - Optimum temp. for growth 42-43 deg C
GRAM STAIN: curved or comma shaped - Antecedent Cause of Gullain-Barre Syndrome
OXIDASE TEST: screening test for genus vibrio
- Naturally found in sea water MEDIA FOR PRIMARY ISOLATION
- Transmitted through consumption of contaminated seafood A) MODIFIED SKIRROW'S MEDIA (blood agar base, 7% horse lysed blood, vancomycin,
- All are HALOPHILIC (8-10 NaCl), except: V. cholera, V. mimicus trimetrophrim and polymyxin B)
- Motile monotrichous B) CAMPY-BAP (Brucella agar base with trimetophrim, polymyxin B, cephalotin,
- Facultatively anaerobe vancomycin and amphothericin B and 10% sheep blood)
- Collected and trasnsported in cary blaire medium C) MODIFIED CHARCOAL CEFOPERAZONE DEOXYCHOLATE AGAR
- Glucose fermenters D) CAMPY-CVA (Brucella base agar with cefoperazone, vancomycin and amphotericin
- Non lactose fermenters: V. mimicus, vulnificus, parahymoliticus B and 5% sheep blood)
- Oxidase positive except: V. meshnikovii
CULTURE MEDIA
- TCBS (Thiosulfate Citrate Bile Salts)
- Fermentable sucrose indicator: Bromthymol Blue pH of 8.6
- Sucrose fermenting = yellow
- (APW) Alkaline Peptone Water- enrichment broth

VIBRIO CHOLERAE
- shooting star motility
- (+) string test
 Uses 0.5% sodium deoxycholate
 (+) result string like SPECIES
- (+) cholera red test CAMPYLOBACTER COLI and CAMPYLOBACTER JEJUNI
 Not specific because any organism positive to indole and nitrate will give a - Asscociated with gastritis and diarrhea
positive result - Thermophilic
 POSITIVE RESULT: red color due to Nitrosoindole
- Causes CHOLERA
- Diarrheal disease characterized by production
- of "Rice Watery Stool"
- CHOLERAGEN – toxin produced by vibrio cholera that promotes hypersecretion of
water and chloride ion preventing absorption of sodium

1) VIBRIO CHOLERAE 01 – the one causing classical epidemic cholera biotypes:

a. Classical
b. El Tor – produces choleragen
2) VIBRIO CHOLERAE 0139 AND VIBRIO CHOLERAE NON 01 – non- toxigenic does not
produce choleragen GRAM NEVATIVE NON-FERMENTATIVE BACILLI
- Pseudomonas
- Burkholderia
- Stenotrophomonas
- Acinetobacter
- Alcaligens
- Moraxella
- Chromobacterium

FAMILY PSEUDOMONADACEAE
PSEUDOMONAS

PSEAUDOMONAS AERUGINOSA
- Causes nosocomial infection
- No. 1 ICU isolate
a. OGAWA or VARIANT F – India - Strict aerobe
b. INABA or ORIGINAL J – Philippines - Motile
c. HIKOJIMA or MIDDLE or INTERMEDIATE – Japan - Non-fermenter, does not ferment carbohydrate;
- TSI=K/K
- Grows well at 37-42 degC
- Oxidase positive
- Beta hemolytic on BAP
- Colonies tend to spread to give off a characteristic odor resembling that of an
overripe grape "Corn Tortilla Like odor"- 2-amino-acetophenone

PRODUCES:
 PYOCYANIN – Water soluble Blue pigment
 PYOVERDIN – Fluorescein- Green
 BOROBIN – Red Pigment
 PYOMELANIN – Brown Pigment

DISEASE
GENUS AEROMONAS 1. found infections in burn patients or blue pus
- Oxidase (+); fermentative gram negative bacilli 2. UTI
- May cause cellulites and wound infections 3. Pneumonia and Sepsis – no. 1 cause of pneumonia for cystic
- Can grow on Mac Conkey, EMB, SSA and CIN 4. Mild otitis externa/ Swimmer's ear or Diver’s ear
- SPECIES: Aeromonas hydrophila water loving organism associated with GIT disease 5. Jacuzzi or hot tub syndrome or Shanghai Fever
 AEROMONAS CAVIAE – most common isolate, can grow at 4-42 degC
TEST
HELICOBACTER Acetamide Utilization test – positive result (Blue)
HELICOBACTER PYLORI Bromthymol Blue as indicator
- Formerly called Campylobacter coli Cetrimide Agar – primary isolation media
HABITAT: human gastric mucosa Toxic to many bacteria but not to P. aeruginosa
CAUSES: Gastritis, Peptic ulcer, Gastric carcinoma
ENDOSCOPY: lab diagnosis to detect H. pylori PSEUDOMONAS FLUORESCENS AND PSEUDOMONAS PUTIDA
VIRULENCE FACTOR: Urease because of its very strong activity - Motile
UREA BREATH TEST: non culture, non-invasive, technique to detect H. pylori that measure - Oxidase positive
urease activity - Pyocyanin negative and pyoverdin positive
COPRO TEST (stool antigen test): can detect only current infection - Can cause transfusion associated septicemia
SPECIMEN: tissue biopsy stained with Warthin Starry
 - P. fluorescens-blood bag contaminant 2. NTM (NONTUBERCULOUS MYCOBACTERIA – causes pulmonary infection, other
than TB

MYCOBACTERIUM TUBERCULOSIS COMPLEX

TB COMPLEX GROUP (MTC)
• M. tuberculosis (var hominis) (Koch's bacillus)
• M. bovis
• M. africanum
BURKHOLDERIA • M. canetti
BURKHOLDERIA CEPACIA
- Found near water sources
- Causes onion bulb rot in plants
- Foot rot in Man

BURKHOLDERIA MALLEI
- Glander's Bacillus
- Causative agent of Glander's disease – a disease of animals like horses transmitted
to man thru direct contact thru skin abrasions, inhalation of organism-infected
livestocks
- able to grow on 42 degC

BURKHOLDERIA PSEUDOMALLEI
- Whitmore's Bacillus
- Causative agent of Meliodosis or Vietnam Time Bomb-glander's like youtheast Asia
disease associated with severe pneumonia
- Colonies with Earthy odor on ashdown medium MYCOBACTERIUM TUBERCULOSIS
MORPHOLOGY
STRENOPTROPHOMONAS - Koch's Bacillus
STENOTROPHOMONAS MALTOPHILIA - Gram positive/ gram-ghost
- May cause nosocomial infections like pneumonia and UTI - Acid fast (because of mycolic acid long chain multiple cross-linked fatty acids in the
- Produces large colonies with yellow pigmentation on TSA (Trypticase Soy Agar) and cell wall); Strict aerobe
Lavender Green Pigment - Slender delicate bacilli with beaded or banded appearance (contains Much
- Able to Grow at 42 degC granules) with tapered end
- Exhibits "cording" (the interwining of bacilli in serpentine coils) when grown in
culture containing

CULTURAL CHARACTERISTICS
- Small, dry, friable, rough, warty and buff-colored colonies
- Later enlarge and develop a "caulif bwer head" center after 2-3 weeks incubation
at 35° Celsius in the dark(no growth at 25° Celsius or 45° Celsius)
- All slants are laid in slanting position with loose caps at 5% to 10% CO2 in the first
two weeks.
- pH 6.5-6.8, enhanced by 5-10% CO2
- Discard culture as negative if no growth appears after 8 weeks (for AFB negative
smear): or after 12 weeks (for AFB positive

OTHER NON-FERMENTATIVE GRAM NEVATIVE BACILLI CULTURE MEDIA

ACINETOBACTER EGG BASED MEDIA
- Colonize most areas of the skin - Lowenstein-Jensen (contains coagulated whole eggs, defined salts, glycerol, potato
- Non-motile flour, malachite green)
- Oxidase negative - Wallenstein's
- Catalase positive - Petragnani's
- Grows well on BAP "Gummy Colonies" and Mac - Dorset's
- American Thoracic Society (ATS)

NON-EGG BASED MEDIA/ CLEAR

- Middle Brook Medium
M7H10
- OADC (oleic acid, albumin, dextrose and catalase enrichment
ALCALIGENS FAECALIS - Glycerol
- Catalase and oxidase positive - Co-factors
- Motile w/ peritrichous flagella - defined salts
- Alpha hemolytic - vitamins
- Apple Like Fruity Odor - malachite green
Disease M7H11
- UTI - M7H10 (without dextrose) plus 0.1% casein hydrolysate
- Wound infection
ENRICHMENT MEDIA
- Diarrhea
- Dubo's albumin
MORAXELLA LACUNATA - Middlebrook 7H9
- Causes Angular conjunctivitis or Bhlepharaconjunctivitis
SELECTIVE MEDIA
- Catalase and oxidase positive
- Mycobactosel (Selective L-J)
- Mac Conkey
- Gruft Mod, Of L-J
- Mistaken as Neisseria
- Selective M7H11 (Mitchison's medium) (with carbenicillin, amphotericin B,
CHROMOBACTERIUM VIOLACEUM Polymyxin B & Trimethoprim lactate)
- Not part of the normal flora, part of the environment  Radiometric Cultures(Bactec TB)
- Contracted by patients with neutrophil deficit, those with CGD (Chronic
TREATMENT
Granulomatous Disease)
PRIMARY TB DRUGS SECONDARY TB DRUGS
- On Dnase agar produces violet pigment-violacein
Rifampin Ofloxacin
- On BAP-beta hemolytic and with "ammonium cyanide odor
Isoniazid (INH) Kanamycin
Pyrazinamide Ciptofloxacin
MYCOBACTERIA Ethambutol
- Gram positive rods, acid fast (due to mycolic acid), nonmotile, Nonencapsulated, Streptomycin
slender, slightly curved or straight rods
- Often have a beaded appearance of Much granules TYPES OF TB
- May have palisade or X,Y,V & L (snapping) formation 1. PRIMARY – type of initial exposure
2. SECONDARY – may happen to those who have tb in the past
TWO GROUPS: 3. MDR (MULTI-DRUG RESISTANT TB) – type that shows resistance to primary tb
1. MTC (MYCOBACTERIUM TUBERCULOSIS COMPLEX) – causes tuberculosis drugs (RIPES)
 4. XDR (EXTENSIVELY DRUG RESISTANT) – type that shows resistance to primary tb 2. PPD (Purified Protein Derivative) – chemical fractionation of OT; preferred for skin
drugs to any fluoroquinolones and to at least one second line of tb drugs testing

POTT'S DISEASE METHODS OF INOCULATION:

- A.k.a Tuberculosis spondylitis  MANTOUX – local injection of PPD
- Skeletal TB of the Spine  VON PIRQUET – scratching
- cause bone destruction, deformity, and paraplegia. Pott disease most commonly  VOLLNER PATCH – patched over skin
involves the thoracic and lumbosacral spine  MOW PERCUTANEOUS TEST – OT/PPD mixed with linoleic ointment
 TINE TEST – multiple puncture test
MILIARY TB
- Small lesions, yellow-white areas of consolidation without gross caseation - NALC & 3-4% NaOH (stronger alkali will decontaminate specimens with
- Seeding of organism in different Organs Pseudomonas and Klebsiella)
- Extrapulmonary TB affecting spleen or liver - 6% oxalic acid (decontaminate specimens with Paeruginosa)
- Potentially fatal form of the disseminated disease due to the hematogenous spread - Hypochlorite chlorox
of tubercle bacilli to the lungs, and other organs
MTB RESISTANCE
GOHN COMPLEX WEEKS: when protected from sunlight, remains in putrifying sputum
- primary focus of pulmonary infection; may be seen in the regional tracheo- 6 TO 8 MONTHS: dried sputum, protected from sunlight
bronchial lymph nodes; lesions are circumscribed caseating or non-caseating 8 TO 10 DAYS: droplets of dried sputum in air
granules KILLED WITHIN TWO HOURS: when exposed to direct sunlight
20 TO 30 HRS.: in sputum, exposed to sunlight before they are killed
LABORATORY CHARACTERISTICS
STAINS KILLING OF MTB IN SPUTUM:
Acid Fast staining BOILING: 10 mins. (easily kills M. tuberculosis)
- Ziehl Neelsen (Hot method) brilliant red DIRECT SUNLIGHT: 20-30 hrs.
- Kinyoun's (Cold method) brilliant red DRIED SPUTUM: 6-8 months
- Fluorescent staining (Truant's, FITC)- yellow fluorescence 5% PHENOL: 24 hrs.
- Auramine- rhodamine (bright yellow color)
CULTURE MEDIA
AGAR-BASED
 Middlebrook 7H10 – has QADC (oleic acid, albumin, dextrose, catalase) &
0.00025% malachite green
 Middlebrook 7H11 – M7H10+2% glycerol (enhances growth of MAC) and 0.0001%
casein hydrolysate, 0.0001% malachite green

EGG-BASED MEDIUM
 American Thoracic Society – fresh whole eggs, glycerol, potato flour 0.02%
malachite green
 Lowenstein Jensen (LJ) – good for Niacin Test- fresh whole eggs, defined salts,
glycerol, potato flour, % 0.025%malachite green
 Petragnani – fresh whole eggs, egg yolks, whole milk, glycerol, potato flour %,
0.052% malachite green
 Dorset Egg medium – Dubois Tween Albumin, Wallenstein's

LIQUID MEDIUM
 M7H12/ BACTEC 12B Medium – broth base, casein hydrolysate, bovine serum,
albumin, catalase, C-labeled palmitic acid, deionized water, (PANTA) Polymixin B,
Amphotericin B, Nalidixic acid, Trimethoprim, Azlocillin & polyoxyethylene stearate
(growth enhancing agent).
 M7H13/BACTEC 13B medium – broth base, casein hydrolysate, bovine serum,
albumin, catalase, 14C-labeled palmitic acid, SPS, Polysorbate 80 deionized water,
(PANTA) Polymixin B, Amphotericin B, Nalidixic acid, Trimethoprim, Azlocillin
 Middlebrook 7H9

MEDIA FOR CONTAMINATED SPECIMENS

 Mycobactosel/ Selective LJ – fresh whole eggs, defined salts, glycerol, potato f bur,
malachite green, cycloheximide, lincomycin, & nalidixic acid
 Gruft Modif cation of LJ – fresh whole eggs, def ned salts, glycerol, potato f bur,
RNA, malachite green, Penicillin & nalidixic acid
 Selective M7H10 – def ned salts, glycerol, OACD, malachite green, cycloheximide,
lincomycin, nalidixic acid
 Selective M7H11/Mitchison's – def ned salts, glycerol, OACD, casein hydrolysate,
Carbenicillin, Amphotericin B, Polymixin B, Trimethoprim lactate

GENERAL RULE RADIOMETRIC CULTURES

1. 2 out of 3 sample must be positive to confirmdiagnosis Principle: Specimen is inoculated into M7H12 broth and measure radioactive CO, production.
2. If none or only the first specimen is positive,additional sample must be added Gives a result after 2 days (17days for conventional methods).
3. 300 fields are required to examine before ruling out a negative diagnosis
4. 5,000-10,000 organisms/mL -needed for positive MYCOBACTERIUM BOVIS
BCG (Bacille Calmitte- Guerin) – on attenuated strain of M.bovis is used to immunize
SEPTI- CHECK AFB individuals
PRINCIPLE
DISEASE
1. The tube is incubated in ambient air at 37 degrees for 6 weeks. - Bovine tuberculosis
2. The Mycobacterium metabolizes the 14C-labeled palmitic acid 14CO2 (radioactive) - Humans develop intestinal TB through ingestion of contaminated milk
product accumulates above the tube. - Pulmonary TB through inhalation of airborne bacteria from the dried-up
3. Radioactivity is measured daily. unpasteurized milk
4. Gl of 10 suggests presence of Mycobacteria.
5. Positive cultures are differentiated into Tb complex group and MOTT by the NONTOBERCULOUS MYCOBACTERIA
addition of the NAP (p-nitro- α-acetyl- amino- B- hydroxypropiophenone) RUNYON’S CLASSIFICATION
- In 1959 Runyon classifed NTM into four groups (Runyon groups I to IV).
Interpretation: ↓radioactivity = positive for MTB complex group (MTBC) - Based on the phenotypic characteristics of the various species, most notably the
↑ radioactivity = positive for MOTT (Mac, M.kansasii) growth rate and colonial pigmentation

ANIMAL INOCULATION
- Injection into guinea pigs; autopsied after 6 weeks for presence of TB

TUBERCULIN TEST
- Tuberculin is injected and read within 48-72 hours (+) previous contact with TB;
wider than 1 cm erythema
- (-) energy, measles, Hodgkin's, sarcoidosis or immunosuppression.

2 TYPES OF TUBERCULIN
1. OT (Old Tuberculin) – concentrated filtrate of broth with tubercle bacilli
 M. scrofulaceum – causes cervical lymphadenitis
M. haemophilus – causes skin ulcer cut non cultivatable
M. ulcerans – causes skin ulcer but is culturable
M. kansasit – 2nd to Mac as cause of NTM opportunistic lung disease

OTHER MYCOBACTERIA THAT ALSO CAUSE PULMONARY TBD

a. M.flavescens
b. M.nonchromogenicum
c. M.shimoidei
d. M.smegmatis-thermoresistable
e. M. malmoense

OTHER MYCOBACTERIUM ASSOCIATED WITH AIDS

a. M.celatum
RUNYON'S CLASSIFICATION OF NTM b. M.malmoense
GROUP I PHOTOCHROMOGENS c. M.xenopi
- when exposed to light for 1 hour & re-incubated in the dark, light yellow colonies d. M.genavense
turn bright yellow)
NON CULTIVATABLE NTM
a. M. kansasii (low catalase activity)
ATYPICAL MYCOBACTERIA
b. M.marinum
MYCOBACTERIUM LEPRAE
c. M. simiae
- Hansen's bacillus- after Gerhard Hansen
GROUP II SCOTOCHROMOGENS - Acid fast
- when expose to light for 2 weeks, the deep yellow to orange color becomes orange - Morphologically similar to M.tuberculosis, globus or cigar pack, picket fence,
to dark red palisade arrangement found in modified mononuclear or epitheliod structure
a. M.scrofulaceum – cervical lymphadenitis M.szulgar chronic pulmonary disease called "lepra cells
b. M. gordonae – meningitis, endocarditis, prosthetic valve infection, cutaneous
CULTURE
infection, hepatorenal M.flavescens
- Never been grown in vitro
GROUP III NON- PHOTOCHROMOGENS - Growth in foot pads & ear lobe of armadillo & white mice ("low temperature")
- colonies remain buff colored even often exposure to light)
DISEASES
a. M.avium complex (including intracellular & Batley's bacillus)
TYPES OF LEPROSY
b. M. gastri (J Bacillus)
1. TUBERCULOID TYPE – skin lesion with loss of sensation (granuloma & hyperactive
c. M.terrae – trivial complex (M.terrae- raddish bacillus)
skin lesion); effective cell mediated Immune response; EASILY treated.
d. M.xenopi
2. LEPROMATOUS TYPE – malignant, chronic, slowly progressive, life threatening:
e. M.ulecrans – a saprophyte (causes Bairnsdale ulcers in Australia, Buruli ulcers in
Ineffective cell-mediated Immune response; skin lesion with symmetric nerve
Africa
damage; (there is absence of immune response) with marked in foldings of the skin
GROUP IV. RAPID GROWERS (GROWTH IN LESS THAN 7 DAYS) called "leonine facie"
a. M.fortuitum-chelonii (formerly M.borstelence)
b. M.smegmatis - Probably transmitted through airborne droplets
c. M.phlei (Hay bacillus) - Infects body parts which are "cold" such as nose and earlobes
d. M.vacae - Natural reservoir host is armadillo
- Low infectivity in humans
MOTT [MYCOBACTERIUM OTHER THAN TUBERCULOSIS] - Not pathogenic to guinea pigs
MYCOBACTERIUM KANSASII
- Thicker and more beaded than M. tuberculosis DIAGNOSIS
- Appears longer, banded or beaded bacilli resembling barber’s pole or shepherd’s - PCR test has been used to identify M. leprae in infected tissues
crook Basis of Diagnosis: based on clinical symptoms
- Also displays cording
Lepromin Test- skin test for M. leprae
CULTURE
Fernandez reaction- used to detect
- Dysgenic growth (smooth, scanty growth)
Ab against the Dharmendra Ag (one of Leprosy antigen)
DISEASE
Mitsuda Reaction- to Differentiate
- TB-like pulmonary infection
tuberculoid to lepromatous type
- M. avium intracellulare (M. avium complex, M. intracellulare)
- Highly prevalent in AIDS patients (PWAS)
Lepromin Test- skin test for M. leprae
MORPHOLOGY
Fernandez reaction- used to detect
- Acid fast
Ab against the Dharmendra Ag (one of Leprosy antigen)
- Coccobacillary
Mitsuda Reaction- to Differentiate
MYCOBACTERIUM GORDONAE tuberculoid to lepromatous type
- Normal inhabitant of tap water 5. LEPROMIN TEST
- Causes prosthetic valve infection, endocarditis and meningitis Delayed type of hypersensitive reaction.
• First described by Mitsuda in 1919.
MYCOBACTERIUM NEOAURUM • Lepromins used as antigens may be of human origin (lepromin H) or armadillo derived
- Bacteria in immunocompromised host with indwelling catheter (lepromin A).
- Mycobacteria causing disseminated disease in AIDS patients Epidemiology - Lepromin

MYCOBACTERIUM PARATUBERCULOSIS This skin test is still used an indicator of CMI response to the organism
- Isolated from the bowel mucosa of patients with Crohn's disease ►Limited use in diagnosis or indicator of protective Immunity
- Highly fastidious Uso killed organisms
- Requires growth factor mycobactin produced by M. phle • Takes as long as 18 ⚫ Fernandez reaction - 48 hours
months to isolate ⚫ Mitsuda
Delayed C response (3-4 weeks)
MYCOBACTERIUM TUBERCULOSIS – slender with beaded or banded appearance
5 mm-fuberculold
MYCOBACTERIUM AVIUM COMPLEX – coccobacillary forms
3.5 mm-borderilno 0.2 mm-lepromatous
MYCOBACTERIUM KANSASII – longer, larger beaded or banded "barber pole"
MYCOBACTERIUM ULCERANS – pale yellow colonies; maybe confused with MTB

DISEASES
MYCOBACTERIUM AVIUM COMPLEX
- High prevalence in patients with AIDS
- Mode of transmission is through the GI tract via infected semen
- Initial symptom is protracted diarrhea
- Pulmonary disease common in elderly and in immunocompromised patients
- Mycobacterium ssp paratuberculosis – causative agent of Johne disease (chronic
diarrhea in cattle, goats, sheeps, and other ruminants)

MYCOBACTERIUM GORDONAE
- Common inhabitant of tap water
- Rarely pathogenic
- Maybe involved in prosthetic heart valve endocarditis and meningitis