OUR LADY OF FATIMA UNIVERSITY COLLEGE OF MEDICAL LABORATORY SCIENCE

SEMINAR 1 – CLINICAL BACTERIOLOGY

NON-ENTERIC GRAM-NEGATIVE BACILLI  Called as the summer diarrhea in Japan

o Pandemic strain: Vibrio parahaemolyticus O3:K6
 Nonenteric- related to water borne
 Virulence: Kanagawa phenomenon (heat stable hemolysin)
VIBRIONACEAE
 Clinical Significance
GENERAL CHARACTERISTICS
o Patients have water diarrhea and vomiting 24 to 48 hours
 Commonly found in wide variety of environments such as fresh water,
after ingestion of contaminated seafood and pneumonia
brackish or estuarine water and salt water
Vibrio vulnificus - VulMa
 Easily isolated in temperatures exceeding 20ºC from algae, plankton, fish and
 Seen in marine environment
shellfish
 Causes primary septicemia and wound infections
Antigenic Structure
Vibrio alginolyticus
 K antigen is seen in Vibrio parahemolyticus
 Least pathogenic for man and not commonly isolated
 O Antigen subgroups
 Can survive at high salt concentration (10%) – 10% NaCl
o Vibrio cholerae O1
Laboratory procedure
 Associated with epidemic cholera
String test
 Vibrio cholerae O1 serotype Ogawa
Halophilic property= salt requirement
 Vibrio cholerae O1 serotype Inaba
TCBS is used for VIbrio identification (containes sucrose)
 Vibrio cholerae O1 serotype Hikojima
Lactose for enterics; sucrose for non-enterics
o Vibrio cholerae O139
 Associated with epidemic cholera
AEROMONADACEAE
 Contains cholera toxin gene (ctx)
GENERAL
o Vibrio cholerae O75 & O141
CHARACTERISTICS
 Can harbor the cholera toxin gene and have been
 Ubiquitous, glucose fermenters, straight rods grow at 10 to 42ºC
associated with sporadic, cholera like diarrhea and
 Classified into 2 groups
bloodstream infection
o Mesophilic group: MesoHydroCaVe optimal temp: 37C
o Vibrio cholerae non-O1
 Motile by single polar flagella
 Resembles Vibrio cholerae O1 but cannot agglutinate O1
 Aeromonas hydrophilia complex
antisera
 Aeromonas veronii complex
Vibrio cholerae O1
 Aeromonas caviae complex
 Causative agent of cholera or also known as Asiatic cholera
o Psychrophilic group:PsychroSalmon optimal temp: 22C can range up
 Virulence: cholera toxin or choleragen
to 42C
 Clinical Significance
 Aeromonas salmonicida
o Presence of rice water stools with numerous flecks of mucus
 Clinical Significance
o Epidemic Vibrio cholerae O1 has 2 biogroups: Classic and El Tor (more
o Aeromonas caviae is most frequently associated with
pathogenic biotype) gastrointestinal infections – A. caviae = AC
 Acute dysenteric diarrhea
CLASSIC BIOTPYE EL TOR BIOTYPE
 Cholera like disease
RBC hemolysis - + LABORATORY DIAGNOSIS
Vogues Proskauer - + Macroscopy
 Sheep blood agar
50ug polymyxin B S R o Large, round, raised, opaque colonies with entire edge and a
Agglutination of chicken smooth surface; incubated for 24hr at 35ºC
RBCs - +
o Aeromonas caviae has a strong beta hemolysis
Lysis by bacteriophage + -  Cefsulodin Irgasin Novobiocin (CIN) II (CIN agar is for Yersinia pestis)
o Contains 4ug of cefsulodin
o Highest recovery of Aeromonas spp
Biochemical Test
 All Aeromonas spp are oxidase positive and glucose fermenters
 Most clinically relevant Aeromonas spp. are indole positive
 Aeromonas spp are O/129 disk resistant or negative
LABORATORY DIAGNOSIS
Vibrio parahemolyticus – K antigen
Specimen  O/129 disk or String test separates Vibrio from Aeromonas
 Swab of body fluids, pus or tissues is accepted if transported by using Cary Blair
 Inositol fermentation separates Vibrio from Plesiomonas
as transport media; buffered glycerol saline is NOT recommended for transport
 Liquid stool should be placed in plastic bags with few drops of saline to  Oxidase reaction separates Vibrio from Enterobacteriaceae
maintain moisture; stored up to 5 weeks
Microscopy V. cholerae Aeromonas Others
 ASPOROGENOUS, gram negative rods that is also pleomorphic when there is TCBS + - +
suboptimal growth condition 6 to 8hrs
 Motility String test + - +
o Most have polar sheathed monotrichous or multitrichous flagella (0.5% Na
seen in broths desoxycholate)
o Some have polar sheathed lateral flagella that can be seen on O/129 disk S R R
culture media as swarming NaCl broth + - -
 Vibrio parahemolyticus 6.5% NaCl broth + + -
 Vibrio alginolyticus Inositol (F) - - -
Additional Mannitol + Mannitol +
Macroscopy
 SBA & CHOC
o Medium to large colonies; smooth, opaque and iridescent with  Vibrios are facultatively anaerobic
greenish hue  All 10 clinically significant Vibrio spp are Catalase. Oxidase, and Nitrate
 MAC Reduction POSITIVE
o Only Vibrio vulnificus can ferment lactose  V. metschnikovii= Catalase. Oxidase, and Nitrate Reduction NEGATIVE
 Thiosulfate citrate bile salt sucrose (TCBS) agar CAMPYLOBACTERIACEAE
o Selective media for patient that has exposure to seafood or
seawater; based on sucrose fermentation
GENERAL CHARACTERISTICS
 Known to cause abortion in domestic animals
o Sucrose fermenter: MFAC = YELLOW COLONIES
 Species known in the genus
 Vibrio cholerae, Vibrio alginolyticus, Vibrio fluvialis, Vibrio
o Campylobacter jejuni
metschnikovii
 Most common cause of bacterial gastroenteritis
o Non sucrose fermenter:
 Acquired through direct contact to animals and
 Vibrio mimicus, Vibrio parahemolyticus,
Vibrio damselae consumption of contaminated water
 Alkaline Peptone Water (APW) – to increase growth of Vibrio o Campylobacter fetus subsp. fetus
 Most frequently isolated from blood culture
o Have a pH of 8.5 and 1% NaCl; incubated for 5 to 8 hours at 35C
before culturing to TCBS  Common among immunocompromised patients
 Wagatsuma Agar o Helicobacter pylori
o Ideal isolation for Vibrio parahemolyticus  Associated with gastric, peptic and duodenal ulcers and
o Detects the presence of Kanagawa toxins gastrointestinal carcinoma
o High salt mannitol media  Major cause of type B gastritis
LABORATORY DIAGNOSIS
Biochemical Test Specimen
 Facultative anaerobes  Blood: recovery of Campylobacter fetus subsp. fetus
 Catalase and oxidase positive EXCEPT Vibrio metschnikovii  Tissue biopsy: recovery of H. pylori and should be stored in cysteine brucella
 Nitrite reducer EXCEPT Vibrio metschnikovii broth with 20% glycerol and frozen at -70C
 All are halophilic EXCEPT Vibrio cholerae & Vibrio mimicus  Stool: transported in CARY BLAIR
 Vibrio spp. are SUSCEPTIBLE to O/129 vibriostatic agent Microscopy
 All Vibrio spp. cannot ferment inositol EXCEPT some strains of Virbio  Campylobacter spp. are curved, gram negative rods and may appear as long as S-
metschnikovii and Vibrio cincinnatiensis or seagull wing shapes; moist, runny looking and spreading colonies
 Gram staining: Carbol fuchsin is recommended as counter stain for 2 to 3 min
o Not well defined in safranin
o Also used in acid fast bacilli
o If not well defined, may be mistaken as gram positive or gram ghost
 Appears coccobacillus in older cultures
 Motility= “darting” motility on hanging drop preparations or when visualized
NOTE: under phase contrast microscopy
o Campylobacter spp.: single polar flagellum
 o Helicobacter spp: single or multiple polar flagellum Cefazolin
 Distilled water and saline seem to INHIBIT motility Nutrient agar
CCDA (Charcoal Cefoperazone, Amphotericin
Macroscopy Charcoal
cefoperazone B
 Enriched selective media Sodium desoxycholate
deoxycholate agar)
o Campylobacter blood agar or Campy blood agar
 commonly used medium to isolate C. jejuni and other enteric Biochemical test
campylobacters.
 contains Brucella agar base, 10% sheep red blood cells, and a C. jejuni C. coli C. fetus
combination of antimicrobials: vancomycin, trimethoprim, (hippurate (growth at
polymyxin B, amphotericin B, and cephalothin. hydrolysis) 37C)
o Other selective media that have been successful in recovering Catalase + + +
Campylobacter spp. are Butzler medium and Skirrow’s medium. Nitrate + + +
 Incubation requirement: Urease - - -
o incubating stool cultures at 42° C = C. jejuni and other enteric Hippurate
+ - -
campylobacters grow optimally at 42° C hydrolysis
o growth of colon microbiota is inhibited at this higher temperature. 42C growth + + -
o C. fetus subsp. fetus, a rare stool isolate, and growth is suppressed at 42° Nalidixic acid S S R
C; therefore to isolate this organism, media should be incubated at 37° C. Cephalothin R R S

 Stool culture: incubated at 42C to recover C. jejuni (42-43C optimal temp)

o Campy-CVA (cefoperazone-vancomycin-amphotericin B) medium has
been reported to provide better suppression of fecal biota, even when this
medium is incubated at 37° C. Incubation at 37° C allows the recovery
of Campylobacter spp. that are inhibited at 42° C. C. fetus subsp. fetus,
C. rectus, and C. curvus can be isolated using routine culture media.
Charcoal-based, blood-free media, such as charcoal cefoperazone
deoxycholate agar (CCDA) and charcoal-based selective media
(CSM), are also available.
o A combination of media that contain either CCDA or CSM can
achieve the highest yield of Campylobacter spp. in stool samples.
To recover H. pylori, a combination of a nonselective medium, such as
CHOC agar or Brucella agar with 5% horse red blood cells, and a
selective medium, such as Skirrow’s agar, may be used. It is important
that the inoculated medium be fresh and moist and that the culture be
incubated in a microaerophilic environment, with increased humidity.
 Blood culture: incubated at 37C to recover C. fetus
ATMOSPHERIC REQUIREMENT
o Microaerophilic and capnophilic - require oxygen, but at a concentration less
than that of room air; 5% is normally optimal.
o Campylobacter spp.: gas mixture of 5% O2, 10% CO2, and 85% N2
o Helicobacter spp.: 5% to 10% O2 and 5% to 12% CO2

BOARDS!!!!
MEDIUM BASE ANTIMICROBIAL
Vancomycin, Trimethoprim,
Campy blood agar Brucella agar
Polymyxin B, Amphotericin
plate 10% sheep RBC
B, Cephalothin
Heart infusion Vancomycin, Trimethoprim,
Skirrow’s
lysed and defibrinated horse RBC Polymyxin B
Butzler Meat extract and peptone Bacitracin, Novobiocin
Defibrinated horse RBC Cycloheximide, Colistin,
  Sexually acquired infection
 known as “the clap” and flow of seed
 acute pyogenic infection of non- ciliated columnar and
transitional epithelium; gonoccoal infection
o Male: acute urethritis with purulent discharge and dysuria;
symptomatic case is reported in association with N. gonorrhoeae
AHU strain [arginine, hypoxanthine, and uracil (AHU) strains
are often isolated from asymptomatic men]
o Female: cervical discharge, dysuria, lower abdominal pain, pelvic
inflammatory disease, ectopic pregnancy or perihepatitis known as
Fitz-Hugh-Curtis Syndrome
o Newborn: ophthalmia neonatorum, a gonococcal eye infection, during
vaginal delivery through an infected birth canal.(vertical transmission)

LABORATORY DIAGNOSIS
 Ideal specimen is swab from genital tracts; urethra in male and endocervix for
female
 Inhibitors of Neisseria gonorrhoeae = DACRON OR RAYON SWABS ARE
USED
GRAM NEGATIVE COCCI o Sodium Polyanethol Sulfonate/SPS
NEISSERIACEAE o Calcium alginate
Aerobic, non-motile, non-spore forming, gram negative diplococci organism o Cotton swabs
Neisseria gonorrhoeae  Transport media are selective media that contains CO2 for optimal
 Pathogenic, not part of normal biota conditions; Z pattern is used as streaking technique
 Fastidious organisms, they can bind transferrin in need for iron supply for o JEMBEC plates – used as transport media for swabs
their growth o Gono-Pak
 VIRULENCE FACTOR o Transgrow
o Receptors for human transferrin
o Fimbriae- makipagsiksikan sa cells  Microscopic test
o Cell membrane proteins o Gram negative intracellular diplococcic with adjacent sides
o Lipooligosaccharides/Endotoxin – exhibits pathogenicity flattened, kidney bean like shape
 Protein I (PorB): protection o More than 5 polymorphonuclear neutrophils without bacteria is
 major outer membrane porin protein (Por) suggested of non-gonococcal urethritis
 forms channels for nutrients to pass into and  Macroscopy test
waste products to exit the cell o CHOC agar as the medium of choice for isolation
 coded for by two genes: porA and porB = Both o Colonies are small, gray to tan in color, translucent and raised after 24
genes are expressed in N. meningitidis, but only to 48 hours of incubation
porB is expressed in N. gonorrhoeae  Biochemical test
 porB is also protective against the host’s inflammatory o Oxidase test
response and serum complement-mediated killing.  Reagent: 1% dimethyl p phenylenediamene dihydrochloride
 Protein II (Opa, for Opacity): adherence  Result: PURPLE COLOR develop within 10 seconds
 Facilitate the adherence to phagocytic and o Carbohydrate Utilization
epithelial cells  Cystine Trypticase Agar with 1% carbohydrate
 Protein III (Rmp): blocks host serum bactericidal (IgG)  Indicator: phenol red
action against the organism.  Positive for glucose
 Immunoglobulin A (IgA) protease: cleaves IgA on mucosal
surfaces Selective Medium Inhibitory agents Suppressed organism
 CLINICAL SIGNIFICANCE Vancomycin Gram (+)
o Gonorrhea (gonococci) Thayer martin Colistin Gram (-)
Nystatin Yeast
 Vancomycin Gram (+)  ROD SHAPED
Colistin Gram (-) Moraxella catarrhalis
Mod. Thayer Martin Nystatin Yeast  Third most common cause of acute otitis media and sinusitis in children
Trimethoprim Swarming
Vancomycin Gram (+)
 LABORATORY DIAGNOSIS
Colistin Gram (-) o Specimen usually from middle ear infusion, nasopharynx, sinuses,
Martin Lewis Anismycin Yeast sputum and bronchial aspirates
Trimethoprim Swarming o Microscopy
Vancomycin Gram (+)  Gram stain shows intracellular, gram-negative diplococci
Colistin Gram (-) o Macroscopy
New York City Amphotericin B Yeast  CHOC: smooth, opaque, gray to white colonies with
Trimethoprim Swarming hockey puck appearance and remains intact if pushed
Vancomycin Gram (+)
Lincomycin Gram (+)  “wagon wheel” appearance in OLDER colonies
GC LECT Colistin Gram (-) o Biochemical test
Amphotericin B Yeast  Oxidase and catalase positive, assacharoloytic and
Trimethoprim Swarming differentiated by DNAse and Butyrate esterase reactions

Neisseria meningitides
 Commensal invasive pathogen
 Transmitted by means of close contact known for its meningitis belt during hot
and dry season
 Encapsulated serogroups
o Serogroup A- Meningitis belt (hot and dry season)
o Serogroup B - most common in the United States and Europe
o Serogroup C - most common in the United States and Europe
o Serogroup Y - most common in the United States and Europe
o Serogroup W-135
 CLINICAL SIGNIFICANCE (N. meningitidis enters the BLOODSTREAM)
o Fulminant meningococcemia
o Disseminated intravascular coagulation (DIC)
o Waterhouse Friderichsen Syndrome: hemorrhage in the adrenal
glands

 LABORATORY DIAGNOSIS
o Ideal specimen is from CSF, nasopharyngeal swabs, aspirates,
joint fluids and sputum
o Inhibited by SPS
o Microscopic test
 Gram stains shows intracellular and extracellular gram-
negative diplococci
 1ml of CSF centrifuged at 1000g for 10 minutes
encapsulated strains are mucoid, green tinge after 18
to 24 hours of incubation
 CHOC: colorless to gray, convex colonies
o Biochemical test
 Carbohydrate test sediment is used for identification
o Macroscopy test FASTIDIOUS MICROORGANISM
 SBA: medium sized, gray and convex, and encapsulated Pasteurelaceae
strains are mucoid  Gram negative, pleomorphic, coccoid shaped to rod shaped
 Positive for glucose and maltose  Non-motile and facultative anaerobes
HACEK GROUP
Neisseria lactamica Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella
 only Neisseria species that uses LACTOSE Haemophilus spp
Neisseria elongata
  Haemophilus in Greek means blood loving o Buboes: enlarged inguinal lymph nodes
 Requires growth factor from blood Haemophilus parainfluenzae
o X factor: hemin or hematin (unknown)  Causes few cases of otitis media and acute sinusitis
o V factor: NAD (vitamin) LABORATORY DIAGNOSIS
Haemophilus influenzae Specimen
 Known as Pfeiffer’s bacillus which causes flu  Blood, CSF, Middle ear exudate, Joint fluids, Lower and upper RT, Swabs from
 Causes intense mucous lining in the nose (coryza), headache, bronchitis conjunctivae, vaginal swabs, abscess drainage
and muscle pain (myalgias) Precautions
 Influenza is originally caused by a virus wherein H. influenzae only acts as a  Lower respiratory tract: bronchial washing
secondary or opportunistic invader  Genital sites: swab with sterile phosphate buffered saline
 Virulence  Direct plating on selective media is preferred
o Capsule MACROSCOPIC
 H. influenza serotype b (Hib): antiphagocytic and anticomplementary  Blood agar= RBC are still intact
activity o X and V factors are found within RBCs
• serotype b capsule is a unique polymer composed of ribose,  X factor is directly available on blood agar
ribitol, and phosphate (polyribitol phosphate).  V factor dependent cannot grow on blood agar due to the
 Non -typable H. influenzae (NTHi): strains that are not encapsulated presence of NADases
• Not all strains of H. influenzae are encapsulated  CHOC agar= RBC are lysed to released X and V factor
o IgA Proteases o Ideally used for better recovery of Haemophilus spp.
 enzyme that has the ability to cleave secretory IgA  Addition of 300mg/L of bacitracin is ideal for Haemophilus spp.
 H. influenzae= only member that produces IgA protease isolation
o Adherence  Addition of 1% isovitalex is ideal for Haemophilus ducreyi
 by fimbriae and other structures  Nairobi biplate
 most NTHi strains are adherent to human epithelial cells, whereas most o Incubated in 5 to 10%CO2 with high humidity
serotype b strains are NOT o Haemophilus ducreyi grows best at 33C with 5 to 10% CO2 for 18 to 24
 lack of this adherent capability in type b organisms may explain the hours in high humidity up to 7 days
tendency for type b strains to cause systemic infections. Species Morphology on CHOC
 The presence of this adherent capability by NTHi strains may explain Translucent, tannish, moist, smooth,
H. influenza
the tendency for these strains to cause more localized infections, such as convex with distinct mousy or bleach like
H. aegyptius
acute conjunctivitis odor
o Lipopolysaccharides- paralyzing effect on the sweeping motion of ciliated H. parainfluenzae Tannish and drier with medium to large
respiratory epithelium H. parahemolyticus size
Small, flat, smooth, nonmucoid,
H. ducreyi transparent to opaque, tan or yellow
colonies
Haemophilus aegyptius
 Known as Koch Weeks Bacillus
 Previously known as Haemophilus influenzae biotype III
 Associated with acute, contagious conjunctivitis commonly referred as “pink
eye conjunctivitis”
Haemophilus influenzae biogroup aegyptius
 Non encapsulated strain
 Causes conjunctivitis primarily in pediatric population and severe systemic
disease known as Brazilian purpuric fever
 severe systemic disease known as Brazilian purpuric fever (BPF) in Brazil in
1984.
Haemophilus ducreyi MICROSCOPY
 The only strict human pathogen  Gram negative, pleomorphic coccobacilli or rods
 Causative agent of chancroid; sexually transmitted disease  Capsules may be observed as non-staining area (halo)
 Chancroid: soft chancre seen in genital skin and lymph nodes  Acridine orange or methylene blue stains is ideally used
 Difficult to pick up and produced clumpy nonhomogenous appearance in  Morphology in gram stain
saline suspension o H. influenzae: small, pleomorphic intracellular
 Communicable sexually transmitted genital ulcer disease (GUD) o H. ducreyi: pale staining, coccobacilli arranged in singly or groups known
o Suppurative: pus forming infection as school of fish or railroad tracks
 Aggregatibacter actinomycetemcomitans
IDENTIFICATION TESTS  Formerly known as Actinobacillus
X and V Factor Requirement  Divided based on surface polysaccharides (a through f)
 Traditional approach for identification of Hemophilus spp  Major virulence factors are COLLAGENASE and LEUKOTOXIN
 Ways on how to determine the X and V factor  Distinct star shape with four to six points colonies after 48 hours of incubation =
o Impregnated strips or disks definitive characteristic
o Haemophilus quad plate contains four zones Cardiobacterium hominis
o X factor only  5% CO2 capnophilic, slow growers
o V factor only  Gram stain results to FALSE gram-positive reaction, forms rosettes, swellings,
o X and V factor long filaments or sticklike structures
o X and V factor with horse RBCs  DOES NOT GROW ON MAC AGAR but exhibits PITTING ON SBA or
CHOC agar incubated with 5% CO2
Porphyrin Test Eikenella corrodens
 Negative porphyrin = cannot make X factor on its own  Associated with traumatic infections
 Alternative method for differentiating the heme-producing species of  Laboratory Diagnosis
Haemophilus spp. o Fastidious: requires hemin, increased CO2 for growth
 PRIN: based on the ability of the organism to convert aminolevunilic acid o Culture media
(ALA) into porphyrins or porphobilinogen which intermediates in the o Corroding bacterium
synthesis of X factor. o Characteristic of human bite or fights
 Porphobilinogen can be detected using Kovac’s reagent o Slight greening effect on blood agar
o Porphobilinogen produced = can produce on its own o Chlorine bleach like odor
 Detected using ultraviolet light at 360nm o Catalase negative, oxidase positive
o Reddish orange: positive; do not require hemin
o No color change: Negative; require hemin
(X factor requirement – positive) Kingella spp
 Important pathogenic in pediatric population that affects bones and joints; most
common cause of osteoarthritis infection in children <4 years old
Biochemical Tests  Laboratory Diagnosis
 Carbohydrate fermenter o Coccobacillary to short bacilli with squared ends that occurs in pairs or
 Oxidase and catalase positive short chains
 Nitrate reducer o Catalase negative, oxidase positive, indole positive, glucose
fermenters
NEEDS X NEEDS V BETA
SPECIES D-ALA
FACTOR FACTOR HEMOLYSIS
H. influenzae + + - -
H. parainfluenzae - + - +
H. haemolyticus + + + -
H. parahemolyticus - + + +
H. aegyptius + + - -
H. aprophilus - - - +
H. paraprophilus - + - +
H. ducreyi + - - -

Aggregatibacter aprophilus
 Former H. aprophilus and H. paraprophilus combined
 Foam loving or needs high concentration of CO2
 Linked to bone and joint infections and also found in dental plaque and gingival
scrapings
 CHOC: Colonies are convex, granular, yellow with opaque zone near the
center on culture media
OTHER FASTDIOUS ORGANISM
Capnocytophaga spp.
 Thin, fusiform, spindle shaped, coccoid and curved filaments
 Gliding motility on solid surfaces with yellow orange pigmentations and
resembles Eikenella corrodens
 Sucrose, glucose, maltose and lactose fermenter (GSML)
 Pasteurella spp. Brucella spp.
 Acquired from exposure to infected animals (zoonosis) B.abortus B.melitensis B.suis B.anis
 Causes respiratory and cutaneous infections Animal
Cattle Goats Pigs Dogs
Source
o Pasteurella multocida: cause pasteurellosis, agent of shipping fever in Urease 1-2hr 1-2hr 0-30m 0-30m
cattles that may acquire through bite or scratch from cats H2S + - - -
 Gram negative, non-motile, facultative anaerobic coccobacilli GROWTH ON MEDIA
 Catalase and oxidase positive Thionine
 Bipolar staining: safety pin appearance when the poles of the cells are more - + + +
intensely stained (wayson stain and gram stain= safety pin appearance) Basic
Fuchsin
+ + - -
 SBA and CHOC shows grayish colonies
Thionine
o Pasteurella multocida shows narrow green to brown halo around the blue
+ + - -
colony after 48 hours
Hemolysis Indole ODC Urease
P. multocida − + + −
P. dagmatis − + − +
P. stomatis − + − +
P. canis − + + −
Francisella tularensis- FranVOUX negative; CBeta positive
 Acquired from exposure to infected animals
 Three subspecies:
o Francisella tularensis subsp. tularensis* (tula a)
o Francisella tularensis subsp. holarctica (type b)
o Francisella tularensis subsp. mediasiatica
 Clinical Significance
o Causes Tularemia, also known as Rabbit fever, Deerfly fever,
Lemming fever and Water Rat Trapper’s Disease
o Acquired through ingestion, inhalation or arthropod bite
 Laboratory Diagnosis
o Small, non-motile, gram-negative bacilli or coccoid bacteria and
strictly aerobic
o Classified as facultative intracellular parasites which requires
supplementation of cysteine for growth on successive passage
o Culture media used are CHOC, MTM and Buffered Charcoal Yeast
Extract agar and Thioglycolate broth
o Oxidase, Urease, Satellite X and V negative
o Catalase and beta lactamase positive

Brucella spp – BroUNOC positive

 Acquired from exposure to infected animals (zoonosis) commonly via oral
exposure of unpasteurized milk
 Known for causing brucellosis or undulant fever
 Laboratory Diagnosis
o Facultative intracellular pathogen
o Small gram negative, aerobic nonmotile, capsulated bacteria that
can appear as coccobacilli or bacilli
o Culture shows smooth, raised and translucent colonies
o SBA, CHOC, MTM, Martin Lewis (18 hrs)
o Biphasic: Castaneda medium (3-4 weeks)
o Catalase, oxidase, urease positive
o Nitrate reducer
 o Does NOT grow in blood agar and mac conkey agar
o Non-motile, oxidase positive, urease negative

Catalase Oxidase Urease Nitrate Motility

B. pertussis + + - - -
B. parapertussis + - + - -
B. bronchiseptica + + + + +
“Kennel cough Bacillus”

Legionella spp.
 Isolated in air conditioning towers and heating systems and grows at 65ºC
 Ubiquitous gram-negative bacilli and has the ability to exist as intracellular
pathogen in mammalian cells
 central portion of young colonies “ground-glass” appearance
 Clinical Significance
o Legionella pneumophilia: Legionnaire’s disease
o Legionella micdadei: Pittsburg pneumonia
o Legionella bozemanni: Wiga’s agent of pneumonia
o Pontiac Fever
o Influenza like febrile disease and asymptomatic
Bordetella pertussis
 Laboratory Diagnosis
 Obligate aerobic bacteria and do not ferment carbohydrates, oxidizes amino acids
o Specimen: bronchial washing or expectorated sputum
and biochemically inactive
o Pleomorphic, weakly staining, gram negative bacilli
 Inhibited by fatty acids, metal ions, sulfides and peroxides
o Legionella micdadei stains best in Kinyoun’s method
o X Bord SulPerFaMet
o Culture media
 Virulence Factor
o Buffered Charcoal Yeast Extract Agar appears as grayish
o Filamentous hemagglutinin: and pertactin (a 69-kilodalton [kDa] outer
white or blue green convex and glistening colonies (ground-
membrane protein) are believed to facilitate attachment to ciliated
glass” appearance)
epithelial cells.
o Feeley Gorman medium shows brown colonies
o Pertussis tox: protein exotoxin that produces a wide variety of responses
o CHOC with L cysteine
in vivo. The main activity of PT is modification of host proteins by
o Saline or buffer inhibits Legionella spp.
adenosine diphosphate– ribosyl transferase, which interferes with
o Best diagnosed with CULTURE and URINE antigen
signal transduction. B. parapertussis and B. bronchiseptica contain the
Streptobacillus moniliformis
structural gene for PT but DO NOT express the complete operon
 Agent of rat bite fever or Haverhill fever
o Adenylate cyclase toxin: inhibits host epithelial and immune effector
 Acquired by ingestion of contaminated milk
cells by inducing supraphysiologic concentrations of cyclic adenosine
 Known for its bread crumb colonies
monophosphate.
o Tracheal cytotoxin: causing ciliostasis, inhibiting DNA synthesis, and
promoting cell death
 Clinical Significance
o Agent of whooping cough
o Pertussis: highly communicable disease that can be prevented by DPT
vaccine
o Three phases of infection
o Catarrhal phase: general flu like
o Paroxysmal phase: repetitive coughing
o Convalescent phase: recovery
 Laboratory Diagnosis
o Ideal specimen is nasopharyngeal swab
o Regan Lowe agar: charcoal cephalexin agar, smooth, glistening and
silver, mercury droplet colonies MISCELLANOUS MICROORGANISM
o Bordet Gengou agar: potato blood glycerol agar
LEPTOSPIRACEAE  Helically coiled bacteria; flexible twisted resembling “stretched spiral”
Leptospira spp.  Transmitted through arthropods vectors including lice and ticks
 Tightly coiled, thin, flexible spirochetes; have hooks on either both ends and  Easily stained and seen in bright field microscope
results to a rapid and rotational motion  Cultured in Kelly medium
 Can be visualized by using SILVER IMPREGNATION  Species known for this genus
 Microscopy: dark field, phase contrast or immunofluorescence o Borrelia recurrentis
 Species known for this genus o Agent of louse-borne relapsing fever
o Leptospira biflexa o Vector: Pediculus humanus (human louse)
o Non-pathogenic, found in water and soil; double hooks o Borrelia burgdorferi
o Leptospira interrogans o Agent of Lyme disease
o Cause of human and animal leptospirosis o Vector: Ixodes dammini (deer ticks)
o Dog, rats and other rodents are the principal animal o Media: Barber Stoenner Kelly 33ºC for 6 weeks
reservoirs o Borrelia anserine, toricatae, parkeri
o Shed in the urine and man acquire infection through direct o Agent of tick-borne relapsing fever
contact with urine of animals o Vector: Ornithodoros ticks
o Clinical significance o Identified by Wright’s/Giemsa stain by blood or bone
 Leptospirosis marrow specimen
 Weil’s disease (severe form)  Jarisch Herxheimer Reaction: large quantities of toxin is released on the
bacteria and dies during treatment
Treponema spp.
SEROVAR DISEASE  Tightly coiled and tightly twisted
Leptospira interrogans serovar canicola Infectious jaundice  Laboratory diagnosis
Leptospira interrogans serovar autumnalis Pretibial fever o Dark field microscopy: corkscrew motility
Leptospira interrogans serovar grippotyphosa Marsch fever o Levaditi silver impregnation
Leptospira interrogans serovar hebdomadis 7-day fever o Serological test
Leptospira interrogans serovar mitis/pomona Swineherd’s disease o Screening: VDRL, RPR, TRUST (regain test)
o Confirmatory: FTA-ABS, TPHA, HATTS, TPI*
Laboratory Diagnosis  Species known for this genus
 Specimen o Treponema pallidum subsp. pallidum
o 1st week: blood, CSF or tissue (Leptospiremia) o Vereneal syphilis known as great pox, evil pox,
o 2nd week: Urine French/Italian/Spanish disease
 Culture media o Transmitted by sexual contact, transplacental
o Fletcher’s semisolid transmission, blood transmission
o Stuart liquid o Non-cultivatable on agar medium, viable in freezing
o Ellinghausen-McCullough-Johnson-Harris temperature but killed in refrigerated temperature
 Other tests o Clinical infection
o Macroscopic Agglutination test  Primary: hard chancre (painless, firm), coiled
o Screening method organism with corkscrew motility on dark field
o Uses KILLED leptospira  Secondary: condylomata (wart like lesion),
o Serum + Antigen = agglutination serological tests needed
o Microscopic Agglutination test  Latent: asymptomatic, serological tests needed
o Gold standard  Tertiary syphilis: gummas, neurosyphilis/Tabes
o Uses LIVE leptospira dorsalis, CSF serological tests
o Serum + Antigen = agglutination

o Treponema pallidum subsp. pertenue

o Yaws/Framboise: chronic nonvenereal disease of skin and bones
o Transmission: direct contain to traumatized skin
o Treponema pallidum subsp. endemicum
o Bejel: lesions in oral cavity, mucosa and skin, bones and nasopharynx
SPIROCHEATACEAE
o Transmission: mouth to mouth by utensils
Borrelia spp.
o Treponema pallidum subsp. carateum o Spore like features
o Pinta: ulcerative skin disease o Metabolically active but no cellular division
o Transmission: direct contact to traumatized skin o Reticulate body
o Non-infectious
o Binary fission for multiplication
CELL WALL DEFICIENT MICROORGANISM  Chlamydia trachomatis
Mycoplasma spp. o Divided into two biovars:
 Smallest free-living organism found in animals and plants o Trachoma
 Lack cell wall, pleomorphic in appearance  Includes serovars (A-K)
o Mycoplasma pneumoniae o Lymphogranuloma venereum
o EATON’S AGENT: Frequent cause of community acquired  Non-infectious
pneumonia and tracheobronchitis in children, young adults  Binary fission
o Primary atypical pneumonia/Walking pneumonia o Clinical Significance
o Laboratory diagnosis o Trachoma
 Selective media: PPLO agar, Edward’s Hayflick  Chronic eye infection that leads to blindness
 Confirmatory: hemadsorption test  Begins with follicular conjunctivitis
 Best method: Inhibition of growth by  Seen in climates with high temperature
using specific antisera o Pelvic Inflammatory Disease
o Reiter Syndrome
o Mycoplasma hominis
LABORATORY DIAGNOSIS
o Agent of salpingitis, post partal fever in female and non-
Chlamydia trachomatis
gonococcal urethritis in male  Specimen: wooden swabs
o Laboratory diagnosis  Transport: sucrose phosphate glutamate buffer
 Genital mycoplasma that produces LARGE  Microscopy: cytological methods
fried egg like colonies  Cell culture: McCoy, Hep-2 HeLa cells, buffalo green monkey kidney cells
 Media: A7/A8, SP4-urea, NYCA Chlamydia pneumoniae
 Previously known as TWAR strain
Ureaplasma urealyticum  Causes sinusitis, acute respiratory disease, bronchitis and pneumonia,
asthma and cardiovascular disease
 Genital mycoplasma, TINY fried egg colony appearance  SPX: sputum, bronchial wash, throat washing and swabs
 Media: A7/A8, SP4-urea, NYCA  CELL CULTURE: selected cell lines, Hep-2 cells
Chlamydia psittaci
 Causes psittacosis;
o Based on a history of exposure to birds
o fourfold rise in the level of antibody to the chlamydial group LPS
antigen
 Parrot fever in avian species or ornithosis
 produces lower respiratory tract infections in humans

CHLAMYDIACEAE RICKETSSIACEAE
Chlamydia spp. Rickettsia spp.
 Deficient in energy metabolism, OBLIGATE INTRACELLULAR PARASITES  Short, non-motile gram-negative bacilli
(cell cultures ae used, NOT culture media)  Grows in yolk sac of embryonated eggs and several cell lines
 Unable to synthesize amino acids, cofactors and nucleotides  Divided into several group according to infections caused
 Identified by its growth cycle SPOTTED FEVER GROUP
o Elementary body 1. Rocky Mountain Spotted Fever
o Infectious o Caused by R. rickettsii
 o Most severe rickettsial infection factor (hemin or hematin), “X for unknown”; V factor (NAD, “V vitamin”; or
o Rashes in the palms and hands both
o Vector: tick bites of Dermacentor variabilis
2. Boutonnerus Fever
o Caused by R. conorii CHOC agar used for isolation of heamophilus = the availability of V factor
o Also known as Mediterranean spotted fever in the media
o Causes Kenya tick typhus, south African and indian tick typhus Blood agar – X factor
o Rashes in the palms, hands and face  Add source of V factor – S. aureus, S. pneumoniae, Neisseria
o Tache noires: black spots produce V factor
TYPHUS GROUP - ARTHROPODS CHOC – X and V factor
Murine Typhus
o Caused by R. typhi
o Rashes in the extremities but rare
Swab sample of haemophilus spp. detection should be moistened with
o Acquired through arthropods; phosphate buffer saline prior to collection. APPLICABLE FOR H. ducreyi
Oriental rat flea (Xenopsylla cheopsis) and Rat (Rattus exulans) Pus = Suppurative seen in genital area
BrillZinsser disease Buboes = lymph nodes
o Caused by R. prowazekii
Louseborne Typhus
o Caused by R. prowazekii Direct plating / bedside culture to avoid contamination
o Also known as Epidemic typhus
o Acquired through arthropods Best isolation for a Haemophilus spp. coming from respiratory samples =
 Human louse (Pediculus humanus) CHOC agar with 300mg/L bacitracin (18 to 24hrs of incubation)
 squirrel louse (Neohaematopinus sciuriopteri)
TRANSITIONAL GROUP
H. ducreyi from genital samples = CHOC with 1% isovitalex
1. Ricketssialpox
o Caused by R. akari Green to brown halo around the colony after 48 hrs of incubation on
o Rashes on face, trunk and extremities Blood agar helps differentiate Pasteurella multocida from what other
o Acquired through mouse mite Liponyssoides sanguineus fastidious organism = HAEMOPHILUS
OTHER MICROORGANISM Pasteurella = Gram stain = safety pin appearance
Orientia tsutsugamushi Growth on SBA in the absence of satellitism or in pure culture combined
o Causes scrub typhus with bipolar staining may differentiate Pasteurella
o Acquired through vector Leptotrombidium deliensis
Yersinia = wayson stain = safety pin appearance
NON-FERMENTATIVE – Pseudomonas

Non-motile, fastidious facultative anaerobe, catalase and oxidase positive, and

considered as obligate parasites, requires the V factor is true for what
pathogen= heamophilus

Catalas positive and Oxidase positive = fastidious (hacek), bordetella,

brucella, capnocytophaga, fanciscella
Haemophilis organisms require preformed growth factors present in blood – X

Non-enteric Gram- Negative Bacilli
Key feature to differentiate Aeromonas spp. from another non-enteric
microorganism = RESISTANCE to 0/129 susceptibility testing
TCBS = only Vibrio grows
String Test – touching the loop in the colony creating a string
Inositol = Plesiomonas
Non-enteric organism is unlikely to grow on salt media or nonhalophilic =
V. cholera and V. mimicus
Positive string test in 0.5% sodium deoxycholate = most Vibrios and V.
cholerae
V. parahaemolyticus = heat stable hemolysin, known as kanagawa
phenomenon, and cultured in a high salt mannitol media (Wagatsuma
agar)
Media allow the enhancement the growth of Vibrio spp. prior to culture
media inoculation = alkaline peptone water in ph 8.5 at 35C and addition
of 1% NaCl or salt
TCBS – subculture here Vibrio (selective- differential media)
 Contains sucrose (enteric) [non-enteric = glucose]
Biochemical test to separate V. cholera from Plesiomonas spp. = Inositol
fermentation
Non-enteric = aeromonas (CIN II agar)
CIN – yersinia pestis
CIN II- Aeromonas spp. = high isolation
Pseudomonas aeruginosa = produces pyoverdin (yellow) and pyocyanin
(blue) and shows a green color pigmentation on colonies
 Pyocyanin combining with pyoverdin produces green color
One definitive characteristics of Pseudomonas aeruginosa is growth at
which temperature = 42C, acetamide utilization, Nitrate reduction, ADH
positive, citrate positive, cetrimide agar to enhance pigment
When green culture in nutrient agar = that is Pseudomonas!!
Burkholderia pseudomallei can appear microscopically as safety pin
appearance on gram stain =
 Selective medium = ASHDOWN MEDIUM
 Wrinkled colonies
 Supplemented with colistin
 Deep pink colonies
 Earthy odor
B. pseudomallei – Melliodosis or Whitmore disease
B. cepacia – correlated to cases with CYSTIC FIBROSIS
B. mallei – Glander’s disease
Acitenobacter abumanii and Acitenobacter iwoffi is seaparated based on
what growth requirement = GLUCOSE REQUIREMENT
Acitenobacter abumanii = Saccharolytic – needs sugar
Acitenobacter iwoffi= Asaccharolytic- DOES NOT NEED SUGAR
Selective and differential to enhance pigment production og Pseudomonas
aeruginosa = CETRIMIDE AGAR
Non enteric – Campylobacter = CCDA
Adjacent with sides flattened, gram negative cocci, kidney bean shape =
Neiserria
Neiserria virulence characteristics = ability to bind transferring, presence
of porin protein, endotoxin or lipid A
 Neisseria Blebs contain oligosaccharide (LOS)
 Neisseria gonorrhoeae = gonococcal infection (sexually
transmitted infection)
o Male = urethritis, purulent discharge; AHU strain
o Female = perihepatitis (severe form of pelvic
inflammatory disease) or Fitz-Hugh-Curtis Syndrome
 Endocervical swab – insert swab up to 2 cm into
anterior urethra
o Newborn = Ophthalmia neonatorum given with
erythromycin
 Neisseria metabolize carbohydrates through oxidation
 Use Dacron or rayon swabs
o Trachoma – infective serovar
 Follicular conjunctivitis = leading to blindness
o Grows on cell lines only?
o McCoy cells, Hela cells =
o Fully dependent on host or cells
o Glycogen in the inclusion among miscellaneous organism
o MOMP – majpr outer membrane protein – most
prominent component

M. catarrhalis – assacharolytic, DNAse positive

Thin, spiral organism with periplasmic flagella observed under dark field
microorganism
 Treponema (two genera: Treponema (dark field) and Borellia
(bright field))
 Leptospira

Borellia : Laboratory diagnosis of relapsing fever or borreliosis = Wright

stained peripheral blood smear
 Specimen of choice is BLOOD CULTURE
 Coiled
o B. recurrentis- Louseborne – epidemic relapsing fever
o B. burgdogferi – Lyme disease
o B. anserine, toricatae, parkeri- Tick-borne – endemic relapsing
fever

Leptospira interrogans
o Animals (dogs, rodents, insects, rats) as reservoir- urine of
infected animals
o GOLD STANDARD – agglutination test (Ag-Ab test)
o Dark field microscope
Treponema
o Syphilis

Laboratory method used to diagnose RICHETTSIA disease =

Immunohistochemical method and Weil Felix test
o Grows on cell lines only?
o Antibodies to rickettial spcies are known to cross react with
Proteus
o GOLD standard = IFA (immunofluorescent antibody

Chlamydia trachomatis = reiter syndrome also known as reactive

arthritis
o Biovars
o Lymphogranuloma – non infective but with binary fission