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MLS 114 MOT: direct contact with contaminated respiratory droplets (cough...

MLS 114 MOT: direct contact with contaminated respiratory droplets (coughing or sneezing) Corynebacterium and Other Non-spore-Forming Gram-Positive Rods MAHON A.S.M, F.J.O Humans are the only natural host for C.diphtheriae NON SPORE FORMING BACILLI VIRULENCE Diphtheria toxin- major virulence factor; produced by strains of C. FACTOR diphtheriae infected with a lysogenic B-phage which carries the tox CLINICALLY IMPORTANT GRAM-POSITIVE BACILLI gene 1. Corynebacterium toxin is nontoxic until exposed to trypsin 2. Erysipelothrix Fragment A & Fragment B 3. Listeria The toxin blocks protein synthesis in eukaryotic cells 4. Actinomycetes-Nocardia Exotoxin (A and B) – heat labile NONBRANCHING, CATALASE-POSITIVE BACILLI CLINICAL 2 forms of disease in humans 1. Corynebacterium Diphtheriae INFECTIONS Respiratory diphtheria 2. Other Corynebacterium spp. Cutaneous Diphtheria 3. Listeria monocytogenes 4. Rothia C. diphtheriae is carried in the URT and spread by droplet infection or hand-to-mouth contact CORYNEBACTERIUM Incubation Period: 2 to 5 days Medically Important Corynebacterium Most Common Site Of Infection: tonsils oropharynx 1. C. diphtheriae Combination of cell necrosis and exudate forms a tough 2. C. ulcerans gray-to-white pseudomembrane which attaches to the tissues 3. C. jeikeium 4. C. pseudotuberculosis LABORATORY SPECIMEN Babes-ernst DIAGNOSIS NPS of the inflamed areas or vigorously swabbed culture /Hungod sa General Genus Characteristics: the pseudomembrane Gram (+) straight or slightly curved bacilli, frequently swollen on one Swabs of skin lesions for cutaneous diphtheria or both ends (Club-shaped) Dacron swabs should be used for specimen collection Non spore formers and non branching CatalasepositiveandOxidasepositive MICROSCOPY Non-motile highly pleomorphic gram-positive bacillus that appears in palisades/ picket fences/cigarette packets Divided into three groups: V and L formation/ Chinese letters 1. Group 1–human and animal pathogens Club-shaped swelling and beaded forms 2. Group 2–plant pathogens staining with methylene blue produces a beaded 3. Group 3–non pathogenic appearance. Unless isolated from the blood or spinal fluid, species other than C. diphtheriae are generally considered to be commensal. Corynebacterium diphtheriae Klebs-Loe er bacillus Agent of diphtheria CULTURE CHARACTERISTICS MOT: direct contact with contaminated respiratory droplets (coughing or sneezing) Facultative anaerobe Humans are the only natural host for C.diphtheriae Optimal growth temp: 37 deg Celsius Require eight essential amino acids; better growth is Corynebacterium diphtheriae => obtained on medium containing blood or serum Plated onto: SBA,CNA,cysteine or potassium tellurite media,and Loe er serum medium Klebs-Loe er bacillus Agent of diphtheria CULTURE MEDIA DESCRIPTION  Loe er Serum Enhances metachromatic granule Black, beta hemolytic, starch/ Medium formation glycogen (-) Contains bovine serum, peptic digest of animal tissue, heart muscle infusion; Dextrose and Sodium chloride Belfanti Resembles the mitis biotype and rarely carries the diphtheria toxin gene Cystine Tellurite For selective isolation and di erentiation blood Agar (CTBA) of Corynebacterium diphtheriae types Modified Tinsdale A selective and di erential media for medium Corynebacterium A potassium tellurite medium -Elek test: definitive -Roemer test: incubates the bacteria in pig -Schick’s test: skin test (Ab’s) BIOCHEMICAL IDENTIFICATION Catalase-positive Nitrate reduction positive Urease Negative Glucose & Maltose fermenter Nonmotile COLONIAL MORPHOLOGY (+) DNase CTBA: black or gray colonies; A brown halo surrounding the Ferments dextrose colony, owing to cystinase activity, is a useful di erentiating feature because only C.diphtheriae, C. ulcerans, and C. pseudotuberculosis produce a brown halo TOXIGENICITY TEST Modified Tinsdale: 1. In vivo: Guinea Pig inoculation Four colonial types or biotypes: gravis, 2. In vitro: Elek Test (definitive test) intermedius,belfanti and mitis Immunodi usion test; controls and unknown organisms are streaked on medium of low iron content. Each organism is streaked in a single straight line parallel to BIOTYPES DESCRIPTION each other and 10 mm apart A filter paper strip impregnated with diphtheria antitoxin is Gravis Largest colonial type (1 – 2 mm laid along the center of the plate on a line at right angles to colonies on BAP) the inoculum lines of control and unknown organisms. Gray, non-hemolytic, The plate is incubated at 35 deg C and examined after 18,24, starch/glycogen (+) and 48 hours. 3. Rapid ELISA & immunochromatographic strip assays 4. PCR assay to detect the tox gene Intermedius Small (0.5 mm on BAP), Black colonies with gray borders on tellurite media, non hemolytic lipophilic colonies Mitis Medium sized, Fried egg appearance on BAP (clear with white centers) May produce bleach-like odor on tellurite medium  Other Corynebacterium Corynebacterium The most frequently recovered Corynebacterium amycolatum Species Part of normal skin microbiota Often associated with prosthetic joint infection and reported to cause bloodstream infection and endocarditis Colonies: flat, dry have matte or wax appearance,and non lipophilic - Corynebacterium Named after Johnson and Kaye jeikeium Most common cause of Corynebacterium-associated NH Strict aerobe prosthetic valve endocarditis in adults Lipophilic and a strict aerobe, nonhemolytic, does not produce urease, and is nitrate reduction negative Corynebacterium A veterinary pathogen Listeria monocytogenes pseudotuberculosis Causes a granulomatous lymphadenitis in humans Can produce diphtheria toxin,produces a brown halo on CTBA Widespread in the environment; recovered from soil, water, vegetation and animal product capnophilic Produces urease and on SBA forms small yellowish white colonies Major source of infection is contaminated food(cabbage, fruit,dairy products) Recognized as an uncommon but serious infection primarily of neonates, pregnant Corynebacterium A veterinary pathogen women,elderly adults, and immunocompromised hosts. ulcerans Isolated from humans with diphtheria-like illness H strict aerobic Grows well Loe er agar and produces a brown halo on CTBA VIRULENCE FACTOR Listeriolysin O–a hemolysin & Produces an arrow zone of Beta Hemolysis Catalase, superoxide dismutase, Phospholipase C, and a surface protein (p60) capnophilic Does not reduce nitrate Urease positive CLINICAL INFECTIONS LISTERIOSIS Infections of newborns and immunocompromised are the Corynebacterium Most commonly associated with UTIs most common urealyticum Lipophilic and strict aerobe During pregnancy, listeriosis is most commonly seen during Presumptive identification for urine isolates: pinpoint, the third trimester; believed to be responsible for NH strict aerobic nonhemolytic, white colonies that have a spontaneous abortion and stillborn neonates & characteristic coryneform microscopic morphology Infection of a neonate with L. monocytogenes is extremely Nitrate negative, rapidly urease positive within serious; fatality rates approach capnophilic minutes 50% if the fetus is born alive. “Perinatal human listeriosis (granulomatosis infantiseptica) Early-onset listeriosis: results from intrauterine infection; Corynebacterium Part of the normal biota of the human nasopharynx most often result in sepsis pseudodiphtheriticum Although infrequent, it is mostly associated with Late-onset listeriosis: occurs several days to weeks after respiratory tract infections in immunocompromised birth; most likely to manifest as meningitis facultative aerobic individuals In immunocompromised hosts, the fatality rate is high. The & Does not show characteristic pleomorphic · most common manifestations are CNS infection and morphology endocarditis capnophilic Reduces nitrate and produces urea Infection of healthy individuals may occur when they eat food contaminated with L. monocytogenes Hoffman bacillus LABORATORY SPECIMEN COLLECTION DIAGNOSIS Diagnosis is often made by culturing L. monocytogenes from blood or CSF Swabs from lesions MICROSCOPY Indirect Smear, it appears as Gram-positive coccobacillus With subculturing, cells become colloidal; whereas older cultures appear gram variable Can resemble Streptococcus when found in coccoid form and Corynebacterium when the bacillus forms prevail Organisms are not usually seen the CSF smear BIOCHEMICAL IDENTIFICATION Catalase-positive Motile at room temperature ○ in wet mount preparation: Tumbling motility (end-over-end motility) when viewed microscopically ○ Motility medium: umbrella pattern at room temperature (22-25 deg Celsius) but not at 35 deg Celsius CULTURAL CHARACTERISTICS Grows well on SBA and chocolate agar, Nutrient agars, BHIB, and Thioglycolate Prefers slightly increased Carbon dioxide Optimal growth temperature: 30 to 35 deg C Can be cultured on McBride’s medium Colony morphology: small, round, smooth & translucent. Narrow zone of beta hemolysis (may be visualized only if the colony is removed) Cold enrichment (4 deg Celsius) may be used to isolate the organism from clinical specimens CAMP reaction: Block type enhanced hemolysis B-hemolytic Bile-esculin hydrolysis positive Hippurate hydrolysis positive Positive CAMP reaction  VIRULENCE TEST LABORATORY Microscopy Anton Test DIAGNOSIS Thin, rod-shaped, gram positive organisms that can form long filaments Arranged singly, in short chains or in a V shape\ E. rhusiopathiae decolorizes easily, so it may appear gram-variable Culture Characteristics Inoculate to nutrient broth with 1% glucose & incubated in 5% CO2 at 35 deg C Can grow on SBA and Chocolate agar Colony morphology: nonhemolytic and pinpoint after 24 hours incubation 2 distinct colony types after 48 hours: (a) smaller, smooth form, transparent, glistening and convex with entire edges; (b) larger, rough colonies that are flatter with a matte surface, curled structure and irregular edges NON-SPORE FORMING, NONBRANCHING CATALASE NEGATIVE BACILLI 1. Erysipelothrix rhusiopathiae 2. Arcanobacterium 3. Gardnerella vaginalis Erysipelothrix rhusiopathiae The only species in the genus known to cause disease in humans Gram-positive, catalase-negative, nonspore forming, Identification pleomorphic rod that has a tendency to form a long Catalase-negative filament Non-motile Found worldwide Pleomorphic human cases typically result from occupational exposure Aerobic or facultative anaerobic Risk factor: individuals whose work involves handling fish Hydrogen-sulfide positive and animal products Urease negative, VP negative, does not hydrolyze esculin MOT: cuts or scratches on the skin Gelatin stab: test tube brush-like pattern at 22 deg Celsius Resistant to salting, pickling and smoking; survives well in environmental sources CLINICAL INFECTIONS Erysipeloid – localized skin disease that resembles erysipelas lesions seen on the hand or fingers; sharply defined, slightly elevated, purplish red zone that spreads peripherally as discoloration of the central area fades incubation period: 2 to 7 days Generalized di use cutaneous infection Arcanobacterium spp Contains nine species; Three of which are considered medically important, including: Gardnerella vaginalis 1. A. haemolyticum (formerly, C. haemolyticum) must be distinguished from C. diphtheriae, C. GENERAL Short, pleomorphic gram-positive rod or coccobacillus that ulcerans and Group A streptococci when CHARACTERISTICS often stains gram-variable or gram-negative recovered from patients 10 to 20 years old with The only species in the genus Gardnerella pharyngitis Found as normal biota in the human urogenital tract. 2. A. pyogenes an animal pathogen that is best known for CLINICAL INFECTIONS Bacterial vaginosis (BV) - a polymicrobial disease; G. vaginalis causing infections in cattle. In humans, it is a rare together with other bacteria such as Prevotella spp., cause of sepsis and wound infection. Peptostreptococcus spp., Porphyromonas spp., Mobiluncus spp., 3. A. bernardiae Atopobium vaginae, and Mycoplasma hominis also a rare cause of infection such as BV is characterized with a malodorous discharge and vaginal pH bacteremia, wound infections, UTIs and septic greater than 4.5 arthritis UTI in men and women DOC: Metronidazole LABORATORY Microscopy DIAGNOSIS Appears as Gram-positive rods, many are pleomorphic and some may demonstrate rudimentary branching LABORATORY Microscopy DIAGNOSIS Pleomorphic, gram-variable coccobacillus or short red; Cultural Characteristics 1.5 to 2.5 um length Colony morphology: small colonies with narrow zone of Can be visualized in wet mounts of vaginal fluid when BV B-hemolysis after 24 to 48 hours on SBA is suspected A black opaque dot is observed on the agar when the Observation of CLUE CELLS – large squamous epithelial colony is scraped away cells with gram-positive and gram-variable bacilli and Identification: coccobacilli clustered on the edges ○ Lipase-positive & Lecithinase-positive NUGENT SCORING SYSTEM –a more accurate means of ○ Reverse CAMP reaction diagnosing BV than cultures. ○ CAMP test positive if S. agalactaie is used in place of S. aureus  Hippurate hydrolysis positive Starch hydrolysis positive HBT agar: Small colonies with B-hemolysis NON-SPORE FORMING, BRANCHING CATALASE NEGATIVE BACILLI 1. Nocardia 2. Other Actinomycetes Nocardia GENERAL Aerobic, branched, beaded gram-positive bacilli CHARACTERISTICS modified acid-fast positive Its colony and microscopic morphology as well as the type of infections caused sometimes resemble those of the fungi. The most commonly encountered species – N. brasiliensis, N. cyriacigeorgica, N. farcinica, and N. nova N. asteroides was considered the most prominent Nocardia human pathogen. VIRULENCE FACTORS Catalase and superoxide dismutase Nocobactin CLINICAL INFECTION Most patient with Nocardia infections have an underlying disease or compromised immune defense; ~10% of Nocardia infection occur in seemingly healthy patients with no obvious immune impairment. Pulmonary infection – occurs from inhalation of the organism present in the dust or soil; The sputum is thick and purulent. Resemble tuberculosis Cutaneous infection – occurs after inoculation of the organism into the skin or subcutaneous tissues. N. brasiliensis is the most frequent cause of this form. Culture Characteristics Usually seen in the hands and feet as a result of Cultures for G. vaginalis are infrequently performed. outdoor activity. Produce a characteristic lesions Often takes longer than 24 hours to develop visible termed actinomycotic mycetomas. As the infection colonies progresses, burrowing sinuses open to the skin surface Grows best in 5 to 7% CO2 at a temperature of 35 to 37 and drain pus. deg Celsius Colony morphology: ○ pinpoint, nonhemolytic colonies on SBA LABORATORY Specimen: Tissue and pus from the draining sinuses ○ on Human blood, Beta-hemolytic, small, gray DIAGNOSIS (for direct examination) and opaque granules can be visualized by separating them from the Medium of choice: Human blood bilayer tween (HBT) pus with an inoculating needle and then washing in agar sterile saline. V agar also contains human blood and is used for the recovery of this organism Microscopy Gram-positive, beaded branching filaments often seen Identification in sputum and exudates or aspirated from skin or Catalase-negative abscesses Oxidase-negative The beaded appearance of Nocardia may be confused as chains of gram-positive cocci, however beads do not  usually touch each other and are not as regular a cocci Filamentous, branching isolate that is partially acid-fast on staining with carbolfuchsin and decolorizing with a weak acid Cultural Characteristic Do not require specific growth factors Grow well on most common nonselective laboratory media Incubate between 22 to 37 deg Celsius; may take 3 to 6 days before growth is seen Colony morphology: a chalky, matte, velvety, or powdery appearance and may be white, yellow, pink, orange, peach, tan, or gray pigmented; dry, crumbly appearance similar to breadcrumbs Identification 1. Substrate hydrolysis – casein, tyrosine, xanthine and hypoxanthine 2. Other substrate and carbohydrate use 3. Antimicrobial susceptibility profile 4. Fatty acid analysis by HPLC Other Actinomycetes 1. Actinomadura 2. Streptomyces 3. Gordonia 4. Tsukamurella 5. Rhodococcus equi 6. Tropheryma whipplei  NON-SPORE FORMING, NON-SPORE FORMING, NONBRANCHING NONSPORE FORMING, BRANCHING NONBRANCHING,CATALASE-POSITIVE BACILLI CATALASE NEGATIVE BACILLI AEROBIC ACTINOMYCETES Corynebacterium Listeria Erysipelothrix Arcanobacterium spp Gardnerella vaginalis Nocardia diphtheriae monocytogenes rhusiopathiae 9 species; 3 medically relevant: - A. haemolyticum - A. pyogenes - A. bernardiae MOT direct contact with cuts or scratches on the inhalation from dust or soil contaminated skin respiratory droplets (coughing or sneezing) VIRULENCE Diphtheria toxin - Listeriolysin O: a none - Lipase and Lecithinase - Polymicrobial association - Catalase FACTOR infected with a hemolysin production in BV with other bacteria - Superoxide dismutase lysogenic B-phage - Catalase (Prevotella spp., - Nocobactin (iron chelator) which carries the tox - Superoxide Peptostreptococcus spp., gene dismutase Porphyromonas spp., - Phospholipase C Mobiluncus spp., Atopobium - Surface protein (p60) vaginae, and Mycoplasma hominis) CLINICAL - Respiratory and -Listeriosis:third-trime - Erysipeloid: localized skin - A. pyogenes: Animal - Bacterial Vaginosis (BV) - Primarily a ects INFECTIONS cutaneous diphtheria ster infection causing disease pathogen, rarely causes - BV: Characterized by immunocompromised patients; spontaneous abortion, - Appears on hands/fingers, sepsis and wound malodorous discharge and 10% in healthy patients stillbirth, and severe with sharply defined, infections in humans vaginal pH > 4.5 - Pulmonary infection neonatal infections; purplish-red zones that - A. bernardiae: Rare - Urinary tract infections -thick, purulent sputum high fatality rates in spread peripherally cause of bacteremia, (UTI) in both men and resembling tuberculosis immunocompromised - Incubation: 2–7 days wound infections, UTIs, women - Cutaneous infection: inoculation hosts -Septicemia: often with and septic arthritis via skin; primarily N. brasiliensis; endocarditis - A. haemolyticum: Must causes actinomycotic - Generalized di use be di erentiated from C. mycetomas, leading to burrowing cutaneous infection diphtheriae, C. ulcerans, sinuses that drain pus and Group A streptococci in pharyngitis cases in ages 10-20 SPECIMEN - Nasopharyngeal swab - Blood or CSF: - Lesions or infected tissue - Throat swabs (especially - Vaginal fluid for wet mount - Tissue and pus from draining (NPS) of inflamed area common specimens from a ected areas in pharyngitis cases) - Preferred method: Wet sinuses - Swab of for diagnosis - Blood cultures for mounts and Nugent scoring - Granules visualized by pseudomembrane in - Swab from lesions bacteremia system (more accurate than separating from pus, washing with respiratory diphtheria - Wound swabs cultures) sterile saline - Dacron swab for cutaneous diphtheria samples MICROSCOPY -highly pleomorphic, -Gram-positive - Thin, rod-shaped, - Gram-positive rods, -Pleomorphic,gram-variable - Gram-positive, beaded Gram-positive bacillus coccobacillus gram-positive organisms often pleomorphic coccobacillus (1.5–2.5 µm in branching filaments (in sputum - Arrangements: - Indirect smears: - Can form long filaments - Some species may show length) and exudates) palisades, picket Gram-variable with - Arranged singly, in short rudimentary branching - Visible in wet mounts - Beads may resemble fences, V & L formations, subculturing chains, or V-shape - Presence of Clue Cells gram-positive cocci but do not club-shaped forms - May resemble - Decolorizes easily, may (squamous epithelial cells touch - Methylene blue: Streptococcus appear gram-variable with gram-positive and - Partially acid-fast with beaded appearance (coccoid form) or gram-variable coccobacilli carbolfuchsin staining Corynebacterium clustered around edges) (bacillus form) CULTURE - facultative anaerobe - grows well on SBA, - Inoculate in nutrient broth - Slow-growing; optimal - Cultures rarely performed, - No specific growth factors CHARACTER - Optimal temperature: CHOC, NA with 1% glucose, incubate in incubation 24-48 hours >24 hrs for colonies needed ISTICS 37°C - Prefers slightly 5% CO₂ at 35°C - Narrow zone of - Optimal growth in 5-7% CO₂, - Grows on nonselective media - Requires essential elevated CO₂ - Grows on SBA and β-hemolysis on SBA 35–37°C - Incubates at 22-37°C; growth amino acids - Optimal temperature: chocolate agar - Medium: Human blood seen in 3-6 days - Medium: grows on SBA, 30-35°C bilayer tween (HBT) agar or V CNA, Loe er, cysteine - Cold enrichment at agar (contains human or potassium tellurite 4°C aids isolation blood) media COLONIAL - CTBA: black/gray - SBA: small, round, - 24 hours: nonhemolytic, - Small colonies, narrow On SBA: pinpoint, non - Chalky, matte, velvety, or MORPHOLOGY colonies with brown halo smooth, translucent pinpoint colonies β-hemolytic zone hemolytic colonies powdery colonies (cystinase activity) colonies - 48 hours: two types: (a) - Black opaque dot seen - On human blood agar: - Color variations: white, yellow, - Tinsdale Medium: - Narrow β-hemolysis smaller, smooth, on agar after colony is small, gray, opaque colonies pink, orange, peach, tan, gray produces four types around colony transparent, glistening, scraped with β-hemolysis - Dry and crumbly, resembling (gravis, intermedius, - CAMP reaction convex with entire edges; breadcrumbs mitis, belfanti) produces block-type (b) larger, rough, flat with a hemolysis matte surface, curled structure, and irregular edges BIOCHEMICAL Catalase (+) - Catalase (+) - Catalase (-) - Lipase (+) - Catalase (-) - Substrate hydrolysis: casein, IDENTIFICATION Nitrate reduction - Motile at room - Non-motile - Lecithinase (+) - Oxidase (-) tyrosine, xanthine, hypoxanthine positive temperature - Pleomorphic - Reverse CAMP reaction -Hippurate hydrolysis (+) - Carbohydrate utilization Urease Negative - Motility test: - Aerobic or facultative - CAMP test positive if S. - Starch hydrolysis (+) - Antimicrobial susceptibility Glucose & Maltose umbrella pattern at anaerobic agalactiae used instead testing fermenter 22-25°C - Hydrogen sulfide (+) of S. aureus - Fatty acid analysis by HPLC nonmotile - B-hemolytic - Urease-negative - Bile-esculin and - Voges-Proskauer (VP) (-) hippurate positive - Does not hydrolyze esculin - Gelatin stab: brush-like pattern at 22°C TOXIGENICITY - In Vivo: guinea pig Anton Test-detects TEST inoculation virulence - In Vitro: Elek Test No specific toxigenicity test (definitive) - ELISA & Immunochromatographi c strip assays - PCR assay for tox gene  SPORE FORMING, NON BRANCHING CATALASE POSITIVE BACILLI General Characteristics Widely distributed in the soil and the environment and one or more additional agents with good CNS Some species are thermophiles → grows best at 55°C or penetration higher Capsule Production: stimulate by incubation in Grows well on SBA; do not grow on Columbia CNA atmosphere containing CO2 Non-pigmented colonies Level A Testing Protocol for the presumptive Confused with Clostridium spp. identification of anthrax → use of PEA agar for Comparison stools suspected to contain B. anthracis, in addition 1. Bacillus spp.: catalase - positive, endospore to SBA and other commonly used media formation on aerobic environment - Confirmation test to rule out B. anthracis: direct 2. Clostridium spp.: catalase - negative, endospore fluorescent antibody assays for a cell wall formation on anaerobic environment - polysaccharide and a capsule antigen Species associated with human infection: B. anthracis A β-hemolytic frosted glass–appearing colony containing and B. cereus spore-forming, gram-positive bacilli that are motile, Koch → development of Koch’s postulate → B. anthracis able to ferment salicin, and lecithinase positive is → anthrax in cattle –? Prove germ theory of disease likely B. cereus & Polypeptide of D-glutamic acid: resistant to hydrolysis B. cereus is biochemically identical to B. thuringiensis unnatural form by host proteolytic enzymes because it is the except that B. thuringiensis, an insect pathogen, “unnatural” form of the amino acid typically produces parasporal crystals → phase Anthrax toxin: individually: non-toxic; together: contrast microscopy or by spore staining synergistic B. cereus non gastrointestinal infection: eye infection → Cmata 1. PA: necessary binding molecule for EF and LF → - endophthalmitis, panophthalmitis, and keratitis attachment to specific receptors on host cell with abscess formation. surfaces and target 2. EF: an adenylate cyclase that increases the concentration of cyclic adenosine monophosphate (cAMP) in host cells → when combined with PA, causes edema 3. LF: a protease that kills host cells by disrupting the transduction of extracellular regulatory signals → when combined to PA, death occurs Cutaneous Anthrax: most common and has the lowest mortality Injectional Anthrax: associated with necrotizing fasciitis, organ failure, shock, coma, and meningitis, and it has a much - higher rate of mortality; not been associated with black - eschar formation Complications: 5% px develop meningitis; greater portion in inhalation and injectional form Treatment: multidrug regimen including a fluoroquinolone  SPORE-FORMING GRAM POSITIVE RODS Bacillus spp. Bacillus cereus group – B. anthracis, B. cereus, B. thuringensis, and B. mycoides (most medically relevant group) Worker Isolated as laboratory contaminants iya other name fungod Sa Blackmanar- Bacteria Type Distinction Morphology Gram Stain Spore Clinical Manifestation Virulence Factor Based On (Carried On Plasmids) Req. ⑧ least Kali ~ Bacillus Aerobic or Possible Large, square - Gram - Round, central spore Cutaneous Anthrax → black eschar Poly-D-glutamic acid capsule anthracis facultative bioterrorism ended positive Characteristic black skin lesion; aerobic anaerobic agent or Observed with a spore malignant pustule Anthrax toxin: Bamboo rods Gram - stain → vegetative Pulmonary Anthrax (Woolsorter’s three component exotoxin ANTHRAY Endospore Most virulent variable cells stain red; spores disease / ragpicker’s disease) → Protective Antigen BACILLUS formation - species; agent stain green Upper RTI-like Edema Factor aerobic of anthrax Gastrointestinal Anthrax Lethal Factor (Plague in (india ink stain) Injectional Anthrax → soft tissue europe) infection (skin popping/injection drug use) Laboratory Diagnosis Treatment Catalase Test Motility Fermentation Medium Lecithinase AST Starch Casein Hydrolysis Hydrolysis Initial: + Non-motile Glucose, Grows in high salt (7% + Susceptible: 10 + + ciprofloxacin sucrose, & NaCl) and low pH (babies - Adheres and colonizes the in adults with chronic ○ May cause disseminated gonococcal infection nasopharyngeal mucosa and enters obstructive pulmonary ○ Incubation period: 2 to 7 days (48 hrs to 1 week) the bloodstream > - which isWhy atl is an disease (COPD) ○ In men: Purulent discharge and dysuria Meningococcemia (Sepsis) 3rd leading cause of acute 3-5% (10% in Mahon) are asymptomatic due to AHU (Arginine, ○ Purpura (hemorrhaging of otitis media and sinusitis in Hypoxanthine, Uracil) strain ~ day maystartMan Sasilhelma ana blood into the skin & mucous children Ascending infections may occur (Prostatitis & Epididymitis) - membranes producing bruises) May cause life threatening ○ In women: common site is the endocervix → result to cervical discharge, ○ Petechial Skin Rash (pinpoint systemic diseases dysuria, lower abdominal pain red spot caused be 50% of women are asymptomatic hemorrhage) Complicates to PID (pelvic inflammatory disease) → sterility, Ectopic ○ Tachycardia, hypotension, and pregnancy, Perihepatitis (Fitz-Hugh-Curtis syndrome) thrombosis Ophthalmia Neonatorum Fulminant form of ○ Eye conjunctivitis of newborn Meningococcemia ○ Gonococcal eye infection ○ Disseminated intravascular ○ Acquired during delivery through an infected birth canal coagulation (DIC) ○ Can lead to blindness ○ Septic Shock Ego , K. & Gu l a, C.  is silver Nitrate any giuse Sanna ↑ ○ In the US, application of antimicrobial eye drops (erythromycin) at birth is ○ Waterhouse-Friderichsen required. syndrome (adrenal gamingo postular / the slin vessels also affects it was Blood dissemination hemorrhage) ○ Occurs in less than 1% of all infections Meningitis ○ Purulent arthritis, septicemia, fever and rash on the extremities ○ Sudden onset of headache, ○ Mostly asymptomatic and have nonspecific symptoms stiff neck, confusion, and ○ Mostly caused by AHU strain and occur in women will stay Not all bacteria sasps photophobia. murag porphyria sa jud ○ In blood culture: Inhibited by SPS Arthritis, pericarditis, pneumonia - mao na careful. - Symptomatic Oropharyngeal Infections (often serogroup Y, affecting older dah BJ pr ○ Pharyngitis is the chief complaint - num individuals with pulmonary issues). lagi dat syn Rectal Gonorrhea traveller jud Rarely, it can cause conjunctivitis New ○ Discharge, rectal pain, or bloody stools may be seen ○ 30% to 60% of women with genital gonorrhea have concurrent rectal infection and urethritis. Sources of Genital sources (urethra in men, endocervix in women and swab 4 to 5 cm in anal Recovered from CSF, blood, Collected from middle ear Specimen, canal) nasopharyngeal swabs or aspirate, effusion, nasopharynx, sinus Host and Mode From other sites (rectum, pharynx, joint fluid) joint fluids, sputum, urogenital and aspirates, sputum aspirates, of In men: when no apparent discharge, swab 2 cm into anterior urethra and rotate rectal sites due to oral-genital or bronchial aspirates Transmission Humans are the only natural host contact Mode of transmission: Close contact of infected person via respiratory droplets Humans are the only known host - againreal e diphtheria Specimen Use Dacron or Rayon swabs (calcium alginate and cotton inhibit N. gonorrhoeae) Inhibited by SPS Collection and Direct plating to gonococcal-selective media preferred (prevents drying/temperature Transport effects) Neizeria for Con purposes - Swab should be rolled in a “Z” pattern on the medium Transport Guidelines (if direct plating is not possible): ○ Use Amies medium with charcoal and transport to lab promptly ○ Plate within 6 hours of collection Noizeria ○ Other transport medium: JEMBEC plates, Gono-Pak, Transgrow (contain selective media and CO₂ atmosphere) Direct Appear as kidney-shaped pairs with flattened adjacent sides Intracellular and extracellular Appears as intracellular Microscope Examination extra Gram Stain mascen To tests s Avirulent (lack ability to cause disease) forms ○ Extracellular, gram-neg diplococci lacking pili gram-negative diplococci with flattened adjacent sides gram-negative diplococci in specimens and extra ○ Recommended for urethral discharge and other urogenital specimens insid intra & extra extra intra Ego , K. & Gu l a, C. -  normal flora many too ○ Presence of commensal Neisseria spp. can interfere with results (not > recommended for pharyngeal specimens) that is mas preserved any urogenial - why ○ >5 polymorphonuclear neutrophils per field without bacteria suggests nongonococcal urethritis (banala nag daghan PMN bassa wald lay bacteria Possible organisms: C. trachomatis or Ureaplasma urealyticum F >although - bacteria pud Culture and Medium of choice: CHOC Agar for fastidious - sila , they just difficult to are observe Media used: si meningitidis Grows on Sheep BAP and Incubation ○ Used as a primary plating medium to recover vancomycin-sensitive strains of ○ Sheep BAP ~ less fastidione CHOC agar same as mening; - gonococci ? BAP alone hemin not are ○ CHOC Agar Colonies are smooth, Not enough nutrients sa , Does not grow well on Sheep BAP why released they as not lysed are ○ For specimens from mucosal opaque, gray-to-white Use of selective media necessary to prevent overgrowth of commensal organisms surfaces with normal flora, use Described as “hockey ○ Vancomycin (inhibits gram-positive bacteria) selective media (as with N. puck” colonies (remain W ○ Colistin (inhibits gram-negative bacteria) gonorrhoeae) to suppress intact when pushed with a ○ Antifungal agents (suppress yeast) contaminants. loop) ↑ ○ Trimethoprim (prevents Proteus spp. swarming) Incubation: Same atmosphere as for Older colonies may have a Can be differentiated by other bacteria on selective media by catalase and oxidase N. gonorrhoeae (humidified with “wagon-wheel” tests 5% CO₂) appearance Incubation: 35 deg C in a 3-5% CO2 up to 3 days Examined daily for 72 hours Can grow at 28°C (tolerates CO₂ Sources: lower temperatures) ○ CO₂ incubator ○ CO₂ generating pouch ○ Candle extinction jar: 7 base- ker nama o Inhibited by colistin on gonococcal-selective agar, but some resistant strains Use white, unscented candles (scented or colored candles may inhibit may grow growth) Oxidase- and Candle burns until oxygen drops, generating ~3% CO₂ catalase-positive (similar to Provision of sufficient humidity they love humid environmentTCO ↑ humid = Neisseria spp.) ○ Moisture evaporating from the media in a closed jar environment ↑ humidity V optical > - ~ humidity control ○ CO2 incubator with humidifier > - ○ Pan of water place at the bottom of the incubator Colonies Small, gray to tan, translucent, and raised after 24-48 hours of incubation Visible within 18 to 24 hours Colony Types: LitERALLY SMALL BUT TERRIBLE On SBA: Medium-sized, gray, ○ T1 and T2: piliated, virulent, smaller & raised, appear bright in reflected light convex colonies; encapsulated ○ T3 to T5: Nonpiliated, avirulent, larger, flatter colonies strains may appear mucoid, with a ○ AHU Colonies: Smaller colonies and grow slowly greenish tinge underneath. Fresh culture is recommended for workup since the organism can produce their own On selective agar: Colorless to autolytic enzyme like strep Pneumoniae. gray, convex, and smooth colonies. Presumptive Oxidase Test Oxidase Positive Test to differentiate from Ego , K. & Gu l a, C. Identification ○ Filter Paper Method: Neisseria: Place oxidase reagent (1% - dimethyl- or tetramethyl-p-phenylenediamine ○ Grows on Nutrient Agar dihydrochloride) on filter paper ○ Fails to utilize CHO in => Rub a colony onto the reagent using an applicator stick or a non-nichrome loop the CTA medium (Asaccharolytic → does Purple color develops within 10 seconds not ferment sugars) ○ Direct Plate Method: ○ DNase positive Drop oxidase reagent directly onto the colony Colony turns deep purple to black Organisms become nonviable, so subculture must be done before applying ○ Butyrate esterase reagent reaction positive Superoxol Test ○ Reagent: 30% Hydrogen peroxide ○ N. gonorrhoeae – vigorous bubbling ○ N. meningitidis & N. lactamica – weak delayed bubbling Definitive Carbohydrate Utilization Test CHO utilization (positive for Identification ○ Uses Cystine Trypticase Agar (CTA) medium Glucose and Maltose) ○ pH indicator: Phenol Red ○ 1% of individual carbohydrates gm Glucose G one is Maltose which gond Sucrose Lactose ○ - Yellow color indicates acid production in 24 to 72 hours as BOTULINUM ○ N. gonorrhoeae: Positive for glucose only same Rapid Carbohydrate Degradation Tests: ○ Results in 2 to 4 hours (faster than CTA) ○ Require pure cultures ○ Weak glucose acid production by certain strains of N. gonorrhoeae Other Tests Chromogenic Substrate Methods: Differentiation from N. lactamica: ○ Example: Gonochek II (EY Laboratories) Perform an ONPG test (positive for ○ Detect bacterial enzymes that hydrolyze colorless substrates to colored end N. lactamica) products - for - N gonorrhoeae - GLUCOSE ONLY (B-galactosidase ○ Identifies Neisseria spp. with aberrant carbohydrate utilization Multitest Methods ○ Multitest conventional chromogenic enzyme method ○ Combine enzyme substrate tests with other biochemical tests · Ego , K. & Gu l a, C.  ○ Can be used for isolates from selective or nonselective media ○ Identifies Neisseria spp. and other genera like Haemophilus Immunologic Assays ~ PORcoded geor ○ Employ monoclonal antibodies against gonococcal protein I (only produced ○ Do not require pure or viable organisms by gono ○ Can be done from the primary plates ○ Not approved for use on clinical specimens ○ Positive reaction: agglutination Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrometry (MALDI-TOF MS) ○ Emerging Technology ○ Microbial proteins are separated based on size and charge in an electric field - - ○ Detects unique protein signatures of the organisms ○ Allows for identificatio of pathogens in minutes - Nucleic Acid Amplification Tests (NAATs) ○ Preferred assay for detecting N. gonorrhoeae due to high sensitivity and specificity ○ Detects nucleic acid sequences through amplification (e.g., PCR) or signal amplification after probe binding ○ Does not require viable organisms ○ Use of noninvasive urine specimens allows for self-collection ○ Can detect both N. gonorrhoeae and C. trachomatis in one sample ○ Not recommended for: & cancausenongnocal nephritise is n Child sexual assault cases involving boys seen Extragenital infections (rectal, oropharyngeal) in prepubescent girls Auxotyping Procedure ○ Auxotypes: require specific nutritional factor in artificial media for growth to occur ○ Requires AHU strain Penicillin Disk Test: ○ Streak organism on a plate with a 10-unit penicillin disk ○ After growth, stain the edge of the inhibition zone to observe morphology ○ Helps confirm diplococci vs. gram-negative rods syn aRed so not anymore ↓ soHose Antimicrobial Previously susceptible to Penicillin Meningitis: Treated with penicillin Most isolates produce Resistance Penicillinase producing Neisseria gonorrhoea (PPNG) resistant to penicillin Meningococcemia: Use β-lactamase, making them (Treatment) Plasmid-mediated: β-lactamase production causing penicillin resistance third-generation cephalosporins resistant to ampicillin and Chromosomal-mediated: Altered penicillin-binding proteins (β-lactamase Chemoprophylaxis: Recommended amoxicillin negative) for close contacts with rifampin or Effective Antimicrobial Gonococcal Isolate Surveillance System (GISP): ciprofloxacin; azithromycin for Agents: Ego , K. & Gu l a, C.  ○ Monitor antimicrobial resistance and guide treatment resistant cases ○ Amoxicillin-clavulanic ○ Resistance observed for tetracycline, spectinomycin (1981), and Quadrivalent vaccines (A, C, Y, acid fluoroquinolones (2007) W-135): ○ Extended-spectrum Current Recommended Treatment: Cephalosporins (Ceftriaxone & Cefixime Plus ○ Recommended at 11-12 years cephalosporins Azithromycin) with a booster at 16 years. ○ Azithromycin, Serogroup B vaccine: quinolones, ○ Recommended for 16-23 years trimethoprim-sulfamet old, offering short-term hoxazole protection. ○ Administered as two- or three-dose series for those at high risk of group B infection. Additional Gonorrhea (Means: “Flow of Seed”) Use a biosafety level 2 cabinet due Information ○ 2nd to Chlamydia trachomatis in the number of confirmed sexually transmitted to aerosol infection risk bacterial infections in the United States. Open-bench handling is not ○ Also called “the clap,” from the French word clapoir meaning “brothel” recommended Commensals,Saprophyte,Non Pathogenic Neisseria spp. Normal inhabitants of the upper respiratory tract ; occasionally isolated from the genital tract; commensal Neisseria spp. rarely cause disease. ORGANISM N. cinerea N. lactamica N. N. flavescence N. mucosa N. sicca N. subflava N. elongata N. waeveri polysaccharea Habitat Nasopharynx Nasopharynx of Nasopharynx of Pharynx (not Nasopharynx of Respiratory tract Upper respiratory Nasopharynx -Normal oral infants and infants and often isolated children or of adults. microbiota microbiota in children children from clinical young adults dogs specimens) - Humans ( dog bite wounds) Description Small (1–1.5 mm), Small, grayish Small, gray Colonies similar Large, often Dry, wrinkled, N. subflava Rod shaped Small, grayish white, white (often with a (sometimes to N. meningitidis adherent to the adherent, and means “less semi-opaque with translucent, raised yellow ring), yellowish), but with golden agar, and very breadcrumb yellow” -Large (up to 3 smooth with entire edge translucent, translucent, yellow pigment mucoid like mm), grayish appearance