Aerobic Non-Spore-Forming Gram-Positive Bacilli

Aerobic Non-Spore-Forming Gram-Positive
Bacilli

Listeria monocytogenes
• Gram reaction & characteristics:
• Gram-positive rod, facultative anaerobic, motile (tumbling motility).
• Habitat:
• Animals, soil, water, and vegetable matter; widespread in these environments.
• Normal flora of the vagina and intestines in humans.
• Virulence factor:
• Listeriolysin O: hemolytic and cytotoxic (survival within phagocytes)
• Internalin: Cell surface protein that induces phagocytosis.
• Act A: induces actin polymerization (cell-to-cell spread).
• Siderophores: Organisms capable of scavenging iron from human transferrin and of
enhanced growth.

• Disease: listeriosis.
• L. monocytogenes causes a variety of infections in neonates, pregnant women, and
immunosuppressed patients.
• CNS infections: meningitis, encephalitis, brain abscess, spinal cord infections.
• Neonatal:
• Early onset: Granulomatosis infantisepticum—in utero infection disseminated
systemically that causes stillbirth.
• Late onset: Bacterial meningitis.
• Food poisoning, bacteremia.
• Mode of transmission:
• Direct contact: Human gastrointestinal tract, ingestion of contaminated food, such as
meat and dairy products.
• Endogenous strain: Colonized mothers may pass organism to fetus. Portal of entry is
probably from gastrointestinal tract to blood and in some instances from blood to
meninges.

• Lab diagnosis:
• Samples: blood, CSF, amniotic fluid, respiratory secretions, placental or cutaneous swabs,
gastric aspirate.
• Culture characteristics:
• Listeria monocytogenes and Yersinia enterocolitica, can grow at 4°C to 43°C but
grow best at temperatures between 20° and 40°C. Grow at refrigerated temp, (cold
enrichment).
• On SBA, colonies are small and white with a narrow zone of beta-hemolysis.
• L. monocytogenes and -hemolytic Streptococci appear similar on blood agar plates;
however, L. monocytogenes can be distinguished because it is catalase +ve.
• Lithium chloride-phenylethanol-moxalactam (LPM) agar (highly selective), blue
colonies.
• Biochemical tests:
• Positive: Hippurate hydrolysis, CAMP test (shovel hemolysis), esculin, and catalase.
umbrella motility in semisolid media at room temperature and end-over-end
(tumbling) motility in a wet mount.
• Negative: oxidase.
• Treatment:
• Ampicillin, gentamicin.
• TMP-SMX.
• Note: Listeria: only Gram +ve with endotoxin. Bacteroides: only Gram –ve without typical
endotoxin.

Corynebacterium spp
• Gram +ve rods, aerobic, facultative anaerobes, non-motile, very pleomorphic, arranged in
"picket fences“ (palisades) or "Chinese letters“, nonparallel sides, club-shaped ends.
• Habitat:
• Opportunistic pathogens.
• Normal on skin & mucous membranes, nasopharynx but only in carrier state; not considered
part of normal microbiota (Isolation from healthy humans is not common).
• Diphtheria toxin: A potent exotoxin that destroys host cells by inhibiting protein synthesis.
• Disease:
• Respiratory diphtheria is a pharyngitis (exudative pseudomembrane-dead cells) that covers
the tonsils, uvula, palate, and pharyngeal wall; if untreated, life-threatening cardiac toxicity,
neurologic toxicity, and other complications occur.
• Respiratory obstruction develops, and release of toxin into the blood can damage various
organs, including the heart, myocarditis, polyneuritis.
• Direct contact: person to person by exposure to contaminated respiratory droplets.
• Contact with exudate from cutaneous lesions.
• Exposure to contaminated objects.

• Lab diagnosis:
• Samples: throat, nasal, membrane, swab, blood.
• On cystine-telluride blood agar (CTBA), potassium-
tellurite: Corynebacterium spp. form black colonies
from hydrolysis of tellurite to tellurium.
• On Tinsdale's agar: Corynebacterium spp. form
brown to black colonies with halos from hydrolysis
of tellurite.
• Loeffler serum agar is a nonselective medium that
supports growth and enhances pleomorphism and
the formation of metachromatic granules.
• On SBA as small, white, dry colonies. Most strains
are nonhemolytic.
• Positive: nitrate and catalase.
• Negative: Urease, motility.
• Staining with methylene blue will reveal metachromatic
granules, which are red to purple intracellular
granules.
• The Elek test (immunoprecipitation test) uses antitoxin
to detect toxin production.

• Prophylaxis:
• DTaP vaccine: Diphtheria toxoid, with boosters.
• Treatment:
• Antitoxin.
• Penicillin or erythromycin for local colonization.
• DTaP booster.
• Note: avoid trying to scrape the pseudomembrane because bleeding and toxin spread
may result.

Nocardia spp
• Gram-positive bacilli in chains pleomorphic, filamentous, branching, produce a beading
arrangement, obligate aerobe, appear fungal-like & nonmotile.
• Habitat:
• soil and water.
• Mycolic acid cell wall allow resistance to intracellular killing, tropism for neuronal
tissue, and ability to inhibit phagosome-lysosome fusion; other characteristics, such as
production of large amounts of catalase and hemolysins.
• Disease:
• Generally found in immunocompromised patients with chronic pulmonary disorders
(invasive & disseminated infections, Immunocompetent: skin infections. N. brasiliensis
most common species to cause skin infections. N. asteroides most common species to
cause lung infections. pneumonia, abscesses in kidney, brain.
• Exudate contains masses of filamentous organisms with pus that resemble sulfur
granules.
• Traumatic inoculation or inhalation.

• Lab diagnosis:
• Samples: sputum, bronchial lavage fluid, exudate, CSF.
• Requires up to 6 weeks for growth,
• On SBA wrinkled, dry, crumbly, chalky white to
orange-tan, beta hemolytic.
• Sabouraud dextrose agar or brain-heart infusion
agar.
• Positive: Partially acid-fast, catalase, urease.
• Treatment:
• TMP-SMX.
• Sulfonamides
• Other primary agents: amikacin, ceftriaxone,
cefotaxime, linezolid, or imipenem
• Minocycline
• Surgical drainage of abscesses.

Aerobic Spore-Forming Gram-Positive
Bacilli

Bacillus anthracis
• Gram positive bacilli, aerobic, capsulated, spore forming (endospore), non-motile, Large
with square ends. May be in chains. Oval, central to subterminal spores that aren’t swollen.
Looks like bamboo, boxcar morphology. Spores may not be seen in direct smear.
• Habitat:
• Soil: contracted by various herbivores (hides, wool, meat).
• Capsule exotoxins (edema toxin EF and lethal toxin LF) swelling and tissue death.
• Disease: anthrax (zoonotic, goat, cow), in three clinical forms:
• Cutaneous anthrax occurs at site of spore penetration 2-5 days after exposure
(erythematous papule to ulceration) and finally to formation of a black scar (i.e., eschar);
may progress to toxemia and death> malignant pustules.
• Pulmonary anthrax (woolsorters’ disease), follows inhalation of spores and progresses from
malaise with mild fever and nonproductive cough to respiratory distress, massive chest
edema, cyanosis, and death.
• Gastrointestinal anthrax may follow ingestion of spores and affects either the
oropharyngeal or the abdominal area; most patients die of toxemia and overwhelming
sepsis.
• Injectional anthrax after the intravenous injection of contaminated drugs; soft tissue
infections that lack the eschar associated with cutaneous anthrax. May result in death of
shock, coma, organ failure, and necrotizing fasciitis.

• B. anthracis is considered a potential bioterrorism agent and was used as such in a series of attacks
in the U.S. in 2001.
• Direct contact: animal tissue or products such as wool or hair (infecting organisms).
• Trauma or insect bites: organisms or spores.
• Inhalation: spores; woolsorters’ disease.
• Ingestion: contaminated meat.
• Injection: contaminated drugs.
• Lab diagnosis:
• Samples: blood, CSF, or material swabbed from cutaneous lesions.
• BSL-2.
• On SBA, it produces large, nonhemolytic colonies with filamentous projections, referred to
as medusa-head, comet tail or ground glass colonies. Stands up like beaten egg white when
touched with loop.
• B. anthracis typically does not grow on PEA agar at 24 hours.
• Positive: catalase, capsules seen in CSF & blood smears.
• Negative: motility, citrate.
• Mcfadyan reaction (my micro).
• Malachite green/safranin (central to terminal spore).
• Treatment: penicillin G. Anti-PA (protective antigen) vaccine.

Bacillus cereus
• Gram positive bacilli, spore-forming, aerobic, motile.
• Habitat:
• Vegetative cells and spores ubiquitous in nature; may transiently colonize skin or the
gastrointestinal or respiratory tracts.
• B. cereus and B. subtilis are also common laboratory contaminants.
• Enterotoxins and pyogenic toxin.
• Disease:
• Food poisoning of two types: diarrheal type, characterized by abdominal pain and watery
diarrhea, and emetic type, which is manifested by profuse vomiting; B. cereus–type species is
the most commonly encountered of Bacillus in opportunistic infections, including
posttraumatic eye infections, brain, bone, endocarditis, and bacteremia; infections of other
sites are rare and usually involve intravenous drug abusers or immunocompromised patients.
• Trauma
• Associated with immunocompromised patients
• Predominantly ingestion of food (rice) contaminated with B. cereus or toxins formed by this
organism.

• Lab diagnosis:
• Samples: stool, CSF, blood, food.
• Colony morphology: Large, flat, beta-
hemolytic colonies with irregular edges.
• Mannitol egg yolk polymyxin agar (MYP),
chromogenic media (Bacara) lecithinase
production, for food samples.
• Positive: Motile, citrate, oxidase.
• Treatment:
• Gentamycin, ciprofloxacin, clindamycin,
vancomycin.
• Rehydration.
• Prevention: good food handling and storage
(e.g., avoid reheating rice).

Neisseria gonorrhoeae
(Gonococci)
• Gram negative diplococci (GNDC), kidney or coffee-bean shaped, only glucose oxidizer,
capnophilic, microaerophilic.
• Habitat:
• Not part of normal microbiota. Only found on mucous membranes of genitalia, anorectal
area, oropharynx, or conjunctiva at time of infection.
• Allows attachment to mucosal surface.
• Antigenic variation to evade host defenses.
• Prevents phagocytosis.
• Disease:
• A leading cause of sexually transmitted infections.
• Genital infections include acute purulent urethritis, prostatitis, and epididymitis in males
and acute cervicitis, salpingitis, endometritis, and peritonitis. in females. These
infections also may be asymptomatic in females.
• Other localized infections include pharyngitis, anorectal infections, and conjunctivitis
(e.g., ophthalmia neonatorum of newborns acquired during birth from an infected
mother).

• Disseminated infections result when the organism spreads from a local infection to cause
pelvic inflammatory disease or disseminated gonococcal infection that includes
bacteremia, arthritis, and metastatic infection at other body sites.
• Pelvic inflammatory disease (PID) may cause sterility, ectopic pregnancy, or
perihepatitis also referred to as Fitz-Hugh–Curtis syndrome.
• Person-to-person spread by sexual contact, including rectal intercourse and orogenital
sex. May also be spread from infected mother to newborn during birth.
• Asymptomatic carriers are a significant reservoir for increased disease transmission.
• Lab diagnosis:
• Samples:
• Isolated from the urethra, cervix, anal canal (anorectal swab), throat swab,
oropharynx, skin lesions, joints, and blood. Conjunctival swab (neonatal
conjunctivitis).
• Yellow mucopurulent urethral discharge, Gram -ve intracellular diplococci as well as
E/C.
• Culture confirmation required for females.

• N. gonorrhoeae is fastidious, requiring enriched media such as chocolate. It does not
grow on SBA.
• Selective media include Thayer-martin, modified Thayer-Martin (MTM), Martin-Lewis,
New York City, and GC-Lect agars (recommended).
• The bacteria require increased CO2 5-10% (capnophilic) at 37 °C with a humidified
atmosphere.
• Colonies are flat, smooth, and glistening gray or tan.
• They cannot tolerate cold; therefore, media must be at room temperature before
plating.
• Because of autolysis, gonococci cannot be incubated for prolonged times.
• Positive: Superoxol (30% H2O2), catalase, oxidase, and glucose.
• Negative: Maltose, lactose, sucrose, DNase, and nitrate.
• O/F: oxidizer.
• Treatment:
• Many strains are positive for beta-lactamase production.
• To prevent newborn conjunctivitis, antimicrobial eye drops (e.g., erythromycin) are
administered to all infants at birth.
• Ceftriaxone ( doxycycline for probable concurrent Chlamydia infection).
• Vaccine development difficult because of pili antigen variations.

Transgrow bottle (Martin-
Lewis medium), +ve
oxidase
Thayer-Martin agar

Neisseria meningitidis
(Meningococci)
• Gram negative diplococci (GNDC), capsulated, kidney or coffee-bean shaped, maltose &
glucose oxidizer, not fastidious as GC.
• Habitat:
• Colonizes oropharyngeal and nasopharyngeal mucous membranes of humans. Humans
commonly carry the organism without symptoms.
• Pili, IgA protease, endotoxin, capsule,
• Disease:
• Causes meningococcal meningitis in adult, meningococcemia with petechial rash, leading
to disseminated intravascular coagulation.
• Headache, fever, chills, nausea, vomiting, photophobia, and stiffness of the neck.
• When fulminant, cause (Waterhouse-Friderichsen syndrome).
• Less common infections include conjunctivitis, pneumonia, and sinusitis.
• Kernig’s sign, nuchal rigidity (neck stiffness).
• Person-to-person spread by respiratory droplets, usually in settings of close contact
(e.g., dormitories, prisons, shelters).

• Lab diagnosis:
• Samples:
• Cerebrospinal fluid (CSF), sputum, blood, and nasopharyngeal swabs. Don’t
refrigerate.
• CSF, ↑ PMNs,↑ protein, ↓ glucose, as well as intracellular kidney bean-shaped
diplococci.
• BSL2,3.
• Colonies are flat, smooth, and gray to white on chocolate agar. N. meningitidis will
grow on SBA, CHOC, incubated in increased CO2 and produce bluish-gray colonies.
• Positive: Catalase, oxidase, glucose, and maltose.
• Negative: DNase and nitrate.
• Treatment:
• Penicillin G, ceftriaxone.
• Rifampin for close contacts as prophylaxis.
• vaccine with capsule polysaccharides.
• Note: N. lactamica may misidentified as N. meningitidis, ONPG use for differentiation.

Moraxella catarrhalis
• Gram negative diplococci, kidney shaped.
• Habitat:
• Normal flora of the upper respiratory tract, occasionally colonizes female genital tract.
• Pili, capsule, Antigenic variation, endotoxin.
• Disease:
• Causes otitis media, sinusitis, and respiratory tract infections (pneumonia, bronchitis)in
elderly patients and those with chronic obstructive pulmonary disease.
• Septicemia, endocarditis, meningitis, septic arthritis, eye, urogenital & wounds infections,
• Catarrh refers to inflammation of a mucous membrane with increased flow of mucus or
exudate.
• Spread of patient’s endogenous strain to normally sterile sites.
• Person-to-person nosocomial spread by respiratory droplets may occur.

• Lab diagnosis:
• Samples: sputum, blood, CSF, ear swab.
• Grows on SBA & CHOC. Some may grow at RT &/or on Neisseria-selective media.
“Hockey puck colonies”
• Positive: Catalase, oxidase, DNase (vs Neisseria spp.), nitrate, and butyrate esterase.
• Negative: Asaccharolytic; all carbohydrate tests are negative.
• DNase & butyrate esterase differentiate from Neisseria spp.
• Treatment:
• Amoxicillin-clavulanate (95% produce beta-lactamase).
• Second- and third-generation cephalosporins.
• TMP-SMX.

Haemophilus influenzae
• Gram negative coccobacilli (GNCB) or rods, non-motile, pleomorphic, small to filamentous. Capsules
may be seen.
• Habitat:
• Normal microbiota: upper respiratory tract.
• Capsule: antiphagocytic, type b most common.
• Additional cell envelope factors mediate attachment to host cells.
• Unencapsulated strains: pili and other cell surface factors mediate attachment.
• Disease:
• Encapsulated strains: Meningitis, Epiglottitis, Cellulitis with bacteremia, Septic arthritis, Pneumonia
• Nonencapsulated strains: Localized infections, Otitis media, Sinusitis, Conjunctivitis (pink eye).
• Acute epiglottitis (obstructive laryngitis), septicemia, septic arthritis, osteomyelitis, and
pericarditis.
• Immunocompromised patients: Chronic bronchitis, Pneumonia, Bacteremia
• Type b common cause of pneumonia & meningitis in children were Hib vaccine not available.
• Person-to-person: respiratory droplets.
• Endogenous strains.

• Lab diagnosis:
• Samples:
• Blood, sputum, CSF, and eye swabs.
• Requires hemin (X factor) and NAD (V factor) on chocolate agar.
• Chocolate agar is routinely used for cultures: Smooth, round, flat, opaque, and tan on
chocolate agar (dew drops).
• Grows at 35-37 ° C with 5-10% CO2 and is susceptible to drying and temperature
changes.
• Quad plate.
• Not growing on SBA.
• Satellitism: Haemophilus spp. can grow around colonies of S. aureus growing on an
SBA plate. S. aureus releases NAD. Therefore, Haemophilus will grow near the S.
aureus colonies, forming tiny clear pinpoint colonies.
• Immunofluorescence.
• Specific detection of Hib capsular antigen is by latex agglutination.
• Quellung test.
• Positive: catalase and oxidase.

• Treatment:
• H. influenzae & other spp:
• ceftriaxone or cefotaxime for life-threatening infections; for localized infections
several cephalosporins, beta-lactam/beta-lactamase inhibitor combinations,
macrolides, trimethoprim-sulfamethoxazole, and certain fluoroquinolones are
effective.
• H. ducreyi:
• Erythromycin is the drug of choice; other potentially active agents include
ceftriaxone and ciprofloxacin.
• Hib vaccine: capsular polysaccharide of type B strain conjugated to diphtheria toxoid.
• Rifampin prophylaxis for close contacts.
• H. influenzae isolates should be tested for beta-lactamase.
• Note:
• HACEK organisms (Haemophilus species, Actinobacillus actinomycetemcomitans,
Cardiobacterium hominis, Eikenella corrodens, and Kingella species) are gram-negative
bacilli that are part of normal oral fl ora and can infect heart valves. They are the most
common gram-negative cause of endocarditis in non-IV drug users.

H. influenzae biotype aegyptius & H. aegyptius:
• Both cause conjunctivitis (pink eye) and respiratory infection. H. influenzae biotype
aegyptius also causes Brazilian purpuric fever
• H. parainfluenzae, H. haemolyticus, & H. parahaemolyticus:
• Normal flora of upper respiratory tract. Low incidence of pathogenicity.
• Haemophilus ducreyi:
• Not part of normal human microbiota.
• Causes genital ulcers, soft chancer, (chancroid) & buboes (swollen lymph nodes).
• Transmission: person-to-person: sexual contact
• Chocolate agar with vancomycin is used to inhibit normal flora and contaminants.
• Difficult to culture (selective medium).
• Note:
• To establish X and V factor requirements, disks impregnated with each factor are placed
on un supplemented media, usually Mueller-Hinton agar or trypticase soy agar.
• Specialized Haemophilus Quad plate also can be used.

Non-fermentative Gram-Negative Bacilli

• General characteristics:
• Obligate aerobes.
• Oxidation-fermentation (OF) medium: either open tube pos/closed tube neg
(oxidizer) or open tube neg/closed tube neg (non-oxidizer).
• Grow on SBA & CHOC in 24–48 hr.
• Most grow on MAC. Appear as non–lactose fermenter.
• Most are oxidase pos. Differentiates from Enterobacteriaceae.
• Resistant to variety of antibiotics.
• Found in water, soil, food, and plants, and a few are normal flora of humans
• Approximately 20% of all gram-negative bacilli isolates are non-fermentative gram-
negative bacilli (NFB).
• They do not form spores and do not metabolize carbohydrates under anaerobic
conditions (fermentation).
• TSI: K/no change

Pseudomonas aeruginosa
• Gram negative bacilli, NLF, non-glucose fermenter, motile, capsulated (In the
CF setting).
• Habitat:
• Environment (soil, water, plants); survives well in domestic environments (e.g.,
hot tubs, whirlpools, contact lens solutions) and hospital environments (e.g.,
sinks, showers, respiratory equipment); rarely part of normal microbiota of
healthy humans.
• Exotoxin A, exoenzymes S and T, endotoxin (LPS), proteolytic enzymes,
alginate, pili, adhesins; intrinsic resistance to many antimicrobial agents.

• Disease:
• In immunocompromised individuals.
• P. aeruginosa is resistant to a number of disinfectants and has been responsible for
serious nosocomial infections.
• It is especially associated with hospital environments and equipment, whirlpools, and
swimming pools.
• It causes eye infections (contact lens keratitis), ear infections and is responsible for
"swimmer's ear" which is an external otitis. Folliculitis.
• Lower respiratory tract (pneumonia) infections in patients with cystic fibrosis (CF),
UTI, burn wound infections.
• Endocarditis (IV drug users); osteomyelitis (diabetics, IV drug users).
• Folliculitis (hot tub infection); many other infections in hosts with weakened
immunity.
• Ingestion of contaminated food or water; exposure to contaminated medical devices
and solutions; introduction by penetrating wounds; person-to-person transmission is
assumed to occur.

• Lab diagnosis:
• Samples: urine, sputum, ear swab, blood, skin lesions, pus.
• On MacConkey: NLF.
• On nutrient agar green color.
• Grows at 42 C
• Grape or corn tortillas like odor.
• Large, irregular colonies with a grapelike or fruity odor.
• Beta-hemolytic colonies with a feathery edge on SBA, and
metallic sheen.
• Mucoid colonies when isolated from patients with CF
• Pigment: only P aeruginosa produces pyocyanin, a blue
pigment. Pyocyanin mixes with fluorescein to produce a blue-
green color.
• Blue-green colonies (pyocyanin + pyoverdine pigment)
• Growth on cetrimide agar, produce a yellow pigment that
fluoresces (pyoverdin).
• Other pigments produced by some P. aeruginosa strains are
pyorubin (red) and pyomelanin (brown).
• Positive: Oxidase, catalase, motility, K/NC on TSI.
• O/F: P. aeruginosa is the most important NFB.
TSA

• Treatment:
• Very resistant to antimicrobial agents.
• Anti-pseudomonal penicillin + aminoglycoside (e.g., piperacillin + gentamicin, mezlocillin
+ gentamicin) fluoroquinolones.
• Note:
• P. aeruginosa is associated with moisture and can be introduced in hospitals through
water in respiratory equipment, visitor’s flowers, or endoscopes.

Vibrio spp
• General characteristics:
• Most are indole positive, and all are oxidase positive.
• Motile.
• O1 and O139 cause epidemic.
• All species are halophilic (salt loving) except V. cholerae and
V. mimicus.

Vibrio cholerae
• Gram negative bacilli, NLF, glucose fermenter, facultative anaerobe motile, comma shaped.
• Habitat:
• Common in saltwater environments, on and in marine animals, on plankton, and in seafood
(shellfish, crabs, shrimp, and prawns). human carriers also are known, particularly in endemic
regions.
• Cholera toxin (CT); zonula occludens (Zot) toxin (enterotoxin); accessory cholera enterotoxin
(choleragen) (Ace) toxin; O1 and O139 somatic antigens, hemolysin/cytotoxins, motility,
chemotaxis, mucinase, and toxin coregulated pili (TCP) pili.
• Disease:
• Cholera: profuse, rice watery acute diarrhea leading to dehydration (electrolyte imbalance),
hypotension, and often death; occurs in epidemics and pandemics that span the globe.
• May also cause nonepidemic diarrhea and, occasionally, extraintestinal infections of wounds,
bacteremia, otitis media, respiratory tract, urinary tract, and central nervous system.
• Fecal-oral route, by ingestion of contaminated washing, swimming, cooking, or drinking
water; also, by ingestion of contaminated shellfish or other seafood.

• Lab diagnosis:
• Samples: stool, blood.
• Thiosulfate citrate bile salt sucrose agar (TCBS) is a selective and differential
(based on sucrose fermentation) medium that supports the growth of most species
and is particularly useful for isolating V. cholerae and V. parahaemolyticus. V.
cholerae is sucrose positive and will produce yellow colonies on TCBS agar,
whereas V parahaemolyticus is sucrose negative.
• Non-halophilic (doesn’t require NaCl for growth). Grows on SBA (beta), CHOC,
MAC (NLF). Large yellow colonies on TCBS (ferments sucrose). Alkaline peptone
water (APW) can be used as enrichment.
• Positive: oxidase, nitrate.
• Treatment:
• Oral/IV rehydration therapy (glucose + Na).
• Tetracycline.
• Killed-cell vaccines available (not very effective).
• Not:
• Blood group O patients are more vulnerable.

V. cholerae (string test
positive) and other Vibrio
spp. (string test
negative).
Leifson flagella stain

Legionella pneumophilia
legionnaires disease-1976
• Gram negative coccobacilli, pleomorphic, obligate aerobe, can survived inside macrophage.
• Habitat:
• Aquatic habitats, found in various water systems, including humidifiers, whirlpools,
showerheads and air conditioning chillers. Virulence factor:
• Adheres to respiratory epithelium via pili → phagocytosed by alveolar macrophages →
survives and proliferates inside nutrient-rich phagosome.
• Disease:
• Pontiac fever (mild form) flulike symptoms.
• Legionnaire’s disease (atypical pneumonia): neutrophils arrive and form micro-abscesses
(can be seen on X-ray).
• Legionella is an important cause of community-acquired pneumonia in elderly smokers.
• Inhalation and aspiration of infectious aerosols are considered the primary means of
transmission.
• Inhaled in aerosols from respiratory devices, air conditioners.
• Exposure to these aerosols can occur in the workplace or in industrial or health care
settings; for example, nebulizers filled with tap water and showers have been implicated.

• Lab diagnosis:
• Gram stains faintly or poorly (appear as thin), and it is better to use 0.1 % basic fuchsin
as the counter stain instead of safranin.
• Visualize with silver stain, crystal violet.
• Other identifying tests: Direct fluorescent antibody test, nucleic acid probes, pale yellow-
green fluorescence with Wood’s lamp.
• Specimens:
• The urine antigen test (radioimmunoassay) is the most common laboratory assay
used for the diagnosis of legionellosis.
• From the lower respiratory tract, lung biopsy, bronchial wash, expectorated sputum,
etc. are sometimes used for cultures for the diagnosis of the pneumonic form of the
disease.
• Whitish green on buffered charcoal yeast agar (BCYE) with iron & cysteine, containing
polymyxin-anisomycin-vancomycin (PAV) and inhibitory dyes should be used for
contaminated specimens. Grow also on Brucella blood agar & chocolate agar (tiny
colonies) but not on SBA.
• Incubated in humidified air at 35C to 37C.
• Positive: oxidase,
• Asacchrolytic.

• Treatment:
• Erythromycin, rifampin, tetracycline, fluoroquinolones.
• Note: common causes of atypical:
• pneumonia:
• Mycoplasma
• Legionella
• Chlamydia
• Viruses

Bordetella pertussis
• Gram negative, pleomorphic, coccobacilli, obligate aerobic.
• Habitat:
• Mucous membranes of the respiratory tract of humans.
• Adhesion to ciliated epithelium: Filamentous haemagglutinin,
• Pertussis toxin inhibiting killing by phagocytosis.
• Secreted adenylate cyclase → inhibits bactericidal activity.
• Tracheal cytotoxin → impairs mucous clearance.
• Disease:
• Pertussis (whooping cough).
• Three stages in children and adult.
• Catarrhal: general flulike symptoms, highly contagious.
• Paroxysmal: repetitive coughing episodes, whooping inspiration
• Convalescent: recovery phase, gradual reduction in symptoms.
• The coughs produce copious greenish phlegm.
• Complications of pertussis include otitis media, pneumonia and CNS dysfunction.
• Person-to-person through inhalation of respiratory droplets. Humans are the only known reservoir.

• Lab diagnosis:
• Samples:
• Nasopharyngeal aspirates or a posterior nasopharyngeal swab.
• Dacron or calcium alginate swabs should be used, cotton will inhibit growth of B. pertussis.
• Preferred media: Bordet-Gengou potato infusion medium and charcoal horse blood (CHB)
medium (also known as Regan-Lowe medium).
• Media are often made selective by adding cephalexin (Modified Jones-Kendrick charcoal,
Stainer-Scholte).
• Does not grow on MAC agar. Require protective substances such as charcoal, blood, or starch.
• B. pertussis colonies are small and smooth; they appear like mercury droplets/pearls and are
beta-hemolytic.
• Gram stain shows minute, poorly stained coccobacilli, single or in pairs.
• Positive: catalase, oxidase.
• Negative: urease.
• Treatment:
• Prophylaxis: DTaP vaccine: acellular Pertussis antigens.
• Treatment: erythromycin, azithromycin, clarithromycin, Trimethoprim-sulfamethoxazole and
fluoroquinolones.
• Anti-FHA Abs generated by vaccine.

• Specimens should be plated immediately or placed into a suitable transport medium (e.g.,
Regan-Lowe transport medium, casamino acids medium, Jones-Kendrick charcoal medium, or
Amies medium with charcoal).
charcoal-horse blood agar

Pasteurella multocida
• Gram negative pleomorphic coccobacilli, capsulated, nonmotile, may show bipolar
staining.
• Habitat:
• Commensal found in nasopharynx and gastrointestinal tract of wild and domestic
animals; potential upper respiratory commensal in humans who have extensive
occupational exposure to animals.
• Endotoxin, cytotoxin, surface adhesins, capsule associated with P. multocida.
• Disease:
• Wounds infections, abscesses, cellulitis but can progress into septicemia, endocarditis,
osteomyelitis, arthritis, peritonitis, meningitis, joint infections, and pneumonia,
lymphadenopathy.
• Humans acquire the bacteria from animal bites (cats and dogs) or by inhalation of dried
animal feces. or contact with infected carcass.

• Lab diagnosis:
• Samples:
• Culture of wound site shows Gram -ve coccobacilli with bipolar staining.
• Respiratory secretions.
• Selective media containing vancomycin, clindamycin, and/or amikacin have been
used to isolate Pasteurella from clinical specimens.
• Grows on SBA (non-hemolytic) & CHOC but not MAC.
• Musty odor.
• Positive: Oxidase, catalase, indole, and nitrate.
• Treatment:
• Penicillin (very susceptible), ampicillin, amoxicillin are recommended agents;
• Doxycycline, amoxicillin-clavulanate are alternative agents;
• ceftriaxone, fluoroquinolones may be effective.
• Clean and drain wound.
• Note:
• Suturing wound may worsen infection by creating a closed anaerobic environment.

Campylobacter and Helicobacter

Campylobacter jejuni
• Gram negative curved bacilli, motile, that may appear spiral, C or S-shaped on Gram
stain (a bird in flight, Seagulls or gull-wings shaped, ), Most species are microaerophilic,
capnophilic & thermophilic.
• Habitat:
• Poultry, pigs, bulls, dogs, cats, birds, sheep.
• Motility and adherence, enterotoxin, LPS, cytotoxic enzymes.
• Disease:
• Enteritis, bloody diarrhea, secretory diarrhea.
• Major cause of food poisoning, causing gastroenteritis, proctitis (inflammation of the
lining of the rectum), most common cause of bacterial diarrhea.
• Extraintestinal infections include septicemia, hepatitis, pancreatitis, abortion and
neonatal sepsis, hemolytic uremic syndrome, nephritis, prostatitis, urinary tract
infection, peritonitis, myocarditis, meningitis, septic arthritis, and abscesses.
• Guillain-Barré Syndrome are thought to be complications of Campylobacter infections.
• By eating undercooked contaminated poultry or other meat products (Sources of
infection: chickens, raw milk, pets).

• Lab diagnosis:
• Samples: stool.
• Optimal condition: (3-5% O2, 10% CO2, 85% N2) & referred to as Campy gas.
• Optimal temperature: 42C. Grow slowly at 37° C. normal enteric flora inhibited by
42°C incubation.
• Selective media (Campy media) e.g., Charcoal cefoperazone deoxycholate agar and
Campy-colistin vancomycin amphotericin B, are available for the isolation of C. jejuni
from stool specimens.
• Campy-BAP: non-hemolytic. Not grow on MAC.
• Hold plates 3 days.
• Carbol fuchsin or basic fuchsin is used as a counterstain.
• Positive: Oxidase, catalase, nitrate & Hippurate hydrolysis, darting corkscrew
motility.
• Negative: urease.
• They do not oxidize or ferment carbohydrates, and most human isolates are catalase
and oxidase positive.
• Oxidase-positive, curved Gram-negative rods that are hippurate hydrolysis positive
should be reported as C. jejuni without further workup.
• Treatment:
• Erythromycin, azithromycin, clarithromycin, ciprofloxacin.

Helicobacter pylori
• Gram negative, curved spiral-shaped bacilli, motile.
• Habitat:
• gastrointestinal and hepatobiliary tracts of mammals (including humans) and birds.
• Adhesins for colonization of mucosal surfaces.
• Motility allows H. pylori to escape the acidity of the stomach and burrow through and
colonize the gastric mucosa in close association with the epithelium.
• The organism produces urease that hydrolyzes urea forming ammonia (NH3),
significantly increasing the pH around the site of infection.
• CagA protein, affects host cell gene expression, inducing cytokine release and altering cell
structure (peptic ulcer disease and gastric carcinoma).
• Disease:
• Acute gastritis (abdominal pain, nausea, vomiting), peptic and duodenal ulcers, gastric
malignancy.
• Chronic: gastric adenocarcinoma and MALT lymphoma.
• Oral-oral (kissing), fecal-oral.
• Zoonotic (cats, dogs) transmission.

• Lab diagnosis:
• Samples: stool, blood, biopsy.
• Can be isolated from gastric biopsy.
• Fecal antigen detection, urea breath test,
and demonstration of urease activity in
stomach biopsy material.
• On SBA, Brucella, and Skirrow's agars
incubated microaerophilically (2-7% O2, 5-
10% CO2, hydrogen 5-8%) & at 37C.
• Doesn’t grow at 42°C. Slow growing.
• Easier to see when carbol fuchsin.
• Positive: Oxidase, rapid urease, and
catalase.
• Treatment:
• PPI + amoxicillin + clarithromycin, or PPI +
BMT (bismuth therapy, metronidazole,
tetracycline).

Anaerobic Gram-Negative Bacilli

Bacteroides fragilis
• Gram negative strict anaerobic bacilli, capsulated, with rounded ends and may be
pleomorphic, nonmotile.
• Habitat:
• Bacteroides species are the most common among normal GI (colon) flora. Normally
makes vitamin K for host.
• Polysaccharide capsule, endotoxin, and succinic acid, which inhibit phagocytosis.
• Disease:
• Occur Below the diaphragm.
• Bedsores, Peritonitis, GI or pelvic abscesses, abdominal and pelvic infections, bacteremia.
• Endogenous strains of normal microbiota gain access to normally sterile sites, usually as
result of one or more predisposing factors that compromise normal anatomic barriers
(e.g., surgery or accidental trauma intrauterine devices) or alter other host defense
mechanisms (e.g., malignancy, diabetes, burns, immunosuppressive therapy).

• Lab diagnosis:
• Samples: stool, blood.
• Some strains hemolytic on anaerobic blood agar (BRU/BA).
• Produces brown to black colonies on Bacteroides bile esculin (BBE) agar.
• Positive: growth in 20% bile, catalase positive, bile-esculin positive
• Negative: indole, lipase negative, lecithinase negative, and gelatinase negative.
• Treatment:
• Resistant to penicillin, colistin, kanamycin, and vancomycin and susceptible to rifampin.
• Drain abscess + repair lesions + antibiotics (clindamycin).
• Highly effective agents include most beta-lactam/beta-lactamase–inhibitor combinations,
imipenem, metronidazole, and chloramphenicol Cefoxitin (cephalosporin) Moxifloxacin
(fluoroquinolone).

Alyazeed Hussein, BSc, SUST
This has been a presentation of Alyazeed Hussein
Thanks for your attention and kind patience
@elyazeed7
@Alyazeed7ussein

Aerobic Non-Spore-Forming Gram-Positive Bacilli

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