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Systematic bacteriology Neisseria and Haemophilus

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Systematic bacteriology Neisseria and Haemophilus Powered By Docstoc
					Culture and Identification of Fastidious Bacteria
Lecture prepared by Dr. M. Watts

Objectives
Become familiar with (yet more) key tests based on bacterial metabolism to identify important Gram negative cocci and GN cocco-bacilli Learn about fastidious pathogens Haemophilus spp and Neisseria spp

Genus: Haemophilus
Respiratory organisms Blood loving small Gram negative, nonmotile cocco-bacilli Facultative & capnophilic harbored in adult URT First isolated in 1892 secondary to infection by flu virus

Normal flora & Pathogens
Normal flora H. parahaemolyticus H. haemolyticus H. parainfluenzae True pathogenic species

H. influenzae H. aegyptus (biotype of H. influenzae) H. ducreyi

H. influenzae
can occur as normal flora of the nose and pharynx Some encapsulated Six capsule types (a,b,c,d,e,f) type b species are clinically significant

Most of infections occur in unvaccinated children <5 yo Why?

H. influenzae type B (HiB) and Human Disease
pharyngitis Epiglottitis airway obstruction in children between the ages of 2 and 4. meningitis most frequent in 1 month to 2 olds; 90% serotype B usually secondary to nasopharyngeal infection fever, nausea, vomiting, diarrhoea; coma; cardiovascular collapse highly contagious, especially in close quarters

Was the most common cause of bacterial meningitis in kids between 5 mo – 5y

Severe pediatric disease

Meningitis, fever, Headache, Stiff neck
Permanent hearing loss in ~20% survivors

Effective Vaccine available

Virulence factor: polysaccharide capsule (PRP) Capsular ags conjugated to protein 90 % efficiency

Vaccine effective

Other pathogenic Haemophilus sp
H. aegyptius H. influenzae biogroup aegyptius conjuctivitis Brazilian purpuric fever (BPF) life-threatening pediatric infection H. ducreyi Chancroid genital ulcers common Asia and Africa (Syphilis: chancre)

Isolation and ID: H. influenzae
Direct antigen tests
latex agglutination EIA CIE
CIE

Antiserum agar shows halos of precipitation
H.Flu antiserum agar
D L Ingram, A M Collier, E Pendergrass, and S H King: J Clin Microbiol. 1979 May; 9(5): 570–574

Culture: Fastidious
require growth factors: X (haematin) and V (NAD) factors provided by heated blood chocolate agar

possible satelliting on blood around S.aureus

Culture: Mueller-Hinton agar

Streak plate with enough H. influenza to form a lawn growth only around the X&V filter

HAEMOPHILUS: Satellitism
Marginal growth occurs on BA as factors can leak out (X significant, V minimal) S. aureus produces V factor and releases X factor by hemolyzing blood Haemophilus colonies will form small colonies called "satellites" in the haemolytic zone around Staphylococccus colonies

"Staph streak" technique.
In this procedure, both Staphylococcus and Haemophilus organisms are grown together on blood agar
Satellitism procedure
H. influenzae heavily streaked on a BAP a single narrow streak of a hemolytic Staphylococcus known to produce NAD (factor V) is made through the area where the H. influenza had been inoculated using an inoculating wire plates incubated in a candle jar at 37C for 24 hours.

Differentiation of Haemophilus spp
H. influenzae requires both X and V

H. influenzae biotype aegypticus
requires X and V factor

Other Haemophilus species require only factor V (NAD+)

Porphyrin synthesis test
Haemin is a porphyrin H. parainfluenzae
has porphobilinogen synthase can convert ALA (aminoaevulinic acid) to protoporphyrin and then haemin

H. influenzae – H. parainfluenzae +

PROCEDURE
ALA tubes inoculated with a suspension of 3-5 similar colonies taken from an O/N Choc plate Incubated at 35º C with 5% CO2 for 4-24 hours. equal volume of Kovac's added Positive: reddish coloration of the lower part of tube after 2- 5 min Negative results: no change in color

Test on a disc Which org is this?

H. influenzae Lab ID: Summary
Tiny gram negative coccobacilli Nonmotile and non-spore forming Like increased CO2 Must have chocolate!! Need both Hemin (X factor) and NAD (V factor) Or satellite around staph on BA Facultative anaerobes Reduce nitrates to nitrites oxidase and catalase positive

Neisseria
FAMILY NEISSERIACEAE NEISSERIA MORAXELLA KINGELLA ACINETOBACTER

Neisseria Species:
Neisseria animalis; Neisseria bacilliformis; Neisseria canis; Neisseria cinerea; Neisseria dentrificans; Neisseria dentiae; Neisseria elongata; Neisseria flava; Neisseria flavescens; Neisseria iguanae; Neisseria macacae; Neisseria meningitidis; Neisseria gonorrhoeae; Neisseria lactamica; Neisseria mucosa; Neisseria perflava; Neisseria pharyngis; Neisseria polysaccharea; Neisseria sicca; Neisseria subflava; Neisseria weaveri; Neisseria sp.

Neisseria spp
PATHOGENS N. gonorrhoeae, N. meningitidis, N. lactamica – POTENTIAL PATHOGEN?
RARE CASES SOME CROSS REACTIVITY WITH N. menigitidis OPPORTUNISTIC INFECTIONS

Neisseria gonorrhoeae gonorrhea known as STD by 1885 second only to chlamydia (CDC) Neisseria menigitidis isolated in 1887 septicemia and meningitis cases estimated to be between 500,000 and 1 million every year (Dietrich et al. 2003).

OTHER SPECIES NORMAL RESPIRATORY AND UROGENITAL FLORA

Neisseria spp. Common features……
Gram negative diplococci; "kidney bean shaped” nonmotile Aerobic
produce acid from carbohydrates oxidatively

Fastidious:
require chocolate agar and cysteine (N.gono)

cultivated in 5-10% carbon dioxide and humidity Susceptibly to drying and cold
Important for specimen collection All are oxidase positive and catalase positive

(except for two of the N. elongata subspecies).

The gonococcus

Neisseria Characteristics
Identification based on growth characteristics, carbohydrate utilization patterns, serological typing, auxotyping enzyme substrate procedures.
N. gonorrhoeae typed by fimbrial antigens (which are??) T1, T2, T3, and T4 Colonial morphology different Can change during culture

Grouped by auxotyping

N. meningitidis grouped by capsular polysaccharide Some groups include A, B, C, W-135, and Y

NEISSERIA GONORRHOEAE
GONOR < Gr = “SEED” RHOIA < Gr = “FLOW” A DISEASE ASSOCIATED WITH SEMEN MEDICAL DOCUMENTS AS EARLY AS 200AD
Virulence factors Fimbria enzymes that cleave iron from transferrin and lactoferrin IgA protease

Neisseria gonorrhoeae: Disease association
Sexually transmitted (males > females) 70% M→F: 30% F→M Males Copious urethral discharge (confined to urethra) Females Cervicitis + discharge, up to 80% asympt Complications Ascending infection (salpingitis and PID) Transmission to neonate (eye) Disseminated gonococcal infection

Clinical manifestations of N. gonorrhoeae infection

N. menigitidis

Clinical manifestations of N. meningitidis infection.

Starts with colonisation of nasopharynx. Spreads, crosses blood brain barrier. Symptoms: massive release of TNF due to LOS, LPS. Leads to deranged clotting mechanisms

NEISSERIA MENINGITIDIS: diverse disease spectrum
SEPTICEMIA, HIGH FEVER, ARTHRITIS PINPOINT HEMORRHAGES –PETECHIAE and BLOOD CLOTS FORMED IN VESSELS GRADUALLY PROGRESS TO PURPURA FULMINANS DISSEMINATED INTRAVASCULAR COAGULATION
INDISCRIMINANT CLOTTING THROUGHOUT VASCULAR SYSTEM DEPLETES CLOTTING FACTORS BLEED TO DEATH INTERNALLY

WATERHOUSE-FRIDERICHSEN SYNDROME
HEMORRHAGIC NECROSIS OF ADRENAL CORTEX ADRENAL APOPLEXY ADRENAL INSUFFICIENCY VASCULAR COLLAPSE

Cause of rash
Organism has lipooligosaccharide (LOS) component LOS activates host inflammatory mediators Septic shock can be produced Clotting aspect of septic shock blocks capillaries Clotting factors consumed leading to subcutaneous bleeding at other sites

Thrombotic emboli Clots lodge in capillaries of extremities Cause infarction and tissue loss.

Capsular serotypes
13 different types, almost all disease caused by A, B, C, W-135 and Y
No vaccine

Type B, the most common type in the Western world, does not yet have a vaccine types A, C, Y, and W have working vaccines that prevent bacterial infection.

Isolation of Neisseria gonorrhoeae and Neisseria meningitidis
Good specimen collection needed transport quickly to lab for cultivation Special transport media required
• do not refrigerate swab

enriched medium with increased carbon dioxide tension for growth is required Plates are incubated at 35-37°C under increased carbon dioxide tension (5-10% CO2) such as that provided by a candle jar or a carbon dioxide incubator.

Neisseria gonorrhea culture
Need an enriched, selective agar to inhibit the growth of commensal bacteria. also requires an atmosphere enriched in C02. absolute requirement for cysteine (N menig doesn’t) Can grow on BA in air, better on chocolate in CO2 Usually 48 - 72 hours on SELECTIVE media

Colony Morphology
four colony forms,
T1, T2, T3, and T4

Initially colonies are pinkish-brown. after 48 h. inc clear, golden-brown pigmentation autolysis of the cells.

T3 colonies 24 and 48 h

modified Thayer Martin (MTM)
antibiotics to inhibit normal body flora:
vancomycin to inhibit gram-positive bacteria; colistin to inhibit gramnegative bacteria; trimethoprim to suppress Proteus nystatin to inhibit yeast.

Added hemoglobin enrichment (chocolate colour) increased carbon dioxide tension
confirmed by doing an oxidase test

Other Media used for specimen plating
NYC – NEW YORK CITY MEDIA

VANCOMYCIN/LINCOMYCIN – ANTI GRAM POS COLISTIN – ANTI GRAM NEG AMPHOTERICIN B – ANTI-FUNGAL TRIMETHOPRIM – ANTI-PROTEUS

MARTIN-LEWIS Media

VANCOMYCIN – ANTI GRAM POS COLISTIN – ANTI GRAM NEG ANISOMYCIN – ANTI-FUNGAL TRIMETHOPRIM – ANTI-PROTEUS

Neisseria gonorrhoeae
Rapid diagnosis in males Direct exam of specimens
definitely Gram stain

Confirmed by culture Treatment
Penicillin no longer appropriate (resistance) IM ceftriaxone or ciprofloxacin PLUS a drug for chlamydia

Tests for presumptive identification of N. gonorrhoeae

Gram-negative diplococci oxidase-positive

Catalase +ve
grow on selective media Recovered from urogenital site

The Superoxol test

30% hydrogen peroxide Emulsify a few colonies with a loop in a drop of 30% w/v hydrogen peroxide. Immediate (within 12seconds) production of bubbles is defined as positive superoxol test.

30% hydrogen peroxide

CHO use
Neisseria can be differentiated by their ability to use the sugars: Glucose Sucrose Lactose Maltose These tests are done using a media with single carbohydrates ( glucose, maltose, sucrose, or lactose) and a pH indicator to detect acid.

Identification of : Carbohydrate Utilization

N gonorrhoeae

N .meningitidis

CHO utilisation

Easy to remember: G, gonorrhea, glucose-only; M, meningititis, maltose-also

CHO utilisation + DNAse + b-lac

New rapid ID methods for N.gono
Immunological testing

fluorescent antibody stains Direct antigen assays: EIA Chromogenic enzyme substrate testing isolation of specific enzymes via hydrolysis of substrate to produce a colored end product; GC is positive for hydroxyprolyl aminopeptidase only Auxotyping test for certain nutritional factors Nucleic acid methods

Immunological tests
Direct Fluorescent Antibody Test

Chromogenic enzyme substrate testing: Gonochek II
A tube test that designed to differentiate between N. lactamica, N. meningitidis, N. gonorrhoeae and B. catarrhalis. The enzymes are detected by the production of colored end products

Auxotyping: to identify strains
The complete Gonococcal Genetic Medium (GGM) contains 8 kinds of amino acids,
L-glutamic acid, L-arginine L-proline L-methionine(Met), L-aspartic acid(Ser), L-serine and L-isoleucine (ILe),

and 2 nitrogen bases, hypoxanthine (Hyx) and uracil (Ura), and agar, soluble starch, vitamins, coenzymes etc

majority of the strains belong to only two auxotypes, i.e. non requiring (NR) and proline requiring (Pro-) strains.

N. meningitidis ID
culture on an enriched, non-selective medium egs??? 3-7% carbon dioxide. MTM Chocolate agar is sometimes used Medium to large, bluegray, mucoid, convex, colonies form in 48 hours at 35-37°C.

Colonies become more golden during incubation

N. meningitidis
Superoxol test

N. menigitidis

Also oxidase positive catalase positive

CHO utilisation

Summary: simple flow chart

ID of Other species
N. lactamica

nonpathogenic inhabitant of upper respiratory tract therefore can be isolated from throat swab differentiate by growth on blood and MTM positive for glucose, maltose, and lactose; in chromogenic assay positive for b-gal and hydroxyprolyl aminopeptidase
Colonization with N. lactamica may be responsible for the increase in immunity to meningococcal disease that occurs during childhood

Other, non-pathogenic neisserias
N. sicca, N. subflava, N. flavescens, N. mucosa: rarely encountered N. cinerea occasional growth on selective media positive hydroxyprolyl aminopeptidase differentiate from GC with colistin (cinerea is sensitive) N. polysaccharea growth on MTM positive for glucose and maltose polysaccharides from sucrose negative for gamma-glutamyl aminopeptidase

both colistin resistant

Production of polysaccharide
Principle: Some bacterial species produce a starch-like polysaccharide from sucrose which stains dark blue-purple to black with iodine.

Production of Polysaccharide from Sucrose by N. polysaccharea, but not by N. cinerea.

Organisms to distinguish from Neisseria spp
Kingella Moraxella Acinetobacter

Kingella
oxidase positive catalase negative coccoid bacilli Kingella denitrificans
colistin-resistant & routinely isolated on selective medium for N. gonorrhoeae. cell morphology may appear to be diplococci and very similar in appearance to N. gonorrhoeae. The nitrate test differentes between N. gonorrhoeae (nitrate-negative) and K. denitrificans (nitrate-positive).

K. kingae
occasionally causes invasive disease, osteomyelitis/septic arthritis in young children, bacteremia in infants endocarditis in school-aged children and adults

Moraxella (Branhamella) catarrhalis
M catarrhalis used to be considered a saprophyte of the upper respiratory tract Now found commonly in otitis media and sinusitis Opportunistic:
nosocomial infections by droplets bronchitis or pneumonia in children and adults with underlying chronic lung disease occasionally bacteremia or meningitis,

mechanisms by which M. catarrhalis causes infections are not understood.

Moraxella (Branhamella) catarrhalis: ID
gram negative diplococci positive for oxidase and catalase DNAse +ve negative for chromogenic enzymes hydrolyze tributyrin (butyrate positive)

M. catarrhalis: Key differentiating tests
Similar colony appearance

but Asaccharolytic

and colistin sens

Cellular elongation test
Moraxella:
plate to blood, add penicillin disk, incubate; pick colony from zone of inhibition, Moraxella will appear as long, filamentous gram negative bacilli

Acinetobacter:
Acinetobacter baumannii, the hospital opportunist serious infections eg pneumonia multi –drug resistant oxidase negative coccoid bacilli

A Baumannii report The Iraq Infection: Forbes Magazine - August 2nd 2005
NEW YORK - Military doctors are fighting to contain an outbreak of a potentially deadly drug-resistant bacteria that apparently originated in the Iraqi soil. So far at least 280 people, mostly soldiers returning from the battlefield, have been infected, a number of whom contracted the illness while in U.S. military hospitals. No active-duty soldiers have died from the infections, but five extremely sick patients who were in the same hospitals as the injured soldiers have died after being infected with Acinetobacter baumannii.

Louis Pasteur
“The microbe is nothing; the terrain is everything.”

Summary: Neisseria meningitidis
Antigenic Structure and epidemiology

have pili for attachment IgA protease Cleavage of iron to free iron Antiphagocytic polysaccharide capsule Serogroups A, B, C, Y most involved in outbreaks
Pathogenicity of N. meningitidis

exposure begins with nasopharyngeal infection sometimes enters blood stream via cervical lymph nodes endotoxin may be produced third stage involves crossing of blood brain barrier to meninges can get DIC and Waterhouse Freidrichsen's syndrome
ID methods

direct exam of body fluid primary media: similar to GC grey round colonies that can grow on blood agar, Two D shaped diplococci oxidase positive catalase positive positive for glucose and maltose, but not fructose, lactose, or sucrose serological typing latex agglutination chromogneic enzyme substrate tests

Summary: N gono
Antigenic Structure and epidemiology
Fimbriated strains cause disease by attachment to mucosal cells and deter ingestion by PMN's serve in process of conjugation produce enzymes that cleave iron from transferrin and lactoferrin; iron is needed for colonization of mucosal surfaces Have IgA protease: ie cleave protective IgA on mucus membranes
Pathogenicity: STD, can invade BS - disseminate Correct specimen collection important Special Media used for specimen plating

Modified Thayer Martin (MTM): New York City Media Martin Lewis Media GC Lect Agar
Identification methods

incubate at 35C in 5-10% carbon dioxide opaque, convex, grey-white colonies on selective media gram negative diplococci oxidase positive catalase positive CHO use New rapid ID methods Auxotyping Chromogenic substrates Immunological methods


				
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