MT 111

CLINICAL BACTERIOLOGY 1 - LECTURE

NONFERMENTATIVE GRAM NEGATIVE BACILLI

● opportunistic pathogen
NONFERMENTERS ● causes nosocomial infection
● Group of Gram-negative bacilli that do not ferment
glucose and other sugars ● Pathogenesis
● Environmental bacteria - produces slime polysaccharide, endotoxin, and
● Not usually found as part of the normal flora of the proteases
human body - inactivate components of complement
● Readily isolated from water, soil, vegetables, plants, - Endotoxin (Lipopolysaccharide)
and hospitals surfaces - Exotoxin- proteases, hemolysins, lecithinase,
● Oxidase positive ( except Acinetobacter spp.) elastase, deoxyribonuclease
● Frequently resistant to many of the antibiotics that ● Exotoxin A: functions similarly to diphtheria toxin,
are effective against members of the promotes cellular damage and tissue invasion and
Enterobacteriaceae is toxic for macrophages, blocks protein synthesis
- Pili: found on bacterial surface, mediate attachment
Pseudomonas to host cells
- Alginate: a polysaccharide polymer that inhibits
● strictly aerobic phagocytosis and contributes to the infection
● catalase-positive potential in patients with cystic fibrosis
● Oxidase-positive (except P. luteolus and P. - Pigments
oryzihabitans) Pyocyanin water-soluble, bright bluish
● motile, some have polar flagella phenazine pigment, damages
● grows on MAC cells by producing reactive
● Pseudomonas fluorescent and Nonfluorescent group oxygen species

Pyoverdin green, water-soluble and

Pseudomonas Fluorescent Pseudomonas fluoresces under
group Non-Fluorescent group short-wavelength ultraviolet
(Produce pyoduce) light

1. Pseudomonas 1. Pseudomonas stutzeri Pyorubin red

aeruginosa
Pyomelanin brown
2. Pseudomonas putida 2. Pseudomonas. mendocina
● Causes Ecthyma gangrenosum
3. Pseudomonas 3. Pseudomonas ● Common cause of lung infection in people with Cystic
fluorescens pseudoalcaligenes fibrosis
● Jacuzzi or hot tub syndrome: necrotizing rash in divers
4. Pseudomonas veronii 4. Pseudomonas alcaligenes
and swimmer

5. Pseudomonas monteilii 5. Pseudomonas luteola

● Laboratory Diagnosis
- Gram staining: Gram negative, medium-size,
6. Pseudomonas mosselii 6. Pseudomonas
straight rods
oryzihabitans
- Cultivation/ Biochemical reaction
● Media of choice: grows well on most routine
Pseudomonas Fluorescent Group laboratory media
● 5% Sheep blood agar, MacConkey agar,
1. Pseudomonas aeruginosa thioglycolate broth, brain-heart infusion broth
● most commonly isolated species from clinical ● formation of sheen and/or pigment on the
specimen slants of TSIA and Pseudomonas P agar,
● found in moist environments, pools, hot tubs, Mueller-Hinton agar, Trypticase soy agar
catheters, and humidifiers in hospitals ● Β-hemolytic, rough or ground glass

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 appearance of its colonies on sheep blood agar ● Oxidase (+), ADH (+), Acetamide (-)
● Cetrimide agar: a selective and differential 3. Pseudomonas oryzihabitans
medium for the identification of P. aeruginosa ● Found in Japanese rice paddies
- Cetrimide acts as a detergent and inhibits
most bacteria
- Enhances the production of pigments Acinetobacter spp.
● (+) Oxidase reaction
● Denitrification of nitrates and nitrites ● short, rod shaped to spherical Gram-negative bacilli
● (+) Arginine dihydrolase (ADH) ● non motile
● (+) Citrate ● strictly aerobic
● alkaline slant/neutral butt reaction in TSIA ● difficult to decolorize in Gram stain
● growth at 42° C ● more that 25 species are known but differentiation
● musty grape-like (or corn tortilla) odor caused by the biochemically is difficult
presence of 2- aminoacetophenone
● often collectively known as Acinetobacter
calcoaceticus–Acinetobacter baumanii complex
● Antimicrobial susceptibility
● associated with nosocomial septicemia, pneumonia,
Susceptible Resistant Multiple bacteriuria, wound infection
Resistant
Strains ● Laboratory Diagnosis
- (-) Oxidase
● Aminoglycosides ● sulfamethoxaz ● Colistin - inability to reduce nitrates
● the ole-trimethopr ● polymy - (+) Catalase
carboxypenicillins im (SXT xin B - Cultivation: purplish colonies on MacConkey’s
and ● Tetracyclines agar
ureidopenicillins ● Tigecycline
● ceftazidime or ● Ertapenem 1. Acinetobacter baumanii
cefepime ● Nitrofurantoin - Glucose-oxidizing (saccharolytic)
● carbapenems ● - Nonhemolytic
(except ertapenem)
● and the quinolones 2. Acinetobacter lwoffi
- Non-glucose oxidizer (asaccharolytic)
- Nonhemolytic
2. Pseudomonas putida
● Low virulence, rarely causes clinical disease 3. Acinetobacter haemolyticus
● Associated with catheter-related sepsis in cancer - Nonsaccharolytic
patients - Hemolytic
● (-) Gelatin hydrolysis
● Produce acid from xylose ● Antimictrobial Susceptibility
- Acinetobacter baumanii complex
3. Pseudomonas fluorescens Susceptible Resistant
● Pseudobacteremia related to contaminated catheters
and catheter-related devices ● Doxycycline ● β-lactam and
● (+) Gelatin hydrolysis ● Trimethoprim-sulfamet aminoglycoside
● Produce acid from xylose hoxazole antibiotics
● Quinolones - Resistance to the
Pseudomonas Nonfluorescent Group ● Ureidopenicillins aminoglycosides is
● Imipenem caused by
1. Pseudomonas stutzeri ● Ampicillin-sulbactam plasmid-mediated
● Rarely isolated, rarely causes infection ● Ceftazidime acetyl-, adenylyl-, and
● wrinkled, leathery, adherent colonies that may ● carbapenems (except phosphotransferases
produce a light-yellow or brown pigment ertapenem) - CRAB
● ADH (-), Starch hydrolysis (+) (carbapenem-resistant
● Can grow in an anaerobic environment in Acinetobacter
nitrate-containing media (differentiates P. stutzeri baumanii)- resistant to
from other Pseudomonads) all classes of
antibiotics, except
2. Pseudomonas mendocina colistin and tigecycline
● Produces nonwrinkled, flat colonies, may appear with
a yellowish-brown pigment
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 medium, supplemented with
Stenotrophomonas maltophilia colistin
- Deep pink colonies because of
● third most common nonfermentative, Gram negative the absorption of neutral red in
bacilli isolated in the clinical laboratory the medium
● Nonmotile - Colonies exhibit an earthy odor
● nosocomial pathogen 2. Burkholderia cepacia complex
● Risk factors for colonization or infection: - Complex comprises at least nine species
- mechanical ventilation (B. cenocepacia, B. multivorans, B.
- use of broad-spectrum antibiotics vietnamienesis, B. cepacia, and B.
- Catheterization doloda)
- neutropenia - Nosocomial pathogen associated with
contaminated equipment, medications,
● Laboratory Diagnosis disinfectants
- (-) Oxidase - Can cause bacteremia, UTI, septic
- (+) DNAse arthritis, respiratory tract infection
- (+) Catalase - Important pathogens in patients with
- (+) esculin and gelatin hydrolysis cystic fibrosis and Chronic
- (+) Lysine decarboxylase granulomatous disease
- Grow on ● Laboratory Diagnosis
● Blood agar plate: lavender green colonies - Culture media: grows well on most lab
● MacConkey agar: colonies may appear bluish media
● Selective media:
● Antimicrobial Susceptibility - Burkholderia cepacia-selective
agar (BCSA)- MOST effective
Susceptible Resistant
in reducing overgrowth while
maintaining good recovery of
● Levofloxacin ● Cephalosporins
B. cepacia
● trimethoprim- ● Penicillins
- Pseudomonas cepacia
sulfamethoxazole ● Carbapenems
selective agar (PC)
(antibiotic of choice) ● aminoglycosides
- oxidation-fermentative
● Ticarcillin-clavulanate
base-polymyxin B, bacitracin
● Minocycline
lactose agar (OFBL)
● MacConkey Agar
● Sheep-Blood Agar
Burkholderia - Biochemical reaction
● (+) oxidase
● Aerobic, non-spore-forming ● Oxidizes glucose, maltose, lactose,
● All are motile (polar flagella) except Burkholderia and mannitol
mallei ● (+) Lysine decarboxylase
● Nosocomial infection ● (+) ONPG
● 2 most important human pathogens: ● (-) Ornithine decarboxylase
1. Burkholderia pseudomallei ● Fail to reduce nitrate to nitrite
- Acquired via inhalation or contact
through cut or abraded skin
- Causes Melioidosis ● Other Burkholderia species
- an aggressive, granulomatous, 3. Burkholderia mallei
pulmonary disease caused by ingestion, - Causes Glanders- respiratory tract
inhalation, or inoculation of the zoonosis
organisms, with metastatic abscess - Primarily affecting livestock such as
formation in lungs and other viscera horses, mules, and donkeys
- Melioidosis is most prevalent in - Rare infection in humans
Southeast Asia and Australia. - May cause severe local suppurative or
acute pulmonary infections
● Laboratory Diagnosis ● Laboratory Diagnosis
- Culture media: - Nonmotile
● Nonfermentative wrinkled colony - Growth on MAC
● Bipolar staining on Gram stain - Variable glucose oxidation
● Ashdown medium: selective - (+) ADH

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 - Reduce nitrates to nitrites
- AST uses broth microdilution with Brucella Aeromonas
broth, inc @ 35˚C in ambient air for 16-20
hours ● Facultative anaerobes
● Widely distributed in freshwater, estuarine, and
4. Burkholderia gladioli marine environments
- Resembles Burkholderia cepacia ● Enteric pathogens
complex ● Extraintestinal infections: septicemia and wound
- Found in patients with cystic fibrosis infections
and chronic granulomatous disease ● (+) oxidase
- Recovered from the blood and tissue of ● (+) catalase
immunocompromised patients (CF px) ● Motile
who had lung transplant ● reduce nitrates to nitrite
● Laboratory Diagnosis ● Glucose-fermenting
- Yellow pigment may be produced after ● Classified into 2 groups:
48-72 hrs of incubation
Mesophilic group Psychrophilic group
- One or two polar flagella
- (+) catalase
-Optimal growth @ 37˚C -Optimal growth @ 22˚C
- (+) urease
-A. hydrophila complex ● A. salmonicida
- (-) oxidase
composed of:
- Grows on MAC
● A. hydrophila, A.
- Oxidizes glucose
bestiarum,
- (+) Mannitol
● A. dhakensis and
- (-) decarboxylase
● certain motile
strains of A.
● Antimicrobial Susceptibility
salmonicida
Susceptible Resistant -A. veronii complex
composed of
Burkholderia ● Chloramphen ● aminoglycosi ● A. veronii biovar
cepacia complex icol des and sobria,
● Ceftazidime polymyxin ● A. veronii biovar
● Piperacillin ● some strains veronii,
● Minocycline are resistant ● A. jandaei,
● some to βlactam ● A. trota,
fluoroquinolo antibiotics ● A. schubertii,
nes ● A. diversa, and
● SXT ● A. encheleia
-A. caviae complex
Burkholderia ● Ceftazidime ● polymyxin composed of
mallei ● Imipenem ● A. caviae,
● Doxycycline ● A. media, and
● tetracycline ● A. eucrenophila

Burkholderia ● SXT
pseudomallei ● Chloramphen ● Culture medium
icol - combined use of ampicillin SBA and a modified
● Tetracycline cefsulodin-Irgasin-novobiocin (CIN) II plate
● semisynthetic ● 4 μg of cefsulodin
penicillins ● pink-centered colonies from the
● ceftazidime fermentation of mannitol

Burkholderia ● Aminoglycosi ● Aztreonam ● Antimicrobial Susceptibility

gladioli des ● the Susceptible Resistant
● Carbapenem cephalospori
s ns ● trimethoprim- ● Penicillins
● Ciprofloxacin ● polymyxin B sulfamethoxazole ● Ampicillin
● SXT (100% ● Aminoglycosides ● carbenicillin
resistant) ● Quinolones

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 ● (+) Phenylalanine deaminase (PDA)
Moraxella ● (+) oxidase
● Nitrate and nitrite (+) with gas formation
● Nonmotile
● Strongly oxidase (+)
● Strictly aerobic Alcaligenes
● Asaccharolytic
● Susceptible to Penicillin ● found in water (e.g., swimming pools, tap water,
● Opportunists that reside on the mucous membranes dialysis fluids)
of humans and lower animals ● resistant to disinfectants, such as chlorhexidine and
● can be isolated from the respiratory tract, urinary quaternary ammonium compounds
tract, and eyes ● obligate aerobe
1. Moraxella catarrhalis ● (+) oxidase and catalase
- most frequent isolate ● Grown on MAC
- Commensal of the upper respiratory tract of ● Motile with 1-12 peritrichous flagella
humans ● Alcaligenes faecalis
- Respiratory and ear specimens - linked to eye infections, pancreatic
- Resembles Neisseria due to Gram-negative abscesses
coccal morphology
- Previously called Neisseria catarrhalis and also
Branhamella catarrhalis Achromobacter
● Laboratory Diagnosis
- Culture media: SBA and CHOC agar ● obligate aerobe
● smooth, opaque, gray-to-white ● Grows on MAC
colonies ● Asaccharolytic species-
● “hockey puck” colonies- because - Achromobacter piechaudii
the colony remains intact when - Achromobacter denitrificans
pushed across the plate with a loop ● Saccharolytic-
● Antimicrobial Susceptibility - Achromobacter xylosoxidans and the
- unnamed Achromobacter groups B, E, and F
Susceptible
● Achromobacter xylosoxidans- most commonly
● amoxicillin-clavulanic acid isolated from this genus
● extended-spectrum cephalosporins
● Azithromycin
● Quinolones
● trimethoprim-sulfamethoxazole Chromobacterium

● Chromobacterium violaceum
2. Moraxella nonliquefaciens - reservoirs are soil and water
- Second most frequently isolated - commonly found in tropical and subtropical
- Normal flora of the respiratory tract of humans climates, in particular Southeast Asia and
- Does not grow on MAC India
- Opportunistic pathogen
3. Moraxella osloensis - immunocompromised patients with
- Normal flora in the genitourinary tract neutrophil deficits, including CGD
- Similar morphologically and biochemically to M. - skin lesion is the typical portal of entry
nonliquefaciens - osteomyelitis, abscesses, septicemia, urine
and gastrointestinal infections
4. Moraxella lacunata - (+) Oxidase, catalase
- Common conjunctival isolate - (-) indole
- Ferments glucose and variably sucrose
- Grows on MAC and most enteric bacteria
Oligella - Reduce nitrate
- Grows at 42˚C
● Small, paired, Gram negative bacilli or coccoid - Motile with polar flagella
● Usually isolated from the urinary tract - Produces a violet pigment called Violacein
● Oligella urethralis- nonmotile - Violacein: ethanol-soluble, water insoluble
● Oligella ureolytica- motile by peritrichous flagella pigment
● Do not grow on MAC
● Nonoxidative
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 ● Chloramphenicol
● SXT
Flavobacteriaceae ● third-generation cephalosporins (e.g., ceftazidime,
ceftriaxone, ceftizoxime), polymyxin B
● Nosocomial infection
● Elizabethkingia meningoseptica-causes meningitis or
septicemia in newborns THE HACEK GROUP OF BACTERIA
- Causes pneumonia, endocarditis, bacteremia, ● Composed of 5 Gram negative coccobacilli
and meningitis in adults ● Part of the normal oral flora
- produce acid from glucose, maltose, and ● Opportunistic pathogens
mannitol ● Causes Bacterial endocarditis
- hydrolize esculin ● Grow in blood culture bottles within 5 days
- (+) Indole ● Composed of:
Haemophilus,
Aggregatibacter (Actinobacillus),
Ralstonia Cardiobactrium,
Eikenella,
● Contaminate hospital fluids Kingella
1. Ralstonia pickettii
- meningitis, endocarditis, and osteomyelitis
- slow growers, requiring more than 72 hours on
primary cultures before colonies are visible Haemophilus
- (+) oxidase and catalase
- Grows on MAC ● facultative anaerobes
- reduces nitrate ● Gram negative, pleomorphic coccobacilli or rods
- oxidizes glucose and xylose ● Nonmotile
- motile by means of a single polar flagellum ● Ferment carbohydrates
2. Ralstonia mannitolilytica ● (+) oxidase and catalase
- Causes infections to patients with CF ● Reduce nitrates to nitrites
● Obligate parasites on the mucous membranes of
humans and animals
● Require growth factors found in blood
Sphingomonas - X factor (hemin or hematin)
- V factor (nicotinamide adenine dinucleotide)
1. Sphingomonas paucimobilis - Found within RBCS
- Isolated from water sources ● Laboratory Diagnosis
- Causes peritonitis associated with CAPD, - Culture media:
septicemia, meningitis, leg ulcers, empyema, ● 5% rabbit or horse blood agar
and splenic and brain abscesses ● Chocolate agar- used in the
- Produce yellow pigment isolation of Haemophilus sp.
- Does not grow on MAC - The lysing of the RBCs by
- requires more than 48 hours for culture on SBA heat in the preparation of
- weakly oxidase positive CHOC agar releases both
- motile at 18° to 22° C but not at 37° C X factor and V factor and
- (-) indole inactivates NADases
- oxidizers

2. Sphingomonas parapaucimobilis ● Haemophilus paraphrophilus

- isolated from sputum, urine, and vaginal
specimens
- H2S positive by the lead acetate method Aggregatibacter (Actinobacillus)
- (+) Simmon citrate
- (-) DNase 1. A. actinomycetemcomitans
● Antimicrobial Susceptibility - Human tissue infections have been attributed to
bites by cattle, sheep, pigs, and horses or
Susceptible
through contact with these animals
- Gram-negative, non–spore-forming
● Aminoglycosides
coccobacillus or short rod - major contributor to
● Tetracyclines
periodontitis

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 - grows both aerobically and anaerobically
- addition of 5% to 10% CO2 enhances growth Kingella kingae
- “star-shaped” colonies, often seen at 48 hours
- (+) Oxidase, glucose fermentation ● coccobacillary to short bacilli with squared ends that
- Catalase variable occur in pairs or short chains
- Do not grow on MAC ● resist decolorization in Gram stain
- Negative for X and V factor requirements ● nonmotile
- (-) urease, indole, esculin, and citrate ● causes indolent, slowly progressive endocarditis
- Usual treatment for endocarditis is with ● most common cause of osteoarthritis infection in
penicillin and aminoglycoside children younger than 4 years of age
● fastidious
2. A. aphrophilus ● (+) oxidase
- one of the most prevalent species in the HACEK ● (-) catalase
group involved in endocarditis ● Can grow on Neisseria selective agar (e.g., modified
- found in dental plaque and gingival scrapings Thayer -Martin medium)
- fever, heart murmur, congestive heart failure, ● SBA: large white-to-beige β-hemolytic colonies,
and embolism spreading, corroding colony
- Choc agar: convex, granular, and yellow ● No growth on MAC
colonies with an opaque zone near the center

Cardiobacterium hominis

● Pleomorphic, nonmotile, non-spore-forming,

fastidious, Gram-negative bacilli
● Normal flora of the nose, mouth, throat, may also be
found in genitourinary and gastrointestinal tracts
● Bacterial endocarditis, periodontitis, peritonitis
● facultative anaerobe (5% CO2)
● growth is enhanced in microaerophilic conditions
● does not grow on MAC
● grows on BA,CHOC
● (+) oxidase, indole
● (-) catalase, urease, nitrate, gelatin, and esculin

Eikenella corrodens

● Formerly classified as Bacteroides corrodens

● “corroding” bacilli- pits or makes a depression or
corrodes the surface of the agar
● Infections occur from human bites or fights
● Poor dental hygiene or oral surgery
● periodontitis, meningitis, empyema, pneumonia,
osteomyelitis, arthritis, and postoperative tissue
infections
● least common isolate of the HACEK group in adult
infectious endocarditis
● fastidious, gram-negative bacilli, nonmotile
● grow best under conditions of increased CO2 with
hemin
● (+) oxidase
● Asaccharolytic
● (-) catalase
● Produce yellow pigment
● chlorine bleach–like odor from the agar surface
● do not grow on MAC agar or eosin-methylene blue
(EMB) agar