Vibrio Species Identification Guide PDF

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T. Rodriguez VIBRIO (COMMA/ CURVED BACILLUS) o Classical = VP ( ); do not agglutinate chicken RBC, susceptible to polymixin B (50µg) It is not part of the human flora. o El tor = VP (+); agglutinate chicken RBC; resistant It is found in brackish or estuarine water, and marine to polymixin B (50µg). water or salt water. Potent enterotoxins: cholera toxin(CT), zot toxin and ace It is temperature sensitive (> 20OC) and It can be Isolated toxin from algae, plankton, fish and shellfish. Virulence factor: choleragen (cholera toxin) It is facultative anaerobic; monotrichous organism. String test: + (mucoid "stringing" reaction) It is a halophilic organism (require the addition of sodium Antigenic structures: somatic O and flagellar H for growth) except V. cholerae and N. mimicus (V.cholerae subgroups) Microscopy: Gram-negative short, curved rod, TSI reaction: A/A, ( ) gas, ( ) H2S. asporogenous Culture: CLINICAL MANIFESTATION AND PATHOGENESIS: o smooth, opaque, Iridescent with a greenish hue CHOLERA CAP o It Is an acute diarrheal disease that is spread o NLF Mac Conkey (except V. vulnificus LF) mainly through contaminated water. o BAP o It is acquired from ingesting Improperly Susceptibility test: 150-µg Vibriostatic disk on MHA or TSA preserved food like seafood (shellfish), milk ice (01/29) cream. Mode of acquisition: consumption of raw or undercooked o hallmark of cholera: rice-watery stool (10-30x of seafood defecation/day) Disease/ Infection: cholera, wound Infection, septicemia o cause of outbreak: Improperly handled and and necrotizing fasciitis preserved seafood, milk, ice cream and meat Species: V. cholerae, V. vulnificus, V. parahemolyticus, V. CHOLERAGEN alginolytlcus, V.metschnikovil, V. hollisae o is a protein composed of two functional units, an Common Isolates: V. cholerae 01 and non-01, V. enzymatic A subunit and an Intestinal receptor- parahaemolyticus, V. vulnificus and V. alginolyticus binding B subunit. o The A subunit enters the Intestinal epithelial cells BIOCHEMICAL TESTS: and activates the enzyme adenylate cyclase by Oxidase (+) and reduce nitrates to nitrite except V. the addition of an ADP-ribosyl group in a way metschnikoviI. similar to that employed by diphtheria toxin. Glucose fermenter, NLF except V. vulnificus (causes the o As a result, choleragen stimulates hypersecretion most severe disease) of water and chloride ions while inhibiting Motility test: absorption of sodium ions, leading to massive o polar sheated flagella (broth) fluid loss (10-15 liters) and electrolytes. o peritrichous, unsheated flagella (solid media) o All antigens are poorly immunogenic, so repeat infection occur. VIBRIO CHOLERAE o Motility and chemotaxis mediate the distribution It is the causative agent of cholera/ asiatic cholera/ of organisms. epidemic cholera (V. cholerae 01). o TCP pili provides the attachment to mucosal cells It has rapid darting or shooting-star motility. for cholera toxin release. The single flagellum Is covered with lipopolysaccharide o Mucinase production allows for the penetration sheath. of the mucosal layers. It has caused Cholera epidemics (O1 and 0139 strains) and seven, pandemics (01 strains). VIBRIO PARAHAEMOLYTI CUS Culture: smooth, medium to large colonies with a Is the second most common Vibrio species Implicated In greenish hue (BAP). gastroenteritis. V. cholerae subgroups: V. cholerae 01, V. cholera 0139 It is the etiologic agent of "summer diarrhea" in Japan. and V. cholerae non-O1 O3:K6'- a pandemic strain; it V. cholerae 01 serotypes: Ogawa (A, B), Inaba (A, C) and has been implicated in numerous food-borne outbreaks. Hikojima (A, B, C) Epidemic V. cholerae 01 biogroups: Classical and El tor apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 93  Çlïñïçål Båçtërïøløgÿ Non-Enteric Gastrointestinal Pathogen | M. T. Rodriguez Mode of acquisition: eating contaminated seafood like STRING TEST 6.5% NA DESOXYCHOLATE oyster, scallops, crabs, lobsters and shrimps, and even It differentiates Vibrio spp. from Aeromonas spp. sardines (+) result: lysis of cells (Vibrio) - releases DNA, which can Virulence factor: heat-stable hemolysin then be pulled up into a string (viscous string) using an Pathogenicity: Kanagawa phenomenon (hemolysin lyses Inoculating loop human RBCs) Selective medium: Wagatsuma agar (high-salt mannitol VIBRIOSTATIC TEST (SUSCEPTIBILITY TEST) medium) O/129 (2,4-dlamino-6,7-diisopropylpteridine) Strains of this organism which can lyse human RBCs is It is used to separate vibrios (susceptible) from other known as Kanagawa toxin positive. oxidase-positive, glucose fermenters like aeromonads (resistant). VIBRIO VULNIFICUS This test uses 150 µg Vibriostatic agent O/129. It was commonly referred to as the -positive" It will also differentiate V. cholerae O1 and non-O1 Vibrio. (susceptible) from other Vibrio spp (resistant). It is second to V. cholerae in terms in producing serious type of Vibrio-associated infection. BIOCHEMICAL TEST Infections: primary septicemia and wound infections LIA: alkaline/alkaline Mode of acquisition: eating raw oysters and fish (Tilapia). (+) Citrate; yellow colonies on TCBS = V. cholerae (+) Indole V. cholerae, V. mimicus and V. vulnificus VIBRIO ALGINOLYTICUS (+) cellobiose V.vulnificus It is the least pathogenic Vibrio for humans; not commonly API 20E best method for Vibrio spp. isolated. It Is a strict halophile 1% to 10% NaCl SEROLOGICAL TESTS It can be an occupational hazard (fishermen and sailors). Strains that phenotypically resemble V. cholera but fail to Related Infections: eye, ear and wound infections agglutinate in O1 antisera are referred to as V. cholerae (extraintestinal infections) non-O1. V. parahemolyticus can also be serotyped by means of Its LABORATORY DIAGNOSIS : O and K antigens. Specimen: stool, rectal swab, pus and tissue Vibrio species should be collected and transported only in AEROMONAS Cary-Blair medium. It is found in fresh water, estuarine and chlorinated water; isolated from meat products. GRAM STAIN It is not part of the human flora; facultative anaerobic. Gram-negative, straight or slightly curved rods "curved It Is responsible for a variety of diseases among warm- and or comma-shaped bacilli" cold-blooded animals including fish, reptiles and amphibians - it Is CULTURE MEDIA (frogs) Culture media: TCBS, alkaline peptone water, Cary-Blair, In humans, it causes as nebulous syndrome. known as the Mac Conkey and BAP "travelers-diarrhea" similar to ETEC. Growth of vibrios requires media containing 0.5% NaCl Microscopy: Gram-negative straight rods except V. cholerae and V. mimicus. Culture: V. alginolytics tolerates up to 10% NaCl. o bulls eye colonies apron-like pattern/-CIN (with 4 Pathogenic vibrio grows as NLF on Mac Conkey agar. µg cefsulodin) Alkaline peptone water with 1% NaCl (pH 8.5) can be o large, round, raised, white and opaque colonies inoculated (at least 20 mL) and incubated for 5 to 8 hours BAP at 35oC before subculturing to TCBS. o LF (Mac Conkey A.caviae) Sucrose fermenters (yellow colonies on TCBS): V. Biochemical tests: oxidase and catalase (+); glucose cholerae, V. alginolyticus and V. metschnikovii fermenters; motile with single polar flagellum but some Non-sucrose fermenters (green colonies on TCBS): V. species are non-motile mimicus, V. vulnificus, V. parahemolyticus and V. damsel It can typically grow from 4O to 42OC. It will grow in media with 0% NaCl but not in 6% NaCl. apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 94  Çlïñïçål Båçtërïøløgÿ Non-Enteric Gastrointestinal Pathogen | M. T. Rodriguez Extraintestinal infections: septicemia, meningitis, CAMPYLOBACTER JEJUNI keratitis and wound infections It is the most common cause of bacterial gastroenteritis 2 GROUPS: worldwide. It is acquired from eating contaminated chicken and 1. MESOPHILIC GROUP A. hydrophila complex, A. veronii turkey (does not multiply in food). complex and A. caviae complex 2. PSYCHROPHILIC GROUP A. salmonicida (fish pathogen); It invades the epithelium of the small intestine, causing non motile group; grows best at 22° to 25°C. inflammation. It also secretes a toxin that is antigenically similar to the Most common isolates: A. caviae cholera toxin. Common Isolates in GI infections: A. caviae It also causes septic arthritis (AIDS). Common Isolate in HUS: A. hydrophila and A. veronii It is slow growing, fastidious and asaccharolytic; darting motility; unable to grow in 3.5% NaCl. A. hydrophila ("water lóving") causes gastroenteritis and cellulitis Optimum growth: 42°C Vibriostatic 01/29 test: resistant (aeromonads and Microscopy: curved or seagull-winged shaped plesionomads) Infective dose: Inositol fermentation: + (plesiomonads are negative) CAMPYLOBACTER FETUS SUBSP. FETUS Indole (+): A. caviae, A. hydrophila and A. veronii It has been Isolated most frequently from blood cultures TSI: (37°C incubation) and is rarely associated with o A/A, ( ) gas , ( ) H2S = A. caviae gastrointestinal illness. o A/A, (+) gas, (+) H2S = A. hydrophila and A. veronii LABORATORY DIAGNOSIS: CAMPYLOBACTER Specimen: feces, rectal swab and blood It is motile by a single polar flagellum; non-spore forming. Campylobacter spp. that causes enteric illness are isolated It grows in 5-10% oxygen (microaerophilic) except C. from stool samples. rectus and C. carvus (both strict anaerobe); oxidase (+). Rectal swab Is a less-preferred specimen for the isolation It is the most recognized antecedent cause of Guillain- of the enteric Campylobacter. Barre syndrome many patients with GBS are tested positive for antibodies to Campylobacter. MICROSCOPY It is also an animal pathogen (cattle and swine) causing Recommended counterstain: carbolfuchsin sterility and abortion. If safranin is used as a secondary stain, it should be applied Microscopy: for 2-3 minutes. o faintly staining Gram-negative, small, curved or Hanging Drop preparation: (+) darting motility S-shaped rod To observe the typical motility, organisms should be o may appear as coccobacilli-old cultures suspended in Brucella or TSB. o long spirals or seagull-wing shaped (enteric campylobacters) CULTURE Culture: Selective media: CAMPY-BAP, Butzler agar and Skirrow's o gray, flat, glistening, irregular, with a "tailing media and charcoal cefoperazone desoxycholate agar effect along the streak line" or "runny spreading" (CCDA) colonial growth Transport medium: Cary-Blair medium Mode of acquisition: ingestion of contaminated water, In blood culture, 2 weeks incubation may be needed for poultry and dairy products handling pets like dogs, birds detection-turbidity is not often visible, so blind cultures and cats (occupational hazard) sexually transmitted may be necessary. Species: C. jejuni, C. coli, C. Iari, C. fetus subsp. fetus, C. Campylobacter is detected effectively by the CO2 sputorum, C. concisus, C. curvus and C. rectus monitoring. Enteric campylobacters: C. jejuni, C. coli, and C. lari EIA is used for direct detection of C. jejuni and C. coli in fecal specimens. C. jejuni are susceptible to the nonspecific bactericidal activity of normal human serum. apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 95  Çlïñïçål Båçtërïøløgÿ Non-Enteric Gastrointestinal Pathogen | M. T. Rodriguez HELICOBACTER Transport media: Stuart's media, cysteine-brucella broth w/20% glycerol (tissue samples) and Isotonic saline with It is found in the GIT of mammals and birds. 4% glucose It is motile (monopolar or multi-bipolar flagella); May require more than 5 days incubation; capnophillc microaerophilic. environment. Most species have strong urease activity. The presence of hemin stimulates further growth. Microscopy: Gram-negative spiral-shaped organisms (S- shaped) rods resembling campylobacter OTHER TESTS: Culture: gray with translucent colonies Urea Breath test-excellent sensitivity and specificity Biochemical test: oxidase and catalase (+) PCR-also sensitive method for detection of H. pylori Transmission: oral-oral and fecal-oral routes H. pylori is susceptible to metronidazole. Species: H. pylori, H. cinaedi, H. fennelliae, H. rappini (formerly Flexispira rappini) weeks) sheep blood, and incubated at microaerophilic HELICOBACTER PYLORI condition and read after 3 days It is the major cause of type B gastritis, peptlc ulcer and Differential Diagnosis gastric carcinoma. Biochemical/ Susceptibility Primary habitat: human gastric mucosa-mucous layer of C. jejuni H. pylori Test the antrum and fundus of the stomach but does not Catalase Test + invade the gastric epithelium; It colonizes the stomach for Nitrate Reduction + v a long. Urease + Motility allows this organism to escape acidity of the H2S Production (TSI) stomach. Hippurate Hydrolysis + Urease enzyme plays a significant role in the survival and Indoxyl Acetate Hydrolysis + growth by creating an alkaline microenvironment Susceptibility to generates ammonium from urea, thus neutralizing gastric Resistant Susceptible Cephalothin (30mcg) acid. Susceptibility to Nalidixic It binds to Lewis antigen (part of the blood group antigens) Susceptible Resistant acid (30mcg) and to the monosaccharide sialic acid O Growth at 15 C Biochemical test: a strong urease producer; oxidase and Growth at 25OC catalase (+) Growth at 42OC + v Routes of transmission: oral-oral route, fecal-oral route V variable HELICOBACTER CINAEDI Enteric Pathogens: It has been Isolated from blood of patients with o Salmonella bacteremia and patlents with HIV. o Shigella o Yersinia LABORATORY DIAGNOSIS o E. coli 0157: H7 o P. shigelloides urine (ammonia testing), feces and dental plaque o Vibrio Tissue specimens should be maintained at 4'C and o Aeromonas processed within 2 hours of collection. o Campylobacter GRAM STAIN 0.1% basic fuchsin counterstain enhances morphology. stains for biopsy specimens: Warthin-Starry, Silver stain or Giemsa stain CULTURE Culture media: CAP, MTM, Skirrow agar, 5% sheep's blood Brucella agar apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 96  Çlïñïçål Båçtërïøløgÿ Non-Fermentative Gram-Negative Bacilli | M. T. Rodriguez NONFERMENTATIVE GRAM-NEGATIVE BACILLI Culture: flat spreading pigmented (metallic sheen) fruity "grape-like" or "corn tortilla" Opportunistic pathogens; environmental bacteria; not odor BAP usually found as normal flora of the human body. It is acquired thru ingestion of contaminated food or They can be Isolated from nebulizers, dialysate fluids, water, exposure to contaminated medical devices; saline, catheters and other hospital devices. penetration of wounds. It can withstand treatment with chlorhexidine and It can exist in distilled water and chlorinated water; hot quaternary ammonium compounds. tubs and contact lens solution; disinfectant and whirlpools It can be isolated from soil, plants, water, vegetables, Virulence factors: endotoxin, pill, exotoxin A (cellular foodstuff and hospital surfaces. damage), capsule, lecithinase, elastase and protease, alginate and pill BIOCHEMICAL TEST: They are motile except B. mallei; oxidase (+); NLF. PIGMENTS: They fail to acidify oxidative-fermentative (OF) media Pyoverdin yellow-green or yellow-brown plgment when it is overlaid with mineral oil or fail to acidify TSI agar Pyocyanin blue (only produced by P. aeruginosa) - no acid production in the slant or butt of TSI or KIA. Pyorubin red Do not ferment CHO by enzymatic reaction but by Pyomelanin brown or black oxidative method produce very weak acids as end products. CLINICAL SIGNIFICANCE: Some group members oxidize carbohydrates to derive It is the leading cause of nosocomial respiratory tract energy for their metabolism (oxidizers) while others do infections. not break down carbohydrates at all (non-oxidizers). Hugh and Leifson medium oxidative and fermentative It is the 3rd most common cause of Gram-negative (OF) medium with 1% carbohydrate and bromthymol blue bacillary bacteremia. indicator. It is causative agent of ecthyma gangrenosum, swimmer's OF medium result: yellow (+) in the open tube and ear, hot tub or Jacuzzi syndrome (necrotizing skin rash), green/blue-green (-) in the closed tube infections of the nail beds and lung infections in cystic TSI reaction: K/K;H2S(-) fibrosis patients. PSEUDOMONAS It is the 3rd most common cause of hospital-acquired The most commonly isolated non-fermentative bacilli. infection - displays intrinsic resistance to a wide variety of Their metabolism is respiratory and never fermentative. antimicrobial agents that facilitates the organism's ability They can be found in cosmetics, swimming pools, hot- to, survive in the hospital setting. tubs, inner soles of sneakers. Microscopy: straight and slender Gram-negative rod DISTINGUISHING CHARACTERISTICS Biochemical test: catalase and oxidase (+); NLF; able to (+) Gluconate production survive with few nutrients such as acetate and glucose (+) Arginine dihydrolase (ADH) They are obligate aerobes, non-spore forming and motile Grow at 42OC with polar flagella. (+) Acetamide and citrate utilization They usually grow on MacConkey agar. Fluorescent Pseudomonads: P. aeruginosa, P. PSEUDOMONAS FLUORESCENS AND PSE UDOMONAS fluorescens, P. putida, P. veronii, P.montellli and P.mosselli PUTIDA They have been Isolated from contaminated blood PSEUDOMONAS AERUGINOSA (AGENT OF "BLUE PUS") products, cosmetics, hospital equipment, urine and The most commonly isolated species of the genus in respiratory specimens. clinical specimens. They can grow at 4°C and have been linked to transfusion- The most commonly encountered Gram-negative associated septicemia. bacterium that Is not a member of the They can produce acid a form xylose (like P. aeruginosa). Enterobacteriaceae. Differential test: Gelatin hydrolysis (P. fluorescence) (+) It has the ability to Invade the vascular walls of blood vessels, which facilitates its spread in the body. It is non-spore forming, monotrichous organism. apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 97  Çlïñïçål Båçtërïøløgÿ Non-Fermentative Gram-Negative Bacilli | M. T. Rodriguez LABORATORY DIAGNOSIS : o A. baumanii glucose-oxidizing; non-hemolytic strains (common isolate) GRAM STAIN o A. lwoffi glucose-non oxidizing; non hemolytic strains (common isolate) Straight and slender Gram-negative rod o A. hemolyticus glucose-non oxidizing; CULTURE hemolytic strains Culture media: BAP, CAP, Mac Conkey, Seller's medium, Cetrimide Agar (Cetrymethyl Ammonium Bromide), STENOTROPHOMONAS MALTOPHILLA Irgasan (2, 2, 2 trichloro-2- hydroxydiphenylether) and It is the 3rd most commonly Isolated non-fermentative Gram- C390 (9 chloro, 9 diethylaminophenyl-10 phenylacridan) negative bacilli. BAP spreading and flat with serrated edges; bluish-green It has been isolated from plant materials, water, milk, frozen red or brown pigmentation food and sewage. Cetrimide agar is a differential and selective medium; it It can contaminate blood-drawing equipment and enhances the pigment production (pyoverdin and disinfectants. pyocyanin) of P. aeruginosa. The grape-like or corn taco-like odor; mucoid colonies contaminated blood collection tubes. (commonly seen in patients with cystic fibrosis) are due to It is strictly aerobic; non-motile with growth at 42°C. large production of alginate, a polysaccharide that Maltophilla surrounds the cell. maltose and not glucose). Microscopy: short to medium size, Gram-negative straight Good growth at 42°C. rods BIOCHEMICAL TEST Culture: o lavender-green to light purple pigment with TSI reaction: alkaline slant/neutral butt (K/N) or K/K BAP Oxidase (+); H2S (-) o brown pigment BH infusion agar with tyrosine P. montellii can be distinguished from P. putida by its Biochemical test: inability to oxidize xylose. o catalase, esculin and gelatin hydrolysis (+) o DNAse and LDC (+); oxidase (-); NLF PHAGE TYPING Antimicrobial tests: broth microdilution and E-test This test allows the identification of bacteria by testing Related Infections: endocarditis, bacteremia and wound their vulnerability to bacterial viruses (bacteriophage). infections. ACINETOBACTER BURKHOLDERIA Is a member of the family Moraxellaceae. It is not considered part of normal human flora; generally It is the 2nd frequently Isolated non-fermenters. non-pathogenic. It has been isolated from hospital equipment (catheters, It is aerobic and non-spore forming; motile (polar flagella) humidifiers and ventilators), foodstuff, soil and water. except B. mallei. They can appear as gram-positive bacilli in smears made It involves human contact with heavily contaminated from blood culture bottles they have tendency to resist medical devices. alcohol decolorization. Microscopy: medium-sized Gram-negative straight rods Microscopy: plump, Gram-negative coccobacilli; appear in Biochemical test: catalase and oxidase (+) pairs It grows well on BAP and CAP. Culture: Species: B. cepacia, B. pseudomallei, B. mallei, B. gladioli o purplish colonies Mac Conkey agar BURKHOLDERIA CEPACIA o gummy colonies BAP Biochemical test: strict aerobic and non-motile; catalase It is a low-grade nosocomial pathogen. (+), oxidase (-) It has been Isolated from Irrigation fluids. anesthetics, They have optimum growth at 30° to 35°C and a pH 5.5- nebulizers, detergents and disinfectants. 6.0. It grows well on most culture media but might lose Related Infections: UTI, pneumonia, endocarditis, viability on BAP in 3 to 4 days. meningitis and cellulitis It grows on MacConkey agar. Species: Culture: apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 98  Çlïñïçål Båçtërïøløgÿ Non-Fermentative Gram-Negative Bacilli | M. T. Rodriguez o Non-fluorescing and non-wrinkled yellow or o small, opaque, whitish (BAP); yellow-green colonies BAP o NLF; non-saccharolytic Biochemical test: Biochemical test: catalase and oxidase (+); PAD (+) o It has a weak, slow, positive oxidase reaction Species: o (+) LDC and ONPG o O. urethralis non motile; urease (-) and oxidase Related Infections: pneumonia in patients with cystic (+) fibrosis o O. ureolytica motile; urease and oxidase (+) BURKHOLDERIA MALLEI MORAXELLA LACUNATA (MORAX-AXEMFELD Agent of Glander's /Farcy disease-severe Infections in BACILLUS) horses and donkeys. It is the agent of Blephanoconjunctivitis/ Angular It is a potential bioterrorism agent. Conjunctivitis It is the only non-motile member of the genus. It is part of the normal human flora that inhabit mucous Oxidase production Is variable; Coccobacillus; non- membranes - nose, throat, other parts of URT, pigmented colonies. conjunctiva. It has variable growth in Mac Conkey agar. Microscopy: Gram-negative coccobacillary to bacillary Culture: BURKHOLDERIA PSEUDOMALLEI growth on Mac Conkey Agent of meloidosis- a glander's-like disease. Biochemical test: catalase and oxidase (+); nonmotile; It has been isolated in muddy soil, streams and surface strict aerobe water such as rice paddies. It can survive within phagocytes-"Vietnamese time CHROMOBACTERIUM VIOLACEUM bomb." Is the only member in the genus Chromobacterium. It is motile with polar tuft of flagella. It is an opportunistic pathogen, affecting the Microscopy: bipolar staining Immunocompromised patient with neutrophil deficits. Culture: dry, wrinkled, deep pink colonies; "earthy It is a "fermentative" Gram-negative bacillus that can be odor"(Ashdown medium with colistin) oxidase positive It ferments glucose and variably Mode of acquisition: through inhalation of contaminated sucrose. debris or direct It is motile with polar flagella. Inoculation through damaged skin or mucous membranes They grow on Mac Conkey agar at 42°C. Microscopy: may appear as curved bacilli ALCALIGENES FECALIS Culture: violet pigmented colonles (violacein pigment) Obligate aerobic Gram-negative bacilli; motile by peritrichous flagella. OTHER NON-FERMENTATIVE GRAM-NEGATIVE It grows well on Mac Conkey agar. BACILLI: It has been isolated in soil and water, including moist Flavobacterium, Spingobacterium, Achromobacter, hospital environments. Shewanella and Comamonas Infection is acquired thru exposure to contaminated medical devices and solutions. Culture: o feather-edged non-pigmented colonies BAP o other colonies may be surrounded by zone of green discoloration o -hemolytic; "fruity odor resembling apples or strawberries" Biochemical test: (+) growth in 6.5% NaCl; oxidase (+); NLF OLIGELLA It may colonize the distal urethra. Microscopy: small, paired Gram-negative bacilli or coccobacilli Culture: apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 99  Çlïñïçål Båçtërïøløgÿ Small Pleomorphic Gram-Negative Bacilli | M. T. Rodriguez HAEMOPHILUS 2 CATEGORIES: TYPEABLE - based on capsular characteristics The genus name Is derived from the Greek words meaning o Types a, b, c, d, e, f (capsular serotypes) "blood lover." encapsulated strains They normally inhabit the URT of humans except H. o Type b the cause of serious infections in ducreyi In adults It is mostly consists of H. humans; leading cause of meningitis in parainfluenzae. unvaccinated children; anti-phagocytic and anti- Are obligate parasites on the mucous membranes of complementary; it is composed of sugar-alcohol humans. phosphate (polyribitol phosphate). Are fastidious and non-motile; non-sporeforming; o Other related infections: septicemia, arthritis, capnophilic and facultative anaerobic bacteria. epiglottitis, tracheitis, osteomyelitis, and They die rapidly in clinical specimen - very susceptible to pneumonia drying and extreme temperature. NON-TYPEABLE Microscopy: Gram-negative, small, pleomorphic, o Are normal inhabitants of URT- adheres to coccobacilli or rods human epithelial cells. Culture: o It causes otitis media with effusion (middle ear o moist, smooth, convex colonies on agar plate infection) the 2nd prevalent etiologic agent BAP after S. pneumoniae. o most species will not grow on pure BAP o It also causes pneumonia in elderly patients and Biochemical test: catalase (+); and oxidase (+) except H. individuals with underlying respiratory Infections segnis. including cystic fibrosis/ Growth factors: X (hemin) and V (NAD). o Do not produce capsule (non-encapsulated Human pathogens: H.influenzae, H. ducreyi, H. strains). parainfluenzae, H. paraphrophilus, H. parahaemolyticus, o Other related infections: conjunctivitis and H. pittmanlae, H. aegypticus, and H. segnis sinusitis localized infections HAEMOPHILUS INFLUENZ AE HAEMOPHILUS DUCREYI It is transmitted by person to person by contaminated is not part of human flora; only found in humans during respiratory droplets. infection. It may be mistaken for S. pyogenes if the gram stain is not It infects mucosal epithelium, genital and non-genital skin, well decolorize. and regional lymph nodes. -lactamase Is the agent of chancroid or "soft chancre" - a highly production. communicable sexually transmitted genital ulcer disease. It is the main cause of meningitis in children - the organism Culture: spreads from the nasopharynx to the regional lymph o transparent, small, flat, smooth, non-mucoid, tan nodes to the blood and finally to meninges. or yellow colonies (CAP) Culture: o saline o translucent, convex, tan mucoid colonies with suspension o mousy or bleach- Suppurative, enlarged, draining, inguinal lymph nodes Principal virulence factor: polysaccharide capsule (buboes) are common in the majority of infected patients. (serotypes a-f) It is commonly seen in socioeconomically disadvantaged Other virulence factors: lgA proteases, fimbriae, OMPs population. and LPS Chancroid-genital lesions; from tender papules to painful Not all strains are encapsulated non-encapsulated ulcers with several satellite lesions. strains are part of the normal microbiota of the URT. The only member of the genus that produce lgA protease. HAEMOPHILUS AEGYPTIC US (KOCH-WEEKS BACILLUS) Do not produce endotoxin; rapidly killed by phagocytes; Is genetically related to H. influenzae. very fastidious. It was observed in conjunctivitis exudates from Egyptians ( ) Porphyrin test this test detects the presence of by Koch in 1883. It is the etiologic agent of pinkeye conjunctivitis. porphyrins. apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 100  Çlïñïçål Båçtërïøløgÿ Small Pleomorphic Gram-Negative Bacilli | M. T. Rodriguez HAEMOPHILUS INFLUENZ AE BIOGROUP AEGYPTI CUS CULTURE (NON-ENCAPSULATED) Culture media: CAP (X and V factors), thioglycollate, BHI It causes conjunctivitis primarily in pediatric populations. Rabbit or horse blood agar is preferred for observing It is the etiologic agent of Brazilian purpuric fever (BPF). hemolysis. Horse blood is preferred than sheep's blood for testing Infection/ because the latter contains growth Inhibiting factors Common Distinguishing Species GF Disease Name Characteristics Associated sheep red cells release NADase which Inactivates NAD Mousy/ bleach- Meningitis, present in the medium. X, H. influenzae bacillus like odor; non- V Epiglottis, The lysing of the RBCs by heat in the preparation of CAP hemolytic Arthritis releases both the X and V factors and Inactivates NADases. Koch- X, Pink-eye H. aegypticus Most strains will not grow on 5% sheep's BAP (hemin Weeks V Conjunctivitis H. influenzae X, Brazilian only). biogroup aegypticus V purpuric fever Most Haemophilus strains require X and/or V factors in X, culture medium. H. haemolyticus -hemolytic V Both X and V factors are found within RBCs, however only H. ducreyi School of fish X Soft chancre X factor is directly available. bacillus Tannish and V-factor producing bacteria S. aureus, S. pneumoniae H. parahaemolyticus - V and Neisseria spp. hemolytic Mannose V-factor dependent Haemophilus (H. influenzae) will grow H. parainfluenzae V as "satellites" around colonies producing the NAD. fermentation Lactose and All clinically significant Haemophilus require V factor H. paraphrophilus mannose V fermentation except H. ducreyi. GF growth factor They grow best between 35°-37°C (except H. ducreyi - 33°C) and in a 5%-10% CO2. LABORATORY DIAGNOSIS: H. aegypticus requires 4 days of incubation while H. Specimens: CSF, sputum, genital lesion/ulcer, joint fluid, ducreyi requires 7 days. vaginal swab, abscess drainage, swabs from conjunctivae, When Haemophilus spp. are grown anaerobically they do bronchial washing and blood CSF samples should be not require heme, only the NAD. centrifuged. No growth occurs on Mac Conkey Agar. Haemophilus spp. are very susceptible to drying and SELECTIVE MEDIA FOR ISOLATION OF HAEMOPHILUS temperature extremes immediate transport and SPP. processing are vital for their Isolation. For recovery of H. ducreyi, the ulcer should be cleaned Horse Blood - Bacitracin Agar selective medium for H. with sterile gauze pre-moistened with sterile saline. influenzae for respiratory secretions of patients with cystic fibrosis Cotton swab pre-moistened with phosphate-buffered saline for collection of ulcerative material (H. ducreyi), CAP with 1% IsoVitaleX or Vitox H. aegypticus and direct plating at the bedside is preferred instead of GC agar base with 2% bovine hemoglobin + 5% fetal calf using transport media. serum on one side and MHA with 5% heated horse blood on the other side and 3 mg/L vancomycin (Nairobi biplate GRAM STAIN medium) H. ducreyi Microscopic: PORPHYRIN TEST (DELTA-AMINOLEVULINIC ACID o Small, Gram-negative, pale pink coccobacilli to TEST) long filament o Pale staining arranged singly or in groups Is a test for differentiating the heme-producing species of ks" or "fingerprints" H. Haemophilus. ducreyi is a means for establishing the organisms' x-factor It can resemble the "amorphous serous material" because requirements and eliminates the potential problem of of its pleomorphic appearance. carryover. Capsules of H. influenzae may be observed in direct Principle: it is based on the ability of the enzyme to smears as clear, non-staining areas ('halos") surrounding convert the substrate delta- aminolevulinic acid (ALA) into the organisms in purulent secretions. apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 101  Çlïñïçål Båçtërïøløgÿ Small Pleomorphic Gram-Negative Bacilli | M. T. Rodriguez porphyrins or porphobilinogen, intermediates in the AGGREGATIBACTER ACTINOMYCETEMCOMITANS synthesis of X factor It was isolated with Actinomyces in a polymicrobic Hemophilus species that need x-factor are unable to infection. synthesize porphyrin from 6- ALA. It is divided into 6 serotypes (a-f) based on its surface It can be performed on agar, in broth or on a disk. polysaccharides. Reagent: Kovac's reagent (p-dimethyl It is a recognized etiologic agent in -development-of aminobenzaldehyde) periodontitis. End product: It has been Isolated from blood, lung tissue, abscesses of o Porphobilinogen red color the mouth and brain, and sinuses. o Porphyrins reddish-orange (UVL 300nm) Virulence factors: collagenase and leukotoxin (+) result: red color H. parainfluenzae, H. Culture: "star shaped" with 4-6 points in the center of parahaemolyticus, H. paraphrophllus, H. aphrophilus (A. colonies after 48 hours aphrophilus) The addition of serum into the medium is necessary to ( ) result: H. influenzae, H. hemolyticus, H. aegypticus, H. demonstrate carbohydrate fermentation. ducreyi Urease ( ) = which differentiates it from Actinobacillus SEROLOGICAL TEST spp. Serotype can be determined through identification of the CARDIOBACTERIUM HOMI NIS distinct capsular antigen through latex agglutination, It infects the aortic valve more frequently than the other capsular swelling or immunofluorescence HACEK organisms. Neufeld Quellung reaction rapid direct Identification of It shows "false Gram-positive" reactions in parts of the the capsular antigen of H. influenza cells. Microscopy: HACEK GROUP o "rosette" formation, swellings and filamentous o Haemophilus spp. BAP o Aggregatibacter aphrophilus (formerly H. o - yeast extract aphrophilus) Culture: "pitting" may be seen o Aggregatibacter actinomycetemcomitans EIKENELLA CORRODENS (CORRODING BACILLI) o Cardiobacterium hominis o Eikenella corodens It causes mixed infection from bites or clenched-fist o Kingella spp (K. kingae HACEK species) wounds. Are small, non-motile, non-spore forming, Gram-negative It causes cellulitis among users of abused drugs - by licking bacilli that will not grow on Mac Conkey agar. the needle. Are part of the normal oral flora; opportunistic pathogens. It is the least common Isolate of the HACEK group in adult infectious endocarditis. Are fastidious and require increased CO2 at least for initial isolation from clinical specimens. They are assacharolytic like the species of Moraxella. They have slow growth (dysgonic) on BAP and CAP (7-14 Culture: yellow colonies; pit or corrode the agar with days); require an additional 1-2 days before they can be may adhere to the sides of the tube Isolated from blood cultures. and produce granules (broth) They caused slow, progressive bacterial endocarditis Biochemical test: ("vegetation"). o Lysine and decarboxylase (+); o arginine dehydrolase ( ) They utilized 6-aminolevulinic acid; all are Indole ( ) except C. hominis. KINGELLA They have tendency to resist decolorization. AGGREGATIBACTER APHR OPHILUS ("FOAM LOVING") Microscopy: Gram-negative plump rods to coccobacilli It is the most prevalent HACEK specles causing with squared ends in pairs or short chains endocarditis. They may grow on gonococcal media (TMA), and may It is isolated from dental plaque and gingival scrapings. resemble N. gonorrheae if the isolate does not pit the agar (as many strains do). It is V-factor dependent. Culture: raised, convex, granular, yellowish apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 102  Çlïñïçål Båçtërïøløgÿ Small Pleomorphic Gram-Negative Bacilli | M. T. Rodriguez Species: K. kingae (most pathogenic), K. oralis, K. Direct inoculation into the bloodstream through abrasion denltrificans in the skin or vaccination. K. kingae is the major Gram-negative bacterium isolated UNDULANT FEVER from degenerative joint and bone Infections is characterized by normal temperatures in the morning (osteoarthritis) in children younger than 3 years old. followed by high temperatures in the afternoon and K. denitrificans might grow at 42°C; superoxol ( ). evening. Other Species C O G M S L LABORATORY DIAGNOSIS: Tests V Factor Specimens: blood, bone marrow, tissues A. aphrophilus V + + + + ( ) X and V it should be handled under biosafety level 3 cabinet due A. actinomyctemcomitans + V + + to aerosol mode of transmission. Factor C. hominis + + + + Indole (+) GRAM STAIN Ornithine E. corrodens + (+) Carbol fuchsin should be substituted for safranin O to K. kingae + + + Nitrate ( ) improve gram stain. CULTURE BRUCELLA (BANG'S BACILLUS) Culture media: BAP, trypticase soy agar Are Important human and animal pathogens - infect Brucella spp. can change from smooth to rough colonies human through contact with infected animals and animal based on the composition of their cell wall products (milk). lipopolysaccharide (LPS). is a category B select biological agents It cause moderate B. abortus requires niacin (nicotinic acid) for growth, but morbidity but low mortality. can be Inhibited by thionine dye. Are strict aerobes, Intracellular-parasites; class III Isolates can be recovered after 7 days, but may require pathogens. prolonged Incubation up to 30 days (culture bottles may It is localized in tissues rich in erythritol (placental tissue) not become turbid). induce spontaneous abortion among animals. BacT/Alert, BACTEC 9240 faster and sensitive detection It is often recovered from blood and bone marrow. of B. melitensis. Are non-motile, non-encapsulated; some species require Castaneda medium (biphasic medium) and TSB Isolation CO2 for growth. of organisms from blood and bone marrow. Microscopy: small, coccobacillary; singly, in pairs or short OTHER TESTS chains; Serum Agglutination Test (SAT) Culture: small, convex, smooth, translucent, non- o 1:160 titer hemolytic, slightly yellow; colonies may become brownish o B. suis ( ) or not detected by SAT with age CO2, H2S production, urea hydrolysis, dye sensitivity, Biochemical test: catalase and oxidase (+); rapid urease phage sensitlvity tests for Brucella spp. identification. producers; assacharolytic Disease: Malta/Crimean/Mediterranean fever or DIFFERENTIAL DIAGNOSIS OF BRUCELLA SPECIES undulant fever (brucellosis) B. B. Species: B. abortus, B. canis, B. suis, B. melitensis Species B. abortus B. melitensis canis suis (common isolate) Goat or Natural Hosts Cattle Dogs Swine Most virulent species: B. melitensis and B. suis sheep It is closely related to Bartonella, Rhizobium and 5-10 % CO2 ± Agrobacterium. H2S production (Lead Acetate + + PRIMARY ROUTES OF HUMAN INFECTION Production) (BRUCELLOSIS): Urease Test + V + + Ingestion of unpasteurized and contaminated milk or Inhibition by cheese from infected animals. + + Fuschin Inhalation of-organisms (animal-carcasses) -aerosol Inhibition by + + infection. Thionine Penetration of ocular or oral mucosa. apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 103  Çlïñïçål Båçtërïøløgÿ Small Pleomorphic Gram-Negative Bacilli | M. T. Rodriguez BORDETELLA After attachment, the bacteria produced several toxins, the most Important of which is the pertussis toxin, which Are obligate aerobic, Gram-negative fastidious causes increased tissue susceptibility to histamine and coccobacilli; non-carbohydrate fermenter. serotonin, and increased lymphocyte response. Are non-motile (except B. bronchiseptica); and with Pertussin toxin also inhibits intracellular signal bipolar metachromatic granules. transduction factors by transferring ADP ribose to G They replicate on ciliated respiratory epithelial cells of protein of cells. humans. Culture: smooth, glistening, silver in color becoming SYMPTOMATIC STAGES: whitish-gray with age CATARRHAL STAGE Biochemical test: o mucous membrane inflammation, mild cold and o Catalase (+); Indole (-) cough with runny nose; high communicable stage o Oxidase (+) = B. parapertussis, B. bronchiseptica PAROXYSMAL STAGE and B. avium o severe and violent coughing (15-25x/24hrs) B. parapertussis is also citrate (+). o associated with vomiting and whooping ("hurried Are mostly inactive to biochemical test systems. deep respiration) Growth factors: nicotinic acid, cysteine and methionine o It may last for 6 weeks Species: B. pertussis, B. parapertussis, B. bronchiseptica CONVALESCENT STAGE and B. avium o symptoms slowly decrease; can last for as long as Virulence factors: pertussis toxin, adenylate cyclase, 6 months after infection tracheal cytotoxin and dermonecrotic toxin, pertactin and fimbriae LABORATORY DIAGNOSIS FOR B. PERTUSSIS: Specimen: nasopharyngeal swabs (Calcium alginate or BORDETELLA PERTUSSIS (BORDET GENGOU BACILLUS) Dacron swab) and bronchoalveolar lavage B. pertussis It is the etiologic agent of whooping cough. Nasopharyngeal swabs should be plated directly onto It only causes disease in humans. culture media or transferred to a suitable transport It requires special collection and transport systems. medium at the bedside. It contains a protective antigen but when combined with antibody, it abolishes its infectivity. GRAM STAIN It does not survive well outside the host. Use of a 2-minute safranin or 0.2% basic fuchsin as Culture: small, shiny colonies resembling "mercury drops" counterstain enhances their visibility. Growth inhibitors: fatty acids, metal ions, sulfides and peroxides. CUTURE Culture media should include "growth protectors" such as Culture media: Regan Lowe, Bordet-Gengou Potato charcoal, blood or starch. Infusion Agar, Modified Jones Kendrick charcoal Virulence factors: pertussis toxin (protein toxin), Regan Lowe agar (charcoal agar + 10% horse blood + filamentous hemagglutinin, tracheal toxin and pertactin cephalexin) transport and enrichment medium; for Specimen for Isolation: nasopharyngeal swab specimens requiring overnight or several day transport. Bordet-Gengou Potato Infusion agar full strength CLINICAL INFECTION: charcoal + 10% horse blood + 40 mg/L cephalexin with methicillin or cephalexin. WHOOPING COUGH (PERTUSSIS) Modified Jones-Kendrick charcoal also contains yeast Is highly contagious, acute Infection of (upper respiratory extract + cephalexin. tract) URT; disease of the children. Plates are Incubated at 35°C without elevated CO2 for 7 It is acquired through the respiratory tract via the aerosol days. route (inhalation of the bacterium). B. pertussis and B. parapertussis are hemolytic on Bordet- It has no known animal reservoir or vector has been Gengou agar. found. Other Bordetella species are less fastidious and will grow Incubation period: 7-14 days on Mac Conkey or media containing blood B. Once inside the URT, the bacteria produced adhesins bronchiseptica. called the filamentous hemagglutinin for attachment to ciliated epithelial cells. apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 104  Çlïñïçål Båçtërïøløgÿ Small Pleomorphic Gram-Negative Bacilli | M. T. Rodriguez TRANSPORT SYSTEM: Disease: tularemia/ deerfly fever/ rabbit fever/ lemming If the transit time is less than 2 hours, the swab can be fever/ water rat trappera's disease placed into a solution of 1% casein hydrolysate (casamino TULAREMIA broth). Amies transport medium with charcoal is appropriate up is a zoonotic disease which can be acquired through to 24 hours. ingestion, inhalation, arthropod bite or contact with infected tissues. SEROLOGIC TESTS LABORATORY DIAGNOSIS: Agglutination test requires a larger quantity of bacteria, so subculture from the primary isolation plate is sometimes specimens: scrapings from infected ulcers, lymph node necessary. biopsies and sputum PCR is a rapid test for Bordetella spp. best specimen: lymph node biopsy PCR assays are regarded as more sensitive than cultures and GRAM STAIN DFA assays. Clinical specimens can only be examined for Bordetella spp. It requires acridine orange stain to visualize organism in a (+) microscopically using DFA staining. blood culture bottle Slides for DFA testing may be prepared directly from swab specimens or following expression of the material from the CULTURE swab to a solution of 1% casein hydrolysate. Culture media: CAP, MTM, non-selective BCYE, MHA and TSB Growth is not enhanced by CO2. DIFFERENTIAL DIAGNOSIS OF BORDETELLA SPECIES Slowly growing organisms require 2-4 days for colony Bordetella Bordetella Bordetella formation Species pertussis parapertussis bronchiseptica It will not grow-on Mac Conkey agar. Nitrate + Reduction PASTEURELLA (ZOONOTIC BACTERIA) Oxidase Test + + Urease Test + + It is the etiologic agent of "shipping fever" In cattle. Motility Test + It is a commensal In the URT of fowl and mammals. BAP Growth + + It is isolated from animal bite (felines) or scratch wounds. It is facultative anaerobic; non-motile Microscopy: small, straight Gram-negative bacilli with FRANCISELLA TULARENSIS safety pin appearance (bipolar staining); non-hemolytlc Is a category A select biological agent-are easily disseminated Biochemical test: oxidase (+) and catalase(+); indole (+) with high mortality rate and posing a risk to national security. weak glucose fermenter It is considered as a potential bioterrorism weapon it should It is phenotypically similar to the Actinobacillus spp. be processed following the biosafety level 3 conditions. It grows well on BAP and CAP but not on Mac Conkey agar. Is a very small, obligate aerobic, coccobacillus; extremely Virulence foctors: endotoxin and capsule invasive. Species: P. multocida, P. stomatis, P. dagmatis, P. bettyae It is non-motile and facultative Intracellular parasite (WBC). and P. canis Microscopy: Gram-negative bacilli with "faint bipolar staining" PASTEURELLA MULTOCID A Culture: round, smooth blue-gray to white, slightly mucoid It is the most frequently isolated species. colonies It has characteristic "mushroom smell" Growth factors: cysteine or cystine, and thiosulfate It grows only on BAP; susceptible to penicillin Biochemical test: o catalase weakly (+); oxidase (-) Biochemical test: oxidase, ornithine decarboxylase and o citrulline uridase (+); glycerol fermenter Indole (+); urease and ONPG (-) Serological test: agglutination tlter of 1:40-diagnostic value PASTEURELLA BETTYAE Reservoir: cottonball rabbit Mode of acquisition: by handling Infected animal carcasses It has been Isolated from amniotic fluid, placenta, blood and or skin of infected animals through insect vector (deerflies urogenital specimens. and ticks); being bitten by carnivores or by inhalation Glucose and fructose fermenters; nonmotile. Virulence factor: capsule Biochemical test: Catalase and indole (+); oxidase (+/-) It may grow on Mac Conkey. apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 105  Çlïñïçål Båçtërïøløgÿ Small Pleomorphic Gram-Negative Bacilli | M. T. Rodriguez LEGIONELLA o Mode of transmission: Inhalation of infectious aerosols (1-5mm) airborne spread The only genus in the family Legionnellaceae. o At risk of pneumonia are immunocompromised It is primarily acquired through inhalation. patients, older than age 50 and heavy smokers and It is fastidious, aerobic and motile. alcoholics. It can Infect and multiply within some free-living amoebae o Symptoms: high fever, nonproductive cough, (Hartmanella, Acanthamoeba and Naegleria spp.), ciliated headache, neurological, and severe protozoa (Tetrahymena spp.) and biofilms. bronchopneumonia. It is capable of surviving at extreme ranges of environmental o Diagnosis depends on isolation of bacteria, rise in conditions for long periods in aquatic sources such as lakes, antibody titer or antigen testing urine samples. rivers, hot springs and mud. it has the ability to adhere to pipes (even when flushed), LABORATORY DIAGNOSIS: rubber, plastics and sediments. preferred specimens: sputum and bronchoalveolar lavage It can tolerate up to 3 mg/L of chlorine-resists water other specimens: urine pleural fluid, blood, and lung, treatment. transbronchial and biopsy material It will not grow on routine primary plating media (BAP). Urine is an important specimen to be collected for antigen Microscopy: faintly staining, thin, Gram-negative bacilli detection. Culture: colonies appear Irldescent with sticky consistency Primary medium: BCYE supplemented wlth L-cysteloe STAINING buffered to.pH 6.9 Microscopy: Faint staining and not usually detectable by Biochemical test: catalase and oxidase weakly(+); Gram stain-extend the counterstain to 10 minutes to gelatinase(+) intensify the staining of cells Major reservoirs: hot water systems, cooling towers and L. micdadei is weakly acid fast with the modified Kinyoun evaporative condensers method. Species: L. pneumophila, L micdadei (Pittsburg pneumonla agent), L. bozemani (Wiga agent) and L. dumoffi CULTURE Culture is the most Important test for LEGIONELLA PNEUMOPHI LA Selective medium: Buffered charcoal yeast extract (BCYE) Is the most commonly Isolated human pathogen in the genus with L- -ketoglutarate Legionella. Semi-selective BCYE with polymyxin B, anisomycin and either It is part of the natural microbial community of soil and vancomycin or cefamandole is used for heavily- aquatic ecosystems. contaminated specimen. It is Isolated in air conditioning ducts, cooling towers, warm- Acid treatment (KCI-HCI) of contaminated specimens water plumbing system, humidifiers, nebulizers (man-made enhances Isolation of the bacteria. facilities), ponds and creeks. Saline or buffer should not be used in processing or It has the ability to Invade the bronchoalveolar macrophages transporting of specimens because of the inhibitory effect of (facultative intracellular pathogen), survive and multiply, sodium to Legionella. producing localized tissue destruction through export of a It can be isolated from Bactec or BacTAlert blood culture cytotoxic exoprotease. bottles. Colony morphology: grayish-white or blue-green, glistening Plates are incubated for 7 days before discarding. convex colonies central portion has "ground-glass" appearance (BCYE) SEROLOGIC TEST Indirect Fluorescent Antibody (IFA)-is the most common PATHOGENESIS: method used for the also known as legionellosis, is a disease. febrile and pneumonic illness. RAPID METHODS 3 PRIMARY CLINICAL MANIFESTATIONS: URINE ANTIGEN TEST can be detected as early as 3 days PNEUMONIA Legionnaire's disease of the infection PONTIAC FEVER nonfatal respiratory infection; resembles DIRECT FLUORESCENT ANTIBODY TEST for detection of an allergic disease; pneumonia does not occur. the common Legionella spp. from the lower respiratory WOUND ABSCESSES, ENCEPHALITIS tract o No person-to-person spread of infection. DNA DETECTION apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 106  Çlïñïçål Båçtërïøløgÿ Aerobic Gram-Positive Bacilli | M. T. Rodriguez BACILLUS Virulence factors: D-glutamic acid capsule and protein exotoxins (edema factor, protective antigen and lethal Are spore forming, rod shaped organisms; can be isolated from soil. factor) They can be aerobic or facultative anaerobes - but only form endospores aerobically. VIRULENT FACTORS: It is motile (peritrichous) except B. anthracis and B. D-GLUTAMIC CAPSULE mycoldes. o It protects the organism from phagocytosis - this Microscopy: resistant to hydrolysis by most proteolytic enzymes. o large, boxcar-shaped Gram-positive cells o The genes that code for the toxin and the enzymes o unstained spores are clear "empty spaces" responsible for capsule production are carried on Biochemical test: catalase (+) and ferments glucose; plasmids. starch hydrolyzers o Antibodies against the capsule do not confer They can survive in temperatures as low as -5°C and as immunity. high as 75°C. PROTEIN EXOTOXINS (EF, PA AND LF) They can survived in extreme environmental conditions o These exotoxins are individually nontoxic but act due to endospores. together to produce damaging effects. Bacillus cereus group is the most clinically significant o Edema results from the combination of PA and EF, species B. anthracis, B. cereus, B. thuringlensis and B. while death occurs when PA and LF combine. mycoides. CLINICAL INFECTIONS BACILLUS ANTHRACIS Humans acquire infections when they are Inoculated with the Anthrax bacillus/ causative agent of anthrax spores, either by inhalation during exposure to contaminated It is not part of normal human flora. animal products, such las hides, or by traumatic introduction (through breaks in the skin or mucous membranes). It will grow aerobically or anaerobically. Person-to-person transmission has not been documented. its endospores can remain viable 1n soil and animal The protein exotoxins are responsible for signs and products for decades. symptoms of anthrax. It is the most likely biologic agent to be used as a weapon Anthrax a disease affecting primarily farm animals; animals feeding on plants contaminated with the spores. of mass destruction, not highly contagious-the centerpiece for counter terrorism planning efforts. 3 TYPES OF ANTHRAX: It is non-motile and a halophilic organism (7% NaCl). CUTANEOUS ANTHRAX/ MALIGNANT PUSTULE Microscopy: o It is acquired thru skin cuts and abrasions. o Gram-positive large, encapsulated and square- o a small pimple or papule appears at the site of spore inoculation 2-5 days after exposure. ended rod o appearance of "black eschar" - black necrotic o unstained central spore "bamboo fishing rod" central area or malignant pustule and it Is painless appearance and does not produce pus. Culture: o flat, irregular w/ swirling projections-"medusa o It is acquired when spores are inhaled into the pulmonary parenchyma. head" colonies BAP o It resembles an upper respiratory tract infection. o non hemolytic; "beaten egg white" appearance o It may progress to chest edema, cyanosis, coma and BAP eventually death. It grows in low pH (10°/mL if It is supplemented with 1%Tween 80 the predominant isolate or > 10°/mL if it is the sole isolate. apmmrmt Bacteriology Handbook for Medical Technologists 2012 Rodriguez | 111  Çlïñïçål Båçtërïøløgÿ Aerobic Gram-Positive Bacilli | M. T. Rodriguez DIFFERENTIAL DIAGNOSIS OF CORYNEBACTERIA SPECIES DISEASE IN PREGNANT WOMEN CTBA It usually occurs during the 3'd trimester. Species U NP EH G Halo It causes spontaneous abortion and stillborn neonates C. diphtheriae + + (stillbirth). C. jeikeium C. ulcerans + +* + Signs and symptoms: flu-like illness, fever, headache, C. pseudotuberculosis + V + myalgia C. urealyticum + C. pseudodiphtheriticum + + DISEASE IN THE NEWBORN U Urease; NP - Nitrate Production; EH Esculin It is associated with intrauterine infection (early onset) Hydrolysis; G Gelatinase; *room temperature; v due to aspiration of infected amniotic fluid. variable Affected infants are full term and "healthy" at birth (late- onset). LISTERIA It leads to meningitis. LISTERIA MONOCYTOGEN ES DISEASE IN THE IMMUNOCOMPROMISED HAST Through the ingestion of contaminated cheese, coleslaw, It is both human and animal pathogen; also Isolated from ice cream, hot dogs, processed meat and chicken. crustaceans, ticks and flies. It is recovered from soil, dust, water, sewage, silage, dairy LABORATORY DIAGNOSIS: products and processed meats. Specimens: blood, swabs of lesions and CSF It is motile with the characteristic tumbling motility It is commonly isolated from blood and CSF. It is aerobic or facultative anaerobic, non-spore forming; (+) catalase. MOTILITY TEST It can grow in high salt concentration (up to 10% NaCl). (+) end-over- (wet mount/hanging It has the ability to survive within phagocytes (Intracellular drop) at room temperature pathogen). (+) mbrella- or inverted Christmas tree It has optimal growth between 30OC and 37OC but also pattern (SIM) at 25°C but not at 35°C grows at 4OC. Microscopy: Gram-positive coccobacillus, in singly or CULTURE short chains resembling streptococci; sometimes may Culture media: BAP, CAP, thioglycollate and BHI, Mc Bride appear as Corynebacteria when the bacillus forms prevail Agar, Nalidixic Acid Medium Culture: small, smooth, translucent grayish-blue colonies It may be confused with group B streptococci-colonies and surrounded by a narrow -hemolysis BAP hemolytic pattern. virulence factors: listeriolysin O (hemolytic and cytotoxic), Growth occurs at wide temperature range between 0.5°C- catalase, SOD, phospholipase C and p60 (surface protein) 45°C. Cold enrichment technique (4°C) is used to enhanced CLINICAL INFECTIONS: recovery from clinical specimens (placental tissues) - Ingestion of contaminated food such as meat, chicken, inoculation of specimens into broth at 4°C for several dairy products and coleslaw. weeks. Colonized mothers may pass