2. Introduction. Neisseria and Moraxella (Branhamella) catarrhalis are Gram negative cocci with the following general characteristicsKidney bean shapedArranged in pairs mostlyFlattened sides of
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1. 1 Neisseria and Moraxella catarrhalis The Gram Negative Diplococci
4. 4 continued Neisseria utilize sugars oxidatively only (acid in “O” tube)
M. catarrhalis is asaccharolytic – no acid produced from sugars
All Neisseria and M. catarrhalis are commensals of humans, except N. gonorrhoeae which is an obligate parasite (rare)
N. gonorrhoeae, N. meningitidis and M. catarrhalis are the major human pathogens in the group.
Other Neisseria species (as well as related genera Kingella and Eikenella) are rarely implicated in human infections
They are generally nutritionally fastidious: capnophilic & requiring X & V factors, and other enrichment. The major pathogens are the most fastidious in this order: N. gonorrhoeae, N. meningitidis, M. catarrhalis.
5. 5 continued The GC and MC grow best on enriched chocolate agar. Most commercial CA contains B vitamins and amino acids.
Some strains of GC (known as the AHU strains) cannot synthesize the amino acids arginine and hypoxanthine, nor the RNA nucleotide uracil.
Actually, all but GC will usually grow on SBA at 37oC, although not well.
If cultured from sites harboring normal microbiota, each of these pathogens are easily overgrown. Therefore, selective media should be used if normal microbiota are expected to be present
All selective and non-selective media should contain the AHU enrichments
6. 6 Selective Media for GC and meningococci
7. 7 Cultural requirements GC and MC are very sensitive to temperatures below 30oC
Clinical specimens suspected of harboring them should never be refrigerated and culture media should be brought to room temperature before inoculation
Plating GC at the “bedside” yields the highest number of positive cultures. Specimens collected away from the clinical lab require special handling
The typical transport media such as Stuart and Amies maintain viability for only a few hours. The inoculated transport media should be incubated at body temperature until they can be taken to the lab. How practical is this?
Body temperature incubation is continued in the lab for one to two more days at elevated [CO2]
8. 8 continued Transgrow and JEMBEC are perhaps the most commonly used GC media. They serve as both a transport enrichment medium and a culture medium. Transgrow is a bottled medium to which CO2 has been added from a cylinder of gas.
JEMBEC® is an agar medium that contains sodium bicarbonate, and is used in a unique plate with a receptacle for CO2 generating tablets. The plate is closed and sealed in a zip-lock pouch & trapped moisture combines with the bicarbonate and releases CO2
Some strains of GC are sensitive to vancomycin which is present in all selective media designed to recover pathogenic Neisseria species from clinical specimens that harbor normal microbiota
For this reason, it is advisable to use a nonselective chocolate medium in addition to one of the selective media
9. 9 Summary of Cultural Methods
10. 10 Colony morphology GC colonies are 1-2 mm in diameter after 24-48h. They are circular, slightly convex, moist, translucent and gray-white. After a few sub-cultures, GC colonies lose the ability to produce pili,causing them to become dryer
Colonies of meningococci are similar to GC except they are a little larger and may be mucoid
Colony morphology of nonpathogenic Neisseria is quite variable
Most nonpathogenic Neisseria produce some form of yellow colonies and tend to be dry and wrinkly
Colonies of M. catarrhalis are smooth gray-white opaque and convex, sometimes with a thin wavy periphery. Colonies are often granular, waxy and therefore difficult to emulsify
When lateral pressure is applied to a colony of M. catarrhalis using an inoculating loop the intact colony slides laterally ( this is known as the “hockey puck” test)
12. 12 N. meningitidis
13. 13 M. catarrhalis
14. 14 Microscopic morphology All Neisseria species and M. catarrhalis have very similar microscopic morphology as previously described
Infections of GC, meningococcus, and M. catarrhalis are usually highly purulent because the infection causes profuse PMN activity. The cells are often present inside the phagocytes
Infections of Gram-negative intracellular diplococci is suggestive of Neisseria. Any Neisseria isolated from a genital specimen, skin lesion, or normally sterile sites must be identified definitively. GC is never commensal.
GC infections are found primarily in genital specimens, but also in the eyes, sinsuses, respiratory tract, and in septic arthritis.
The meningococcus is found in CSF, blood specimens and skin lesions associated with dissemination. It is considered commensal in the throat and URT.
M. catarrhalis is most often associated with bacterial pneumonia
15. 15 Gram Negative Intracellular Diplococci
17. 17 Microscopic morphology Since each of the 3 pathogens discussed here can, on rare occasions, be found in any of these sites, the absolute identity can only be established by observing cultural and biochemical characteristics or performing genetic “probes”
18. 18 Presumptive identification The presence of oxidase positive Gram negative diplococci in pairs from cultures of selective media is very good presumptive evidence of Neisseria species or M. catarrhalis
N. gonorrhoeae is the only species in this group that gives a positive superoxol test, basically a modified catalase test
A drop of 30% hydrogen peroxide is added to a 18-24h culture of the suspected colony a non-blood containing medium
Instantaneous vigorous bubbling is a positive test
Other Neisseria will produce bubbling but it will be delayed by a second or two and will be a somewhat weaker response
M. catarrhalis is the only member of this group that gives a positive “hockey puck” test – remember the waxy colonies
19. 19 Definitive identification Carbohydrate utilization tests (profiles) are conducted:
Cystine-trypticase agar (CTA) basal medium with phenol red containing filter sterilized glucose, maltose, lactose, and sucrose is used
A heavy inoculum of a pure culture no more than 24h old (?) is stab inoculated into the upper half of the CTA
Tubes are incubated without added CO2 at 35oC. After 24h the upper portion of the tubes are observed for acid
This procedure will identify the classically pathogenic Neisseria, presumptively identify M. catarrhalis and group the nonpathogenic Neisseria – see table on next slide
20. 20 Carbohydrate Utilization of Neisseria
26. 26 Definitive identification Additional tests are required if CTA results are not equivocal
GC and meningococcus will not grow on T-Soy agar at 35oC whereas M. catarrhalis and nonpathogenic Neisseria will grow
GC and meningococcus will not grow at room temp period but M. catarrhalis and nonpath. Neisseria will grow on SBA or CA
Remember the superoxal test the GC – only GC is positive
N. lactamica produces beta galactosidase, are therefore is ONPG (o-nitrophenyl beta galactopyranoside – a lactose analogue) positive whereas all the others are negative
M. catarrhalis is the only member of the group that produces DNAase and lipase (tributyrin is a lipid used in a lipase test)
With rare exception, GC and meningococci are the only “Neisseria” types that grow on TM, MTM, or other selective “Neisseria” medium
27. 27 Supplemental tests for Neisseria
28. 28 Commercial products Several identification “kits” are available for these bacteria
Some are miniaturized versions of the carbohydrate utilization tests (e.g. Minitek, Quadferm & Biolog)
Most of the carbohydrate utilization tests function by simple pH indicator reactions (Minitek, API, etc). Some are chromogenic tests that detect specific enzymes via cleavage of a substrate analog yeilding a colored product. Others are based upon increased turbidity from growth in the presence of the carbs (Biolog).
Several products are based on detecting bacterial antigens in an isolated colony with a specific monoclonal antibody (ELISA, coagglutination, or immunofluorescence - FlAB)
DNA probes for GC are also commercially available
29. 29 Virulence factors N. gonorrhoeae - ?infectivity ?severity (fortunately)
Capsule (???): attachment & anti-phagocytosis
Fimbriae/pili: specific attachment to urogenital columnar epithelial cells
Lipopolysaccharide (endotoxin) = tumor necrosis factor
Cell wall proteins I, II, and III (interfere with phagocytosis)
IgA protease (cleaves IgA on mucosal surfaces)
ZERO conferred immunity – how?
30. 30 Pathology – N. gonorrhoeae GC is normally acquired by sexual contact, and is normally limited to mucosal columnar epithelial cells including cervix and urethra (dysuria), but also rectum, pharynx, and conjunctiva (corneal scaring & perforation, usually perinatal). Mucosal infections are usually characterized by a maked local neutrophilic response = copious purulent discharge, burning and itching.
GC LPS stimulates the production of tumor necrosis factor, which causes cell damage. Endocervical infection can lead to scared fallopian tubes leading to tubal (ectopic) pregnancy. Progression can lead to sterility.
GC dissemination and bacteremia manifest most commonly as a dermatits-arthritis syndrome, but in rare cases can result in endocarditis and meningitis
31. 31 Pathology continued N. meningitidis: The meningococcus is the #1 cause of meningitis in teenagers and young adults - a highly acute condition that usually follows a mildly symptomatic nasopharyngeal carrier state. Acute manifestations result from intracranial pressure (-itis), and the infection itself – mechanism is thought to be similar to GC.
Early symptoms include fever, malaise, headache & vomiting, progressing with seizures, altered mental status and coma. Fulminant cases have a high mortality rate.
M. Catarrhalis is the 3rd most common cause in the US of sinusitis and otitis media in children (3-4 million cases annually), and rarely progresses to pneumonia. It appears that pathology is related to endotoxin similar to the Neisseria.
32. 32 Antimicrobial Therapy GC was generally susceptible to penicillin in the US until 1976 when a pen-resistant strain was imported from Southeast Asia. This strain proliferated and spread within a few years. These strains are now detected using the cephalosporin (Cefinase) test
These strains are referred to as CMRNG (chromosome mediated resistant N.gonorrhoeae). Tetracycline and Spectinomycin chromosomal mediated resistance strains also occur.
Ceftriaxone, a third generation cephalosporin, is recommended for therapy by CDC.
Penicillin is still the drug of choice for treatment of infections caused by N. meningitidis
Most M. catarrhalis strains produce beta lactamase, and are therefore pen-resistant.