GRAM-NEGATIVE COCCI

Neisseria
- OBLIGATE AEROBES, NON-MOTILE, NON-HEMOLYTIC
- Fastidious, capnophilic, grow optimally in moist temperature
- Natural habitats: mucous membranes of the respiratory tract and urogenital tracts.
- Grow best in media with blood and cholesterol
- SENSITIVE TO HEAT AND DRYING, THUS REQUIRING A DIRECT INOCULATION OF SPECIMENS “AT THE
BEDSIDE”
- Microscope: gram-negative diplococci that are coffee or kidney bean shaped except for N. bacilliformis, N. elongata, and N.
weaveri which are rod-shaped.
- Culture: colonies are glistening small to large, grayish-white convex, some are mucoid with sticky granular appearance.
- Biochemical tests: OXIDASE (+), CATALASE (+) except N. elongata and N. bacilliformis

1. Neisseria gonorrhoeae
- Leading cause of sexually transmitted diseases
- Found on mucous membranes of urogenital tract, anorectal area, oropharynx, and conjunctiva
- Can be transmitted by an infected mother to a newborn during birth.
- GLUCOSE FERMENTER
- Principal virulence factor: common pili
- Culture: CAP = colonies appear small, shiny, gray to tan-colored, translucent, and raised
- Colonial types: T1 and T2 are virulent with pili and T3 to T5 are avirulent without pili.
- Diseases
- Gonorrhea, Purulent urethritis and cervicitis, pharyngitis, anorectal infections, ophthalmia neonatorum (newborns)
- Specimen collection guidelines:
- Specimen collection and transport is the single most important factor in the accurate diagnosis of pathogenic species of N.
gonorrhoeae and N. meningitidis
- If sample cannot be processed immediately, it should be held at room temperature and avoid refrigeration since Neisseria species
are sensitive to cold temperature
- Swabs should be placed in transport system like Amies medium with charcoal if direct plating cannot be performed
- Cottons swabs should not be used due to presence of toxic fatty acids in the cotton fibers = inhibitory to growth of Neisseria
- TEMPERATURE-DEPENDENT: prefer immediate incubation at 35C; require incubation in increased carbon dioxide after
polating; require iron as growth enhancers
- Laboratory Diagnosis
- SPECIMENS: PUS SECRETIONS FROM URETHRA, CERVIX, PROSTATE, RECTAL MUCOSA, PHARYNX, AND JOINT
FLUID
- PREFERRED: SPX FROM URETHRA AND ENDOCERVIX
- Microscopy: If more than five polymorphonuclear leukocytes (PMNLs) per immersion field is observed but without any bacteria,
it suggests non-gonococcal urethritis which is commonly seen in chlamydial infections
- Culture: CAP, TMA, MTM, ML, NYC, GC-Lect
- N. gonorrhoeae is sensitive to sodium polyanethol sulfonate (SPS) and will not be recovered from routine blood culture
due to possible inhibition
- N. gonorrhoeae does not grow on BAP
- Specimen Transport
- Transport Media: Cary Blair and Amies with charcoal
- Transport System: Transgrow, JEMBEC, Gono-Pak
- Inoculation: swab spx should be rolled onto an agar medium following a “Z” pattern
- Selective Culture Media for Neisseria gonorrhoeae
a. Thayer-Martin Agar (TMA)
- Chocolate agar base with a supplement (IsoVitaleX) and antibiotics
- Inhibitors: vancomycin, colistin, nystatin (VNC)
- Vancomycin: inhibits gram positive bacteria
- Colistin: against gram negative bacteria except Neisseria
- Nystatin: antifungal agent
- N. meningitidis, N. lactamica, and N. flavescens grown on TMA.
b. Modified Thayer Martin Agar (MTM)
- Has all TMA component and trimethoprim lactate (prevents swarming)
- Vancomycin, colistin, nystatin, trimethoprim (VNCT)
 c. Martin-Lewis (ML) Medium
- Contains all ingredients of MTM agar except nystatin which is substituted by anisomycin; it has an increased
vancomycin concentration
- Vancomycin, colistin, trimethoprim, anisomycin (VACT)
d. New York City (NYC) Medium
- Transparent medium with lysed horse blood and yeast dialysate
- Vancomycin, nystatin, colistin, trimethoprim, amphotericin B (antifungal agent) (VNCTB)
e. GC-LECT Medium
- Vancomycin, nystatin, colistin, trimethoprim, amphotericin B, lincomycin

BIOCHEMICAL TESTS

Biochemical Test Positive Result Positive Bacteria Notes

Carbohydrate Utilization
Test (Cystine Trypticase
Agar Test)
Yellow color within 24 to
72 hours of incubation at
35 C
N. gonorrhoeae and N. Medium: CTA with 1% Standard method of
meningitidis carbohydrate identifying N. gonorrhoeae
pH indicator: Phenol red
Control: Carbohydrate free
medium

Oxidase Test Dark purple color within N. gonorrhoeae Reagent: 1% tetramethyl-p- Detects presence of
10 seconds phenylenediamine cytochrome oxidase that
dihydrochloride degrades the substrate
cytochrome c.

Superoxol Test Vigorous bubbling N. gonorrhoeae Reagent: 30% H2O2

DNase Test Clear halo around the POSITIVE: M. Dnase agar with methyl If QUADFerm+ is utilized,
colonies after 18 hours to CATARRHALIS green positive results indicate
24 hours of incubation yellow color.
NEGATIVE:
NEISSERIA

B-lactamase Test Deep pink or red within Substrate: nitrocefin

10 minutes

- Antimicrobial Susceptibility Test

- N. gonorrhoeae and N. meningitidis are resistant to Vancomycin, colistin, nystatin, and lincomycin, these antibiotics can be
added into the culture media to inhibit the growth of normal flora and other bacteria while promoting the selective isolation of the
two species,
- Cephalosporins and quinolones are utilized
- CLSI Reco: Disk diffusion or Agar dilution
- Immunodiagnosis
- Does not require pure selective culture plate
- Fluorescent Antibody Test (FAT)
- Highly specific and sensitive
- Uses monoclonal antibodies that recognize epitopes on the principal outer membrane protein (Por) of N. gonorrhoeae
- Coagglutination
- Confirmatory test for the biochemical characteristics of N. gonorrhoeae
- (+) result: exhibits agglutination
- Molecular diagnosis
- Nucleic Acid Amplification Test (NAAT)
- Currently the recommended and reliable method for the identification of N. gonorrhoeae in clinical samples including
urine specimens
- Specimens: endocervical or urethral swabs and urine
- Methods: Abbott RealTime CT/NG, BD ProbeTec ET, Cobas CT/NG, and Xpert CT/NG
- High sensitivity and capability to detect Chlamydia trachomatis together with N/ gonorrhoeae
- Chemiluminescent Nucleic Acid Probe
- Rapid test for directly detecting gonococcal rRNA in genital and conjunctival specimens
- Specimen: endocervical or urethral swabs
 2. Neisseria meningitidis
- Causative agent of meningococcemia or spotted fever
- LEADING CAUSE OF FATAL BACTERIAL MENINGITIS
- Both considered a commensal and true pathogen of the upper respiratory tract
- Spreads by respiratory droplets easily through close contact especially in crowded housing facilities
- BOTH A GLUCOSE AND MALTOSE FERMENTER
- Secretes beta-lactamase; requires iron for growth
- Principal Virulence Factor: Lipooligosaccharide (LOS)-Endotoxin Complex
- Microscopy: Encapsulated strains can have a halo around the organism
- Culture
- BAP: colonies appear large, smooth, bluish-gray, and convex with greenish dots under the colonies
- CAP: colonies appear small gray to tan colored, convex mucoid
- TMA: colonies exhibit smooth, convex, with colorless to gray appearance
- Major Serogroups: A, B, C, Y, and W-135
- Diseases
- N. meningitidis has the ability to invade serous membranes and joint tissues, resulting in pleuritis, pericarditis, and
arthritis
- The LOS-endotoxin complex produces hemorrhage in the adrenals known as the Waterhouse-Friderchsen Syndrom
- Penicillin is the drug of choice for the treatment of meningococcal meningitis
- Meningococcemia
- Develops with or without meningitis
- Laboratory Diagnosis
- Specimen: CSF, blood, nasopharyngeal swabs, petechial skin lesions
- Microscopy: sediments of CSF after centrifugation is best for staining and culture
- Culture: BAP, CAP, TMA
- Nasopharyngeal swabs should be plated immediately in the JEMBEC system or placed in transport media
with charcoal
- Sensitive to SPS, so the content in the blood culture broth should not exceed 0.025%
- Oxidase Test (+): purple color
- Gamma-glutamyl Aminopeptidase (GGAP) Test
- N. meningitidis produces this enzyme that creates a positive reaction
- Reagent substrate: Gamma-glutamyl-p-nitroannilide (GPNA)
- (+): yellow color

Non-pathogenic Neisseria Species (Growth on CAP)

a. Neisseria cinerea: similar to T3 colonies of N. gonorrhoeae
b. Neisseria elongata: large, flat with clay-like appearance and has tendency to “corrode” the agar
c. Neisseria lactamica: small translucent colonies with a “yellow ring”
d. Neisseria mucosa: large, smooth, with mucoid-sticky appearance
e. Neisseria sicca: colonies are dry and wrinkled exhibiting “bread crumbs”

3. Moraxella catarrhalis (Branhamella catarrhalis)

- Resembles Neisseria species by exhibiting gram-negative coccal morphology
- NON-MOTILE, FASTIDIOUS, BETA LACTAMASE PRODUCER, ENCAPSULATED WITH COMMON PILI
- Causes upper respiratory tract infections in otherwise healthy children and the elderly
- THIS MOST COMMON CAUSE OF OTITIS MEDIA AND SINUSITIS IN IN CHILDREN
- Microscopy: small, gram-negative cocci that tend to grow in pairs from end to end with their adjacent side flattened
- Culture BAP: colonies are smooth, opaque, grayish-white with “HOCKEY PUCK” appearance; old colonies with “WAGON WHEEL”
appearance
- Biochemical Test: Oxidase and Catalase (+)
- Laboratory Diagnosis
- Specimen: Ear discharge, nasopharyngeal swab, sputum, and bronchial and sinus aspirates
- Culture: BAP and CAP
- On CAP: pink to brown colonies and the same hockey puck consistency observed in BAP
- M. catarrhalis is inhibited by gonococcal media by colistin
- Biochemical Test
- Carbohydrate Utilization Test
- Does not utilize any sugar in the CTA test
- DNase TEst/ DNA Hydrolysis test
- DEFINITIVE Test for the identification of M. catarrhalis
 - (+) result: clear or colorless halo around the colonies
- In QUADFerm+ test, positive rxn = yellow color
- Butyrate Esterase/ Tributyrin Test
- DEFINITIVE Test
- Reagent substrate: Bromo-chloro-indolyl butyrate or tributyrin
- (+) Blue/ Indigo Color
- (-) no color change on the disk
- AST
- M. catarrhalis isolates are sensitive to cephalosporins and trimethoprim- sulfamethoxazole
- Molecular diagnosis
- PCR: reveals production of beta-lactamase BRO-1 and BRO-2 encoded by bro-1 and bro-2 genes
- These bro-1 and bro-2 genes are responsible for the penicillin resistance of M. catarrhalis

TEST N. gonorrhoeae N. menigitidis M. catarrhalis

Superoxol + - -

Growth on MTM, ML, NYC + + Variable

Growth on BAP at 25 C - - +

Growth on Nutrient Agar at 35 C - - +

Acid Production

Glucose + + -

Fructose - - -

Maltose - + -

Sucrose - - -

Lactose - - -

DNase - - +

Nitrate reduction - - +

Tributyrin hydrolysis - - +

B-galactosidase - - -

Gamma-glutamyl aminopeptidase - + -
GRAM-NEGATIVE BACILLI: ENTEROBACTERIACEAE
- FACULTATIVE ANAEROBE, NON-SPORE-FORMING
- Culture: BAP/CAP = colonies appear as smooth, large, and gray EXCEPT Klebsiella and Enterboacter with mucoid colonies; they are
NON-HEMOLYTIC EXCEPT some strains of E. coli, which are Beta-hemolytic
- CATALASE (+), OXIDASE (-) EXCEPT Plesiomonas Shigelloides
- Antigen-determinants for Serological Identification
- Somatic O antigen: heat-stable; located in the cell wall; E.coli and Shigella serotyping
- Flagellar H antigen: heat-labile; found in the flagellum; Salmonella serotyping
- Capsular K antigen: heat-labile; found as K1 antigen of E coli and Vi antigen of S. enterica

Escherichia coli (Colon bacillus)

- Primary indicator of fecal contamination in water purification
- Leading cause of nosocomial UTI
- Has both the adhesive fimbriae and sex pili
- Antigenic determinants: O, H, K antigens
- Culture
- MAC: colonies appear flat and dry, exhibit pink color (LF); some may be NLF
- BAP: most are non-hemolytic though some are beta-hemolytic
- EMB: greenish metallic sheen
- IMViC: + + - -
- TSIA: A/A, (+) gas, (-) H2s
- Diseases: Bacteremia, UTI, Hemolytic Uremic Syndrome, Neonatal meningitis

Escherichia hermanii
- Formerly called E. coli atypical or enteric group II
- Isolated from CSF, wound infection, and blood
- Culture: yellow pigmentation

Differential Characteristics of E. coli Strains

1. Enteropathogenic E. coli (EPEC)
- Infantile diarrhea (stool without blood)
- Causes loss of microvilli; no toxin production
- Serotypes: O55:H6
- Lab test: HeLA cell assay
2. Enterotoxigenic E. coli (ETEC)
- Traveler’s diarrhea
- Colonization occurs in the proximal small intestine
- Persons with achlorhydria are at high risk
- Serotypes: O8:H9
3. Enteroinvasive E. coli (EIEC)
- Dysentery-like or Shigella-like infection
- Watery diarrhea with WBCs
- Penetrates and multiplies within the intestinal epithelial cells
- Virulence Factors: Invasin
4. Enterohemorrhagic E. coli (EHEC)
- Hemorrhagic colitis
- Hemolytic uremic syndrome (HUS)
- Bloody diarrhea without WBCs
- Virulence Factors: Verotoxin I and Ii or Shiga-like toxin
5. Enteroaggregative E.coli (EAEC) and Diffusely adherent E. coli (DAEC)
- Persistent watery diarrhea (EAEC)
- Pediatric diarrhea and UTI (DAEC)
- EAEC adheres to HEp-2 cells which form clumps of bacteria
- Virulence Factors: Fimbriae
- EAEC has a “stacked-brick appearance)
6. Uropathogenic E.coli (UPEC)
- Most common cause of UTI in Humans
EHEC 0157:H7
- Most virulent serotype causing Hemolytic Uremic Syndrome (HUS)
- Appears colorless on MAC and produces heavy growth compared to other strains which are pink colored

Klebsiella
- Usually found in the GIT of humans and animals
- Culture: MAC = colonies exhibit pink color (LF) with mucoid consistency
- Growth on media with Potassium cyanide (KCN): POSITIVE
- IMViC: - - + +
- TSIA: A/A, (+) gas, (-) H2s

Klebsiella pneumoniae (Friedlander’s bacillus)

- Frequent cause of lower respiratory tract infection among hospitalized patients
- Agent of ventilator-associated pneumonia due to carbapenemase-producing K. pneumoniae
- Produces a CURRANT JELLY-LIKE sputum among infected individuals
- Virulence Factor: Polysaccharide capsule
- Differential Test: (+) String Test
- Neufeld-Quellung Test: Positive
- Growth on media with KCN: POSITIVE
- IMViC: - - + +
- TSIA: A/A, (+) gas, (-) H2s

Klebsiella granulomatis
- Formerly known as Calymmatobacterium granulomatis
- Etiologic agent of granuloma inguinale or donovanosis
- Pleomorphic gram-negative bacillus that is encapsulated and non-motile
- Best cultivated in a yolk sac or in a fresh egg yolk medium
- ONLY MEMBER OF THE ENTEROBACTERIACEAE THAT WILL NOT GROW IN THE PRIMARY PLATED MEDIA OR
SYNTHETIC MEDIA
- Diagnostic Marker: Blue or bluish-purple rods with a SAFETY PIN appearance; surrounded by a pink capsule and the presence of
DONOVAN BODIES in mononucleated endothelial cells
- Preferred specimen: Tissue from the Ulcer (wound)
- Preferred stains for microscopy: Wright-Giemsa stain and Warthin-Starry

Enterobacter
- Resemble those of Klebsiella when grown on MacConkey agar
- MAC: colonies exhibit a pink color and are sometimes mucoid (LF)
- Growth on media with KCN: POSITIVE
- Ornithine decarboxylase test: POSITIVE
- Lysine decarboxylase test: POSITIVE
- Sorbitol fermentation: POSITIVE
- Urease and Malonate test: POSITIVE
- IMViC: - - + +
- TSIA: A/A, (+) gas, (-) H2s

Cronobacter sakazakii
- Formerly known as Enterobacter sakazakii
- Contaminant of powdered infant formula
- Isolated from individuals with brain abscess and respiratory and wound infections
- MAC: pink color (LF)
- BHIA: yellow pigmentation with mucoid appearance
- IMViC: - - + +
- TSIA: A/A, (+) gas, (-) H2s