MICRO 1_LEC 4_Family Neisseriaceae.pdf

LESSON 4: Family Neisseriaceae Gonococci and meningococci are closely related (70% LECTURER: Dr. Jordan Joy G. Arpilleda, RMT DNA homology)....

LESSON 4: Family Neisseriaceae Gonococci and meningococci are closely related (70% LECTURER: Dr. Jordan Joy G. Arpilleda, RMT DNA homology). ○ Their clinical spectra can possibly overlap. I. GENERAL CHARACTERISTICS (Family Neisseriae) Differentiated by a few laboratory tests and specific characteristics. Neisseria Simonsiella GONOCOCCI MENINGOCOCCI GENERA Kingella Alysiella Eikenella (-) no polysaccharide capsule (+) have polysaccharide capsule Reported as Gram (-) intracellular Most have plasmids Rarely have plasmids cocci seen as dots or cocci in pairs Found in the Upper Respiratory Genital infections STAINING (diplococci). tract and cause meningitis REACTION / ○ They are kidney or coffee-bean APPEARANCE shaped stained pink in pairs. ○ The flat/concave sides are adjacent. PATHOGENICITY Pathogenic for humans. N. gonorrhoeae (gonococci) and N. meningitidis (meningococci) are pathogenic and typically are found associated with or inside the Neisseriae Polymorphonuclear cells (PMNs). ○ PMNs are cells involved in A. MORPHOLOGY AND IDENTIFICATION LOCATION phagocytosis that have >1 nucleus (e.g. nuetrophils). 1 Gram-negative. Some Neisseria are normal Nonmotile diplococcus approximately 0.8 um in 2 inhabitants of the respiratory tract diameter. but rarely cause disease. 3 Individual cocci are kidney-bean shaped. Also occur extracellularly. When organisms occur in pairs, the flat or concave 4 sides are adjacent. ADDITIONAL INFO [DAMIANOS] MOTILITY Nonmotile diplococcus. B. CULTURE SIZE Approximately 0.8um in diameter Gram stain reaction of Neisseria (Gram neg) inside the PMNs (with segmented nucleus) Culture LECTURER’S NOTES: ON STAINING REACTION Enriched media used: All cocci are Gram (+) except Neisseriae. 1 Modified Thayer-Martin 3 GC-Lect ○ According to the basic principles of Gram staining, 2 Martin Lewis 4 New York City agar Neisseria Gram (-) cell wall does NOT have the ability to retain the crystal violet dye during the Colonies formed after 48 hours on enriched media solvent treatment or acetone alcohol stage. Monococci and Gonococci form convex, glistening, elevated Gram (+) organisms have higher peptidoglycan mucoid colonies. content while Gram (-) like Neisseria have higher lipid ○ Diameter: 1-5 mm content, ○ Colonies are transparent or opaque, nonpigmented, ○ Therefore the CVI complex exits the cell and nonhemolytic. retains only the Safranin’s (counterstain) pink color. This transcript is strictly confidential and is intended for AVR 5TH FLOOR group members only; please don’t share or distribute! 1  MICROBIOLOGY | Lesson 4: Family Neisseriaceae May have yellow pigmentation: When bacteria are spotted on a paper soaked with the ○ Neisseria flavescens ○ Neisseria subflava oxidase reagent (tetramethylparaphenylenediamine ○ Neisseria cinerea ○ Neisseria lactamica dihydrochloride oxidase) ○ the Neisseria rapidly turns dark purple. B1. ENRICHED MEDIA MEDIA CONTAINING— COMPLEX ORGANIC SUBSTANCES Technique used to enhance the population density of a Grow best on the following: particular group of microorganisms within the total microbial ○ heated blood population of a sample. ○ Hemin Achieved by: preferentially stimulating the growth of the ○ Animal proteins target group of microorganisms by judicious manipulation ○ 5% CO2 (Candle Jar) of the physiological conditions during the enrichment phase. Contains: the nutrients required to support the growth of a C1. CANDLE JAR METHOD wide variety of organisms, including some of the more fastidious ones like Neisseria. Neisseria sicca Produces: opaque, brittle, wrinkled colonies. Have dry, wrinkled breadcrumb-like colonial morphology on blood agar (48-hour culture). Produces: non-pigmented or Moraxella pinkish gray opaque colonies. Candle jar method catarrhalis Sometimes the bacteria cannot be differentiated just solely on colonial A container with a burning candle that is introduced before morphology. sealing the container’s airtight lid. Confirmation test: when reporting It is the conventional technique microorganisms in sending results, ○ principally used for capnophilic material like Nesseriae. biochemical tests should confirm The candle’s flame burns until it is extinguished by oxygen the identification of the bacteria deprivation ○ creating a CO2–rich; O2–poor atmosphere. C. GROWTH CHARACTERISTICS ○ That is when CO2 will be used by the capnophilic bacteria. Grow BEST — AEROBIC CONDITIONS but some grow in an anaerobic environment. GROWTH INHIBITED BY— FATTY ACIDS AND SALTS Saturated fatty acids inhibit oxygen consumption by the Grow on — ENRICHED MEDIA log phase cells of Neisseria gonorrhoeae. Complex growth requirements. RAPIDLY KILLED OXIDIZE CARBOHYDRATE – PRODUCING ACIDS By drying, sunlight, moist heat, and many disinfectants. Most Neisseriae oxidize carbohydrate patterns Neisseria gonorrhoeae is a fastidious organism. ○ Means of distinguishing them producing acids and not gas. PRODUCE – AUTOLYTIC ENZYMES In vitro at 25°C an alkaline pH POSITIVE (+) OXIDASE TEST; KEY TO IDENTIFY THEM All Neisseriae will be (+) SENSITIVE TO PENICILLIN Produce oxidase all representative organisms are sensitive to penicillin. Do not ferment carbohydrates USED TO: Grow organisms requiring increased↑ CO2 1 concentrations like capnophiles The jar increases the CO2 concentrations but still leaves some O2 for aerobic capnophiles. Neisseria also grows in aerobic environments. 1 Incubate the culture to achieve 5-7% of CO2 atmosphere alternative to a CO2 incubator Oxidase test This transcript is strictly confidential and is intended for AVR 5TH FLOOR group members only; please don’t share or distribute! 2  MICROBIOLOGY | Lesson 4: Family Neisseriaceae SUMMARY: FAMILY NEISSERIAE C. ANTIGENIC STRUCTURE - N. Gonorrhoeae Oxidase Rep organisms Morphology Glucose Penicillin Test Neisseria Gram (-) Cocci + + Moraxella Gram (-) Cocci + - Sensitive Acinetobacter Gram (-) Rods - - Kingella Gram (-) Rods - - THINGS TO TAKE NOTE!!-- FAMILY NEISSERIAE 1 Oxidizes glucose except N. flavescens and N. cinerea Antigenic structure of N. Gonorrhoeae Oxidize and NOT ferment Because Neisseria has the ability to only oxidize Antigenically heterogeneous glucose and produce yellow colored or utilize glucose Capable of changing its surface structures in vitro and without gas. presumably in vivo 2 ONLY 2 are PATHOGENS ○ To avoid host defenses, a means for the bacteria to n. Gonorrhea and n. Meningitidis survive in vivo. the rest have doubtful pathogenicity Pili (Fimbriae) II. NEISSERIA GONORRHOEAE Its surface structure Hairlike appendages that extend up to several micrometers A. GENERALITIES - N. Gonorrhoeae from the gonococcal surface Enhance attachment to host cells Resistance to phagocytosis Made up: stacked pilin proteins ○ basic unit of pili D. CULTURAL CHARACTERISTICS - N. Gonorrhoeae FASTIDIOUS ORGANISMS They have a complex nutritional requirement for them to grow N.Gonnorhoeae Require an enriched medium (blood agar or chocolate agar) Oxidize only glucose (G= Gonnorhoeae, G= Glucose) ○ For cultivation under increased CO2 (3-10%) for its ○ Main carbohydrate pattern of your neisseria recovery from specimen. So you put it in a candle jar. Produce smaller colonies Isolated from clinical specimens CHOCOLATE AGAR Basically the same as blood agar except that during MAINTAINED BY: preparation, the RBC are lysed (gives medium a choco SELECTIVE SUBCULTURE NON-SELECTIVE SUBCULTURE brown coloration) when added to molten agar base. As a result, since there is cell lysis, there will be an small colonies containing larger colonies containing intracellular release of your hemoglobin, hemin, x factor, piliated bacteria non piliated gonococci NAD or V factor. other culture media that doesn't It will go into the agar and will be used by your bacteria. employ an enrichment culture MODIFIED THAYER MARTIN MEDIUM Luxuriant growth occur in chocolate agar supplemented B. MORPHOLOGY - N. Gonorrhoeae with yeast extract or a similar enrichment or Modified Gram (-) Diplococci the paired cells have flattened adjacent Thayer Martin medium walls Made selective for: gonococcus and meningococcus Achieved by the addition of certain antibiotics Coffee-bean shaped Shape Kidney- bean shaped Colistin to kill gram (-) bacilli Non Sporing, non motile Characteristics but piliated organisms Nystatin to kill yeast cells Vancomycin to kill the gram (+) cocci This transcript is strictly confidential and is intended for AVR 5TH FLOOR group members only; please don’t share or distribute! 3  MICROBIOLOGY | Lesson 4: Family Neisseriaceae It has the coverage of all organisms that could possibly grow Require: in the media. ○ Arginine ○ Hypoxanthine CULTURES - PROCESSED RIGHT AWAY ○ Uracil These 3 are known as AHU form. After removal from the incubator Nutritionally Small typical colonies sensitive to penicillin ○ As organisms tend to autolyze in the absence of CO2. Fastidious (PCN). Autolyze - self digest. Gonococci Associated with both asymptomatic OTHER MEDIA USED - N. Gonorrhoeae urethritis and disseminated gonococcal infection. Substitution of anisomycin Frequently associated/isolated from Martin Lewis ○ Instead of nystatin. homosexual men. medium Antifungal ○ Has a longer half life. F. IDENTIFICATION - N. Gonorrhoeae New York City There are two parts of the identification of N. gonorrhoeae: (NYC) medium ○ Presumptive Identification Has trimethoprim lactate ○ Confirmatory Identification ○ Inhibits swarming growth of Modified Thayer Proteus due to its peritrichous F1. PRESUMPTIVE IDENTIFICATION Martin medium flagella. Presumptively, all members of the genus Neisseria are ○ Gives selective coverage of N. Oxidase (+). gonorrhoeae. PRINCIPLE - Oxidase Test COLONIES In the reaction, the enzyme indophenol oxidase, oxidizes redox dye which results in a color change in the bacterial Colonies are seen after a minimum of 20 hours incubation in colony. candle jar at 35°C. Typical colonies are: PROCEDURE ○ Small, translucent, raised, moist, grayish white with Use freshly prepared or refrigerated (for no longer than an entire to lobate margin. 1 1 week) 1% solution of tetramethyl-p- ○ Usually mucoid and tend to come off as whole phenylenediamine dihydrochloride is used. colonies when picked from the agar surface. Memorize this reagent for the practicals. Colony size varies sometimes: This is the oxidase reagent used in the oxidase test. ○ Depending on the age of the culture. 2 Use a platinum loop. ○ Crowding of the plate. A nichrome wire may cause false positives. There is more competition in terms of the ○ It contains iron that may catalyze the oxidation utilization of nutrients in the agar plate. of the oxidase reagent causing a false (+) result. Will appear smaller due to scarcity of nutrients. 3 A portion of the colony is rubbed on a filter paper strip. Plates without growth should be returned to the Upon placing a drop of the reagent, incubator for 48 hours before it can be reported as ○ Color changes into pink, violet, and then black negative. within 10 seconds. E. COLONIAL MORPHOLOGY - N. Gonorrhoeae Beyond 10 seconds will yield colonies not typical of Neisseria. 4 DISTINCT COLONY TYPES The test is repeated on an 18-hour culture on blood agar or other medium not containing any With a stereoscopic microscope on a clear carbohydrates (CHO). medium, the colonies reflect light and have ○ If no characteristic colonies are observed, the agar bright highlights. plate may be flooded with oxidase reagent to Both are piliated - has pili. detect inapparent colonies that could produce T1 & T2 T1 Piliated, small, raised. positive result. 4 Oxidase (+) colonies will be subjected to Gram staining. Piliated, slightly larger, umbonate After producing pink, violet, and black colors, a small T2 colony centers. portion of the Oxidase (+) colonies will be subjected to Gram staining reaction. Non-selective subculture. Colonies are flatter, larger, and less T3,T4, T5 opaque and will not reflect light. Composed of non-piliated cells - no pili. This transcript is strictly confidential and is intended for AVR 5TH FLOOR group members only; please don’t share or distribute! 4  MICROBIOLOGY | Lesson 4: Family Neisseriaceae BASIS FOR A PRESUMPTIVE IDENTIFICATION OF N. PROCEDURE - Carbohydrate Degradation GONORRHOEAE - Oxidase Test Cystine Tripticase Agar (CTA) are inoculated with heavy If observed, these are sufficient for presumptive suspension of organism from a subcultured chocolate identification of Neisseria gonorrhoeae and can be reported 1 agar (CA) plate 18-24 hours old in about 0.5mL of saline already: or Trypticase Soy Broth (TSB). 1. Typical Gram (-) diplococci Has flattened coinciding sides from gram staining You prepare a heavy suspension then inoculate it to reaction. the CTA. 2. Specimen is from the genitourinary tract. Deposit 2-3 drops of the suspension on the surface of 2 3. Inoculated into Modified Thayer-Martin medium or the medium. any enrichment media. Stab the upper third of its depth using a sterile cotton 3 4. Shows a typical Oxidase (+) colony. plugged capillary pipet. 4 Incubated with added CO2 at 35°C in a candle jar. DRAWBACKS - Oxidase Test (+) Production of acid will turn the medium turbid. If specimens are either from: 5 There will also be a production of a yellow color on the ○ Other sites like pharyngeal culture. upper layer of the medium. ○ On special social and other medico-legal situations. They should be identified by more definitive or CTA SUGARS - Carbohydrate Degradation confirmatory procedures before reporting the results. Should not be reported yet, but suspicion is already there. RESULTS - Oxidase Test CTA Sugars (Left to right: (+) glucose, (-) maltose, (-) lactose, (-) sucrose) Positive (+) Glucose A Negative Oxidase (left) & Positive Oxidase (right) test. Maltose Negative (-) Lactose The color change is seen. Sucrose ○ It turns to black (end color) within 10 seconds If there is a positive reaction in glucose, but a negative F2. CONFIRMATORY IDENTIFICATION reaction for maltose, lactose, and sucrose, then it is F2.1 CARBOHYDRATE DEGRADATION positive for Neisseria gonorrhoeae. The most common way of confirming the identification of N. gonorrhoeae. In a positive carbohydrate dehydration test, there will be: PRINCIPLE - Carbohydrate Degradation ○ Turbidity N. gonorrhoeae oxidizes glucose without gas since it is ○ Yellow color on the upper layer more of an oxidation process rather than a fermentation. Positive Reaction of the medium The recommended base medium is Cystine Trypticase The positive reaction is due to the Agar (CTA) with pH 7.6. production of acid. ○ They readily support the growth of Gonococci and The phenol red (indicator) will turn Meningococci. yellow once the pH is