WEEK-8-Gram-Negative-Cocci-Prof.-Lozada PDF

PRELIMS | BACTE (LEC) Week 8 | Prof. Lozada | Gram Negative Cocci (Neisseria spp. And Moraxella catarrhalis) ● ◆ Neisseria Species and Moraxella catarrhalis All Cocci are gram positive except: o Neisseria spp. o Veilonella spp. o Moraxella catarrhalis o Acidaminococus spp. o Megasphaera NEISSERIAC...

PRELIMS | BACTE (LEC) Week 8 | Prof. Lozada | Gram Negative Cocci (Neisseria spp. And Moraxella catarrhalis) ● ◆ Neisseria Species and Moraxella catarrhalis All Cocci are gram positive except: o Neisseria spp. o Veilonella spp. o Moraxella catarrhalis o Acidaminococus spp. o Megasphaera NEISSERIACEAE Neisseria Kingella Eikenella Cocci Adjacent sides flattened Cell division – two planes Catalase + Oxidase + Bacilli Pairs and Chains Cell division – one plane Catalase – Oxidase + Bacilli Catalase Oxidase +   Simonsiella – Bacilli Gliding motility Normal human oral biota Large flat multicellular filaments Catalase – Oxidase + Kingella, Eikenella, and Simonsiella are all bacilli. Kingella kingae, and Eikenella corrodens are part of the HACEK group, which is a group of fastidious organisms or fastidious bacilli. o In the HACEK group, there is also Haemophilus spp., Aggregative bacter, cargobacterium, Eikenella corrodens and Kingella kingae NEISSERIAE ➔ ➔ ➔ ➔ PATHOGENIC NEISSERIA ➔ ➔ GENERAL CHARACTERISTICS ➔ CPOP – Catalase positive, Oxidase positive o Catalase positive means that the organism produces catalase. o In order to determine if the organism is catalase positive, a catalase test is conducted using Hydrogen peroxide as a reagent. o In reality, the role of catalase in bacteria is to hydrolize the hydrogen peroxide, which is a biproduct of the oxidative process in the aerobic respiration. It is produced through aerobic respiration and if H2O2 accumulates it will become toxic to the organism, so one way to protect the bacteria is to produce catalase, which will break down H2O2 into water and oxygen, which is why there is the formation of bubbles. o In oxidase test, we detect for the presence or production of cytochrome c oxidase, microdase test is the test used. Cytochrome c oxidase is necessary in ATP production. ➔ Aerobic, non-motile, non-spore-forming gram-negative intracellular diplococci ◆ Intracellular – capable of invading the cells of their host Except : N. elongate (catalase negative), N. weaveri, N. bacilliformis (catalase variable) (bacilli) rod shaped ◆ Most are capnophilic – They require the presence of carbon dioxide in order to grow and have optimal growth in a humid atmosphere. ◆ They can grow anaerobically if alternative electron acceptors (e.g., nitrites) are available. All are oxidase positive. All are catalase positive. ◆ except for N. bacilliformis and N. elongata subsp. nitroreducens. Natural habitats mucous membranes of respiratory (meningococci) and urogenital (gonococci) tracts Primary Human Pathogens : N. gonorrhoeae and N. meningitidis ➔ ➔ Neisseria gonorrhoeae ◆ Agent of Gonorrhea Neisseria meningitidis ◆ Agent of meningococcemia and meningococcal meningitis o Meningitis is the inflammation of the meninges, meningococcemia means the infection is disseminated or already in the blood of the organism. o Their major virulence factors would be the adhesins which include the pili and the capsule, opacity proteins can also be included. The major role of pili and capsule is the adhesion on host epithelium. o For opacity proteins we have Opa and Opc, they bind with the CEACAM (Carcinoembryonic Antigen related Adhesion Molecule), and can also bind with HSPG (Heparan Sulfate Proteoglycan). o What is important in the pathogenesis of Neisseria is the attachment of the organism to the cell of the host, because if not, they will not be able to invade or infect. Both N. gonorrhoeae and N. meningitidis require iron for growth (all pathogenic strains require iron for growth). They compete with their human host by binding human transferrin to specific surface receptors. Their ability to bind transferrin may be a primary reason that they are strict human pathogens. Pathogenic Neisseria spp. possess receptors for human transferrin, pili, cell membrane proteins, and a Lipooligosaccharide Page 1 of 11 o o  The difference with other gram negative organisms is that lipopolysaccharide is much simpler, and they lack the repeating O side chain found in the lipopolysaccharide of gram negative enterics. Lipooligosaccharide is an endotoxin. ○ ● We also have minor adhesins which include: o NhhA = Neisseriahia homologue A o MspA = Meningococcal serine protease A o APP = Adhesion and Penetration Protein o NadA = Neisserial Adhesion A o All of their primary roles is adhesion, they bind with different receptors on the host cell. Unlike the Opc and Opa, they can only bind with HSPG Neisseria gonorrhoeae EPIDEMIOLOGY AND CLINICAL SIGNIFICANCE ● ● Agent of the sexually transmitted disease Gonorrhea ○ Acute pyogenic infection of nonciliated columnar and transitional epithelium found in the genitourinary tract of the host. ■ Infection produces pus ○ Occurs primarily in the urethra, endocervix, anal canal, pharynx, and conjunctiva ○ Dissemination is possible ■ Can cause arthritis, septicemia Humans are the only known natural host. ○ Asymptomatic carriers are a significant reservoir for increased disease transmission ■ Asymptomatic carriers possess AHU auxotype strain ■ AHU auxotype - Neisseria strain which needs arginine, hypoxanthine and uracil for growth. CHARACTERISTICS ● ● ● Very Fastidious ○ Capnophilic, requires X and V factors, cysteine, and iron (haemoglobin can also be a source of iron) ○ Will NOT grow on BAP ■ BAP has only V factor Catalase and Oxidase positive Gram-negative ○ Intracellular diplococci ● Avirulent forms that lack pili are seen as extracellular diplococcic Virulence Factors ○ Pili ■ Colony types 1 and 2 have Pili that is why it is virulent because pili is needed for cellular adhesion ■ Colony type t3 to t5 avirulent due to missing pili ○ *IgA protease■ breaks the heavy chain of IgA ■ Only seen is some strains ○ *Beta-Lactamase ■ break the beta lactam ring in beta lactam drugs ■ Only seen in some strains ○ Tracheal cytotoxin ■ initially detected in Bordetella pertussis. ■ Damages the ciliated cells of the fallopian tube, can cause scarring and will lead to infertility. ○ Outer membrane proteins ■ OM protein 1 ● AKA porin proteins ● Form pores or channels for passage of nutrients and waste products. ● Por A and Por B genes ● N. gonorrhoeae can only express Pore B genes ● N. meningitis can express both pore A and B genes ● Por B honest inflammatory and compliment-mediated killing. They block phagosome-lysosome fusion in the neutrophils. They can bind with compliment components (e.g C3b and C4b) ■ OM protein 2 ● AKA opacity protein (Opa and Opc) ● For adhesion ● N. gonrrhoeae can only express Opc ● N. meningitidis can express both Opc and Opa ■ OM protein 3 - RMP ● Reduction modified protein ● Create pored on cell surface ● Block bactericidal property of IgG *only in some strains Page 2 of 11 Extra reproductive organ infection ● Rectal Gonorrhea ○ Discharge, Rectal pain, Bloody Stools ○ Approximately 30-60% of women with genital gonorrhea have concurrent rectal infection ○ Anal sex ● Oropharyngeal Gonorrhea ○ Pharyngitis – chief complaint ○ Oral sex Ophthalmia neonatorum ● ● ● ● OM proteins Protein I - porin proteins  Form pores or channels for passage of nutrients and waste products  Por A and Por B genes  N. gonorrhoeae - only expresses por B genes   ● ● ● Adult Ocular Gonorrhea ● ● N. meningitidis - both por A and por B genes GONORRHOEAE ● ● ● ● MOT : Sexual contact ; passage through birth canal Incubation period : 2-7 days; they require moist environment with temperature of 32 to 35 degrees C Males : Acute Urethritis ○ Signs and Symptoms : Dysuria, Purulent discharge ○ Complications : Prostatitis, Epididymitis ■ May lead to urethral strictures due to scarring in the urethra ○ 10% of the cases are asymptomatic ■ Usually associated with AHU strains Females: Endocervicitis ○ Signs and Symptoms : Dysuria, Discharge, Lower abdominal pain ○ Complications : Pelvic Inflammatory Disease, sterility, ectopic pregnancy, salpingitis, Fitz-Hugh-Curtis Syndrome (perihepatitis) ■ If it goes up it causes salpingitis ■ Causes endometritis, tuboovarian abscess ■ Fitz-Hugh-Curtis Syndrome causes upper right quadrant pain in the abdomen ○ 50% of the cases are asymptomatic Blood-borne dissemination ~ about 1% of all infections ○ Gonococcal arthritis ■ Seen in synovial fluid ○ Septicemia Newborn gonococcal eye infection Acquired during vaginal delivery through an infected birth canal Results into blindness Prevented through antimicrobial eye drops (usually erythromycin) “Sumabit lang ang bacteria during labor” 2% silver nitrate (crode’s method?) ○ Agglutination and inactivation of gonococcus? Occurs when eye is inoculated with infected genital secretions Rarely happens as a result of laboratory accident LABORATORY DIAGNOSIS Specimen collection ● ● ● ● Primary Technique : Swabbing using Dacron or Rayon swabs Urethral Swab (men), Endocervical swab (women) Other Sources : Rectum, Pharynx Disseminated infections : Blood, CSF, Synovial Fluid Specimen Transport ● ● ● Transport systems are crucial because the gonococci is extremely susceptible to drying and temperature changes. ○ Amie’s Medium with Charcoal ○ Swabs must be plated within 6 hrs of collection JEMBEC System, Gono-Pak, Transgrow (John E. Martin Biological Environmental Chamber) ○ Contains Theyer-Martin agar ○ Allows direct plating of swab ○ Contain selective media and a CO2 atmosphere ○ Swab is rolled in the media in a Z pattern. Page 3 of 11  o o o o Content: All thayer martin components, trimethoprim Martin Lewis : Anisomycin with Trimethoprim  Nystatin is replaced by anisomycin New York City Agar : Amphotericin B with Trimethoprim  Primary antifungal is amphotericin B Incubation : 35 deg C in 3-5% CO2 held up to 72 hours  CO2incubator, CO2 generating pouch, Candle Jar Plates must be examined daily.(every morning for 3 days) Colony Morphology  Size : Small (avirulent strains larger) : up to 1 mm JEMBEC® System Consists of a plated medium, CO2 generating tablet, and a resealable polyethylene bagUpon receiving in the lab, the media can be crossed-streak with a loop diameter Color : Gray to Tan Pigmentation : golden-brown after 48 hrs Density : Translucent Elevation : Raised/convex(avirulent strains are flat) Margin : Smooth to irregular The colonies are glistening/shiny. Five Colony types : T1 to T5  T1 and T2 – strains with pili, smaller (Virulent)  T3 to T5 – strains without pili ; larger and Flatter (Avirulent) Oxidase Test : +         Gram staining - Intracellular gram (-) diplococci, not definitive especially for female patients Initial inoculation - Possible Neisseria spp. Inoculate in MAC (+) - Non-Fastidious gram-negative, crystal violet inhibits gram positive bacteria BAP (+) CAP (-) - No growth. Neisseria spp. requires X and V factor = The organism is not Neisseria, expected results should be MAC (-), BAP (-), and CAP (+) Colony morphology - gray-translucent colonies Perform gram stain and oxidase testing - after isolation, only diplococci Definitive identification  Carbohydrate utilization test  Nucleic Acid Amplification Test (NAAT) - Gold standard, according to WHO Direct Microscopic Examination  Gram Stain – Presumptive identification  Look for gram negative diplococci (may be intracellularly located in neutrophils Culture  Medium of Choice : Chocolate Agar with Vancomycin, Colistin, and antifungal agents o Thayer-Martin : Nystatin as antifungal agent  Content: vancomycin, colistin, and nystatin o Modified TM : Nystatin with Trimethoprim (to inhibit Proteus *and wider range of gram neg rods*) Detects bacterial cytochrome oxidase Reagent : 1% dimethyl-p phenylenediamine dihydrochloride (Gordon and McLeod reagent) or tetramethyl-p-phenylenediamine dihydrochloride (Kovac’s oxidase reagent)  Positive result : purple (Kovac), red(10-30 mins) to black (60 mins) for Gordon McLeod  Methods : o Paper-Disk (Microdase Method) o Filter Paper Method o Direct Colony Testing Superoxol Test : +     30% Hydrogen Peroxide (+) result : bubbling Higher concentration of hydrogen peroxide Only N. gonorrhoeae can be positive in this test. Catalase Test : +  3% hydrogen peroxide Carbohydrate Utilization Test : +    They don’t ferment but they OXIDIZE Traditional identification of Neisseria spp. Uses Cystine Trypticase Agar with 1% particular carbohydrate (glucose, sucrose, maltose, lactose, fructose)  Indicator : Phenol red  + result = yellow coloration within 24-72 hrs  Expected Results for N. gonorrhoeae o + for Glucose only Rapid Carbohydrate Utilization Test (wag daw discuss)    require pure cultures read in 2 to 4 hours Detect acid production from various carbohydrates, but they are based on the presence of preformed enzymes Page 4 of 11 for carbohydrate utilization rather than on bacterial growth. Problems with this method include :  Weak acid production from glucose by certain strains of N. gonorrhoeae  Misidentification of sucrose-negative strains of Neisseria subflava as N. meningitidis  Strains of N. cinerea that give positive glucose reactions Chromogenic Assay          Detects B-galactosidase, Gamma-glutamylaminopeptidase, and proline-iminopeptidase(Pip) which are specific for N. lactamica, N. meningitidis, and N. gonorrhoeae, respectively. o N lactamica produces B-galactosidase o N. meningitidis produces Gamma-glutamylaminopeptidase o N. gonorrhoeae produces prolineiminopeptidase(Pip) No color changed after the introduction of the organism; we can suspect N. subflava, N. mucosa, N. meningitidis if it results in yellow. Color Blue means N.lactamica, capable of producing beta-galactosidase. Colorless to yellow indicates B. catarrhalis. Red indicates N. gonorrhea, K. denitrificans. The substrates used are bromo-chloro-indolyl-bgalactoside (or -galactopyranoside), g-glutamylnitroanilide, and proline-methoxynaphthylamide, o change their colors after positive reaction to blue, yellow, and red, respectively. All can be assayed in a one-tube format in the two commercially available kits, GonoCheck II and Neisseria PET In both tests, b-galactosidase and g-glutamylaminopeptidase are assayed first. o requires an incubation step at 37°C for 30 min. Pip is tested by the addition of the appropriate reagent (Neisseria PET test) or replacement of the screw cap and subsequent inversion of the tube (GonoCheck II test) IMMUNOSEROLOGICAL TEST  Employ monoclonal antibodies directed against gonococcal protein I Coagglutination Method (Phadebact Monoclonal GC Test)    + result : red spot - result : white to plae pink ring Problem: false positive results of detecting N. lactamica Enzyme immunoassays        uses fluorescein isothiocyanate-labeled antibodies for confirmation of N. gonorrhoeae.  + result : apple-green fluorescent diplocci under a fluorescence microscope Colorimetric Assay (GonoGen II))   uses antibodies adsorbed to metal sol particles. When the culture is emulsified in the solubilizing buffer, the outer membrane of the organism is stripped off releasing the OMP containing complexes into solution. These released OMP complexes are then captured by the antibody/metal-sol particles. NUCLEIC ACID AMPLIFICATION TEST Gold standard according to WHO Sample : First Morning Urine Extremely sensitive and do not require viable organisms Situations where NAAT is not recommended o Child sexual assault involving boys and girls*  NAAT will not be suggested, instead culture is needed and extragenital samples. o Extragenital infections* o Monitoring effectiveness of treatment* o Monitoring antimicrobial resistance NAAT extragenital specimens from ADULTS are recommended. Examples are rectal swab, pharyngeal swab. o Only for patients that are in MSM category and female sex workers NOT *as of 2021 US FDA cleared NAATs for extragenital samples for gonor TREATMENT     Monoclonal antibodies directed against gonococcus are attached to killed S. aureus cells.  Distinguishes the three serotypes WI, WII, and WIII  Cross-reaction with N. cinerea, N. lactamica, and N. meningitidis Fluorescent Antibody Testing (MicroTrak) Quick identification of gonococcal urethritis with good sensitivity and specificity cocci under a fluorescence microscope  Uncomplicated urogenital, rectal, and pharyngeal gonorrhea o Ceftriaxone 500 mg IM single does for patients weighing below 150 kg  CDC recommended o Ceftriaxone 1 g IM single dose for px weighing 150 kg and above If chlamydia has not been ruled out, include : o Doxycycline 100 mg oral twice a day for 7 days o Azithromycin 1 g oral = if px is pregnant Patients with history of ceftriaxone allergy o Gentamicin 240 mg IM single dose plus Azithromycin 2 g oral single dose o 2 g azithromycin may cause nausea, vomiting, and/or diarrhea If ceftriaxone is contraindicated and/or not available : o Cefixime 800 mg oral single dose (plus cotreatment for chlamydia if not ruled out) *Test for cure NAAT swab test or culture ; 7-14 days after treatment “We SHALL not dwell upon this for we are not doctors.”   ANTIMICROBIAL RESISTANCE “BASAHIN NALANG DAW TO” Penicillin o Penicillinase (B-Lactamase) production became common (PPNG strains)  Plasmid mediated o PPNG strains were largely imported from Southeast Asia or Africa Page 5 of 11 o   Chromosome-mediated penicillin resistance  Non-B-Lactamase producers  Altered Penicillin binding proteins Tetracycline o Used as alternative drug for penicillin for px who were allergic o Chromosomally mediated resistance o Plasmid mediated resistance – high level (MIC > 32 ug/mL) Fluoroquinolones o Recommended treatment from 1993 – 2006 o gyrA and gyrB mutation – decreased binding affinity of DNA gyrase to fluoroquinolones Neisseria meningitidis EPIDEMIOLOGY AND CLINICAL SIGNIFICANCE Agent of meningitis and meningococcemia  Meningitis- inflammation of meninges  Meningococcemia- Disseminated in the blood.  Rarely causes pneumonia, purulent arthritis, or endophthalmitis o Caused due to it being disseminated to the blood.  Also found on urogenital and rectal samples Humans are the only known natural host.  Invasive and Commensal  It may cause death  Normal flora of mucosal surfaces of oropharynx and nasopharynx in about 10% of the population  Transformation into invasive form is unclear  Increased risk of invasive disease occurs in individuals with asplenia or individuals with complement deficiencies.  Behavioral risks : smoking, kissing  Mode of transmission: inhalation of respiratory droplets. 13 encapsulated serotypes  6 globally important serotypes – cause invasive meningococcal disease (IMD) o A, B, C, X, Y, and W (W-135)  Meningococcal infection is mostly associated with serotype Y African Meningitis Belt o Areas that frequently experience epidemics during hot and dry seasons o Most cases are caused by serogroup A  Meningococcal carriage involving nonencapsulated strains increases protective antibodies against pathogenic strains. CHARACTERISTICS        Gram negative encapsulated diplococci Not as fastidious as N. gonorrhea o Can grow on Blood Agar Plate Non-Hemolytic Oxidizes Glucose and Maltose Oxidase and Catalase positive Capnophilic (requires CO2) Non-motile VIRULENCE FACTORS Capsule   Composed of sialic acid moieties except for serotype A (polymer mannosamine-1-phosphate) Outer Membrane of Nacetyl-    Porin proteins (porA and porB) Opacity proteins (Opa and Opc) Lipooligosaccharide o Lipid A disaccharide  For attachment Pili CLINICAL INFECTION Transmission  Mode : Droplet Transmission, Contact Transmission  Route : Respiratory  Portal of Entry : Nasopharynx Incubation Period  1 to 10 days Colonization Area  Nasopharyngeal mucosa Infection (invasive meningococcal disease)  Meningococcemia o With or without meningitis o It directly go to the circulation of the patient  Meningitis o With or without bacteremia o Common complication of meningococcal bacteremia Initial symptoms  Flu-like symptoms Meningitis symptoms  Intense headache, vomiting, photophobia, confusion, and stiff neck May progress into coma Bactermia / Meningoccemia  Purpura with petechial skin rash (due to thrombosis) o Meningococcal LOS can activate coagulation cascade which elevates fibrin formation, and thus can lead to clogging of the blood vessels causing rupture or leak and resulting in purpura.  Tachycardia, Hypotension may develop Fulminant meningococcemia  High fever, hemorrhagic rash  Spreads rapidly  May lead to Disseminated Intravascular Coagulopathy, circulatory collapse, and Waterhouse-Friderichsen Syndrome  Thrombosis can lead to hypoxia and eventually ends up with necrosis  Thrombosis can also be observed in the adrenal glands, renal cortex and other parts of the body.  Malalang version ng meningococcemia can lead to Waterhouse-Friedrichsen Syndrome. Waterhouse-Friedrichsen Syndrome  Bilateral hemorrhagic necrosis of adrenal glands with subsequent adrenal failure  Not limited to meningococcal infections  Leads to necrotic adrenal glands Other Complications:  Pneumonia – Serogroup Y  Arthritis  Pericarditis  Conjunctivitis  Urethritis LAB DIAGNOSIS Specimen Page 6 of 11     CSF (and Blood) o Blood culture for meningococcemia but low concentration of SPS. Nasopharyngeal Swab – for carriers Joint fluids, sputum, urogenital samples Petechiae biopsy o In the area where there is pupura Smears   Gram stain the sediments of centrifuged spinal fluid or of petechial aspirate o At least 1 mL CSF (centrifuge at 1000 g for 10 mins) Typical gram-negative intracellular diplococci Culture    Can grow on BAP Plate on BAP and CAP If sx is from mucosal surface, add a medium selective for Neisseria  Incubate (same protocol with N. gonorrhoeae)  examine daily for 72 hrs Colony Morphology  medium sized, gray, and convex  encapsulated strains are mucoid  Not translucent Presumptive Diagnosis  Gram-stain : gram-neg diplococci o Diagnosis is not definitive  Colony Morphology  Oxidase : +  Catalase : +  *Superoxol : o (early differentiation from N. gonorrhoeae but not definitive because other Neisseria are also negative for superoxol) Definitive Diagnosis  Carbohydrate Utilization o + for Glucose and Maltose o Maltose negative serotypes : B (rarely in C and Y)  Chromogenic Assay o Y-glutamyl aminopeptidase +  MALDI-TOF  NAAT  Serotyping – performed after confirmation of isolate o Latex agglutination o only performed in reference laboratories. o No longer required if first shot was given at age 16 or older unless they are at high risk for IMD Serogroup B Vaccine  Two to three doses  Capsular B antigen is weakly antigenic and has similar structure with human glycoproteins o Can cause hypersensitivity rxn due to molecular mimicry “Basahin niyo nalang to ahh mag-LIBRO kayo” Moraxella catarrhalis  Family : Moraxellaceae o Genera : Moraxella, Acinetobacter, Psychrobacter  Also a diplococci which is commonly mistaken for Neisseria.  Cellular morphology resembles the morphology of Neisseria  Commensal of Upper Respiratory Tract  Mainly an opportunistic pathogen o Can cause URTI in an otherwise healthy children and/or older adults o Causes LRTI in adults with COPD  Oxidase and Catalase positive  Assacharolytic and non-pigmented  Aerobic, non-motile, non-spore-forming gramnegative diplococci  Third most common cause of acute otitis media and sinusitis in children.  Most produce B-Lactamase  Typical specimens : o Middle ear effusions, Nasopharyngeal swab, sinus aspirates, sputum aspirates, bronchial aspirates  Grows well on BAP and CAP o Colonies are gray-white, smooth and opaque o Hockey puck colonies – colony remains intact when pushed across the plate  Matigas ang colony. If poked with the inoculating stick the whole colony will move. o Old Colonies – wagon wheel appearance  Optimum growth at 28 deg C.  Specimens would depend on the location of the infection. TREATMENT Penicillin G  Drug of Choice Alternatives for px allergic to Penicillin  Chloramphenicol  Third Gen Cephalosporins o Cefotaxime o Ceftriaxone Ciprofloxacin – chemoprophylactic agent  Alt : Rifampicin, Azithromycin VACCINE Quadrivalent vaccine  Serotypes A, C, Y, and W-135  For 2-55 years of age  First dose – recommended to be given at age 11-12  Booster shot – age 16 Page 7 of 11 ○ Differentiation from Neisseria  Moraxella catarrhalis is DNAse and Butyrate esterase positive.  Exception : N. ovis (DNAse positive) Tributyrin  Substrate used to detect butyrate esterase activity  It is a triglyceride which is hydrolyzed into butyric acid and glycerol in the presence of the enzyme butyrate esterase.  Has several variations, majority use an acidobasic indicator which relies on color changes.  Fluorogenic variation : 4-methylumbelliferyll butrytate (MUB) test DNAse Test  performed on DNAse agar or on test strips ○ ○   DNASE AGAR TEST DNAse Agar Composition  Tryptone, Soya Peptone, DNA, Sodium Chloride, Agar, indicator (usually methyl green)  pH : 7.5 Principle:  Deoxyribonuclease allows the organisms that produce it to break down DNA into smaller fragments (oligonucleotides).  When the DNA is broken down, it no longer binds to the methyl green, and a clear halo will appear around the areas  where the DNase-producing organism has grown. Expected results:  Positive: Medium is colorless around the test organism. (M. catarrhalis)  Negative: If no degradation of DNA occurs, the medium remains green.  Glucose, Maltose, Lactose, and Sucrose Fermentation Beta-Lactamase Activity DNAse Commensal neisseria Commensal Neisseria that can grow on selective media o Saccharolytic : N. lactamica, N. polysaccharea o Asaccharolytic : N. cinerea Commensal Neisseria that are unable to grow on selective media o Saccharolytic : N. mucosa, N. sicca, N.subflava, N. flava, N. perflava o Asaccharolytic : N. flavescens, N. elongate, N. weaver, N. bacilliformis Microbiologists need to be familiar with these because even though they can rarely cause human infections, diagnosis and identification must remain accurate. RAPID DNASE TEST (QUADFERM+ TEST) ● ● When DNase is produced by organisms in the QuadFERM+ test, an acidic end product is formed and the pH indicator changes from red (alkaline) to yellow (acid). Other tests in the QuadFERM+ Test Page 8 of 11 PRELIMS | BACTE (LEC) Week 8 | Prof. Lozada | Gram Negative Cocci (Neisseria spp. And Moraxella catarrhalis) Infections Reported to be Caused by Neisseria Species Other than N. Gonorrhoeae and N. Meningitidis Infection Meningitis Endocarditis Prosthetic valve infection Bacteremia Pneumonia Empyema Bacteriuria Osteomyelitis Ocular infection Dog bite Neisseria Species N. lactamica N. Sicca N. subflava N. mucosa N. flavescens N. sicca N. subflava N. mucosa N. elongate N. flavescens N. bacilliformis N. sicca N. lactamica N. flavescens N. cinerea N. sicca N. mucosa N. subflava N. sicca N. cinerea N. mucosa N. weaveri Page 9 of 11 PRELIMS | BACTE (LEC) Week 8 | Prof. Lozada | Gram Negative Cocci (Neisseria spp. And Moraxella catarrhalis) Traditional Tests Additional Tests Reduction of Acid Produced from Organism Growth Catalase Oxidase Glucose Maltose on ML, MTM, or NYC Group 1: Traditional Pathogens + + + + N. gonorrhoeae + + + + + N. meningitidis Group 2: Commensal Species – Possible Growth on Selective Agar Media Saccharolytic V + (+) K. denitrificans + + + + + N. lactamica + + + + + N. polysaccharea Asaccharolytic V + + N. cinerea V + + M. catarrhalis Group 3: Commensal Species – No Growth on Selective Agar Media Saccharolytic + + + + N. mucosa + + + + N. sicca N. subflava biovars + + + + N. subflava + + + + N. flava + + + + N. perflava Asaccharolytic + + N. flavescens + N. elongate + + N. weaver V + N. bacilliformis Lactose (ONPG) Sucrose Fructose Growth on Blood or Chocolate Agar at 22 degrees C Growth Nutrient Agar at 35 C Nitrate Nitrite DNase - - - - - - V - + - V - + V - + + + + - V V V - - - - V + + + + + + - + + + + + + + + + - + + - + - + + + + + + + + + - V V V - - - - + + + + + + + V + + + V - Page 10 of 11 PRELIMS | BACTE (LEC) Week 8 | Prof. Lozada | Gram Negative Cocci (Neisseria spp. And Moraxella catarrhalis) Page 11 of 11